WO2005021744A1 - デジタル細胞 - Google Patents
デジタル細胞 Download PDFInfo
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- WO2005021744A1 WO2005021744A1 PCT/JP2004/009404 JP2004009404W WO2005021744A1 WO 2005021744 A1 WO2005021744 A1 WO 2005021744A1 JP 2004009404 W JP2004009404 W JP 2004009404W WO 2005021744 A1 WO2005021744 A1 WO 2005021744A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/48—Automatic or computerized control
Definitions
- the present invention is in the field of cell analysis technology. More particularly, it relates to a method for providing a profile of cells in the same environment and a system therefor, as well as data and data array technology and digital cell technology obtained by such technology.
- a method for providing a profile of cells in the same environment and a system therefor as well as data and data array technology and digital cell technology obtained by such technology.
- screening for functional interferences can be used to identify peptides that disrupt regulatory protein-protein interactions.
- This strategy (often referred to as dominant disturbing or dominant negative genetics) is favorable in some model organisms. Used successfully (where high-throughput screening methods are readily applied, and to a lesser extent in mammals (traditionally, mammals have One of the capabilities of the dominant negative strategy is that it accurately pinpoints the “pillar points” of functionally-related protein-protein interactions, and thus, is driven by external factors. The idea of exposing a small number of central points within a large network of protein networks subject to functional regulation, therefore, the consequences of a dominant-negative strategy are that crucial for regulatory components that define specific pathways. Important proteins that can provide information and are suitable for targeting by drug screening programs It may elucidate the protein interaction.
- the analysis is performed by focusing on only a specific point in time before and after a certain change, and it is not a method for analyzing a process of a temporal change taken by a certain point (gene). Absent.
- An object of the present invention is to provide a method for obtaining cell information (profile) data.
- the present invention also provides a method for obtaining information and data on the state of a cell under the same environmental conditions, and a method for accurately presenting such data.
- ⁇ Provide a system.
- a system or method presents information at the cell level under the same environmental conditions directly or directly from the viewpoint of a complex system, we will provide such data and data array technology itself. Make it an issue.
- Another object of the present invention is to provide a digital cell and a method of using the same. Summary of the Invention
- the above object has been achieved by immobilizing cells on a support and monitoring biological factors or aggregates thereof on or in the cells over time to generate data on the profile of the cells. .
- This allows for continuous collection of cell profiles.
- the generation of this data made it possible to reproduce the continuous state of cells, and it became possible to generate digital cells.
- the above object has also been attained by providing a support on which a plurality of cells can be placed under the same environment.
- Such a support has been achieved, for example, by fixing the cells using both a salt or an actin agonist, preferably a salt and actin agonist.
- the cell is fixed to the support by a composition comprising: a) a complex of a positively charged substance and a negatively charged substance; and b) a salt, and an actin acting substance.
- a composition comprising: a) a complex of a positively charged substance and a negatively charged substance; and b) a salt, and an actin acting substance.
- the above-mentioned profile is a cell morphology, and the above-mentioned method is selected from the group consisting of over-expression of a gene, under-expression or knockdown, addition of an exogenous factor, and changes in the environment. 2.
- the profile includes a profile file of the interaction between molecules present in the cell, and the method includes the steps of: The method of claim 1, further comprising the step of:
- the cell is selected from the group consisting of a stem cell and a somatic cell.
- step c) The method according to Item 52, wherein the determining step c) includes a mathematical processing selected from the group consisting of a signal processing method and a multivariate analysis.
- monitoring the profile in the step (b) includes obtaining image data from the array.
- the extrinsic factor is selected from the group consisting of temperature change, humidity change, electromagnetic wave, potential difference, visible light, infrared ray, ultraviolet ray, X-ray, chemical substance, pressure, gravity change, gas partial pressure and osmotic pressure.
- biomolecule is selected from the group consisting of a nucleic acid molecule, a protein, a lipid, a sugar, a proteolipid, a lipoprotein, a glycoprotein and a proteoglycan.
- biomolecule includes at least one biomolecule selected from the group consisting of a hormone, a cytokine, a cell adhesion factor, and extracellular matrix.
- a method for identifying an unidentified foreign factor given to a cell from a profile of the cell comprising:
- step (e) determining a profile corresponding to the profile obtained in step (e) from the profiles obtained in step (b);
- a method for identifying an unidentified external factor given to a cell from a cell profile comprising:
- the recording medium relates to at least one piece of information selected from the group consisting of information on conditions in the monitor, information on the open file, information on the state of the cells, and information on the biological factors.
- a support capable of maintaining a plurality of cells in the same environment
- the above-mentioned monitoring means includes an optical microscope, a fluorescence microscope, a phase microscope, and a laser microscope. Reader using the single light source, surface plasmon resonance (SPR) imaging, electrical signal, means using one or more of chemical or biochemical markers, synchrotron radiation, confocal microscope, non-confocal Item 89.
- the system according to Item 89 comprising at least one means selected from the group consisting of a microscope, a differential interference microscope, a stereo microscope, a video monitor and an infrared power camera.
- a support capable of maintaining a plurality of cells in the same environment
- the monitoring means may be any one of an optical microscope, a fluorescence microscope, a phase microscope, a reader using a laser light source, surface plasmon resonance (SPR) imaging, an electrical signal, a chemical or biochemical marker.
- Item 93 including at least one means selected from the group consisting of: or using multiple species, synchrotron radiation, confocal microscope, non-confocal microscope, differential interference microscope, stereo microscope, video monitor and infrared camera. The described system.
- the means for presenting the data is a display, the system described in item 93.
- the system according to item 93, wherein the means for presenting the data is a speaker.
- a support capable of maintaining a plurality of cells in the same environment
- a support capable of maintaining a plurality of cells in the same environment
- a support capable of maintaining a plurality of cells in the same environment
- a recording medium storing data relating to a correlation between a known foreign factor and a profile of the cell corresponding to the known foreign factor with respect to a biological factor or a collection thereof on or in the cell.
- f means for determining, from the profiles stored in the recording medium (a), a profile corresponding to a profile obtained for an unknown external factor, wherein the unidentified external factor is Said known extrinsic factor corresponding to the determined profile, means;
- a system. (103) A support capable of fixing a plurality of cells and maintaining the same environment of the cells.
- the support according to item 103 comprising a salt and a complex of a positively charged substance and a negatively charged substance, or an actin acting substance.
- the support according to item 103 comprising a salt and a complex of a positively charged substance and a negatively charged substance, further comprising an actin agonist.
- the above salts are calcium chloride, sodium hydrogen phosphate, sodium hydrogen carbonate 115.
- the support according to item 114 comprising a salt selected from the group consisting of thorium, sodium pyruvate, HEPES, calcium chloride, sodium chloride, potassium chloride, magnesium sulfide, iron nitrate, amino acids and vitamins.
- the gene introduction reagent includes at least one reagent selected from the group consisting of a cationic polymer, a cationic lipid, a polyamine-based reagent, a polyimine-based reagent, calcium phosphate, oligofectamine and oligofecta, 114.
- a cationic polymer a cationic polymer
- a polyamine-based reagent a polyimine-based reagent
- calcium phosphate oligofectamine and oligofecta
- actin acting substance comprises at least one protein selected from the group consisting of fibronectin, laminin, and vitronectin, or a variant or fragment thereof.
- nucleic acid molecule includes a sequence encoding a protein selected from the group consisting of cytokines, hormones, cell adhesion factors, cytoskeletal proteins, and enzymes.
- a mixture of the salt, the gene introduction reagent as the positively charged substance, the actin acting substance, the nucleic acid molecule as the negatively charged substance, and the cell is prepared by: See item 121, including securing in an array. The method described.
- An apparatus for producing a support comprising a plurality of fixed cells and capable of maintaining the same cell environment
- An apparatus comprising:
- the apparatus according to item 125 wherein the fixing means includes a printing means. (127) The apparatus according to item 125, wherein the support providing means includes means for molding the support from a support material.
- the stimulation parameters include a parameter indicating a reporter and a parameter indicating chemical stimulation.
- the stimulus response result includes data of the profile of the cell obtained by monitoring a biological factor or an aggregate thereof on or in the cell over time. The method described in.
- f) means for generating at least one set of experimental data for the cells by repeating steps a) to e) as necessary, and providing the set of at least one experimental data as digital cells;
- each of the at least one digital cell is represented by at least one set of experimental data for a cell to be tested;
- Each of the one experimental data includes a cell parameter for specifying the cell, an environment parameter for specifying an environment for culturing the cell specified by the cell parameter, and a puncture applied to the cell specified by the cell parameter.
- a stimulus parameter specifying the intensity and a result of the response of the cell specified by the cell parameter to the stimulus specified by the stimulus parameter under the environment specified by the environmental parameter.
- the service requester receiving the cell parameter, the environment parameter, and the stimulus parameter, and generating a request including the cell parameter, the environment parameter, and the stimulus parameter;
- the service requester providing the request to the service provider
- the service provider searches the database in response to the request, and stores in the database the stimulus response result related to the cell parameter, the environmental parameter, and the stimulus parameter included in the request. Determining whether it is present;
- the service provider determines the stimulus response result. Providing to the above service requester;
- the service requester displaying the stimulus response result
- each of the at least one digital cell is represented by a set of at least one experimental data for a cell to be tested;
- Each of the at least one experimental data includes a cell parameter specifying the cell, an environmental parameter specifying an environment for culturing the cell specified by the cell parameter, and the cell specified by the cell parameter.
- a stimulus parameter specifying a stimulus to be given and a result of the cell specified by the cell parameter responding to the stimulus specified by the stimulus parameter under the environment specified by the environmental parameter A stimulus response result;
- the service requester receiving the cell parameter, the environmental parameter, and the stimulus parameter, and generating a request including the cell parameter, the environmental parameter, and the stimulus parameter;
- the service requester responds to the request by the service register Retrieving a bird and determining whether there is a service provider capable of providing the service of the request among the plurality of service providers;
- the service requester providing the request to the service provider if it is determined that there is a service provider that can provide the service of the request among the plurality of service providers;
- the service provider searches the database in response to the request, and determines whether the database has the stimulus response result related to the cell parameter, the environmental parameter, and the stimulus parameter included in the request. Determining whether;
- the service provider determines the stimulus response result. Providing to the above service requester;
- the service requester displaying the stimulus response result
- a service provider configured to access a database containing at least one digital cell, wherein each of said at least one digital cell is represented by a collection of at least one experimental data for a cell under test.
- each of the at least one experimental data includes a cell parameter specifying the cell, an environment parameter specifying an environment for culturing the cell specified by the cell parameter, and the environmental parameter specified by the cell parameter.
- a service provider including a stimulus response result indicating a result of the response;
- the stimulus response result is provided to the service requester. Means to do;
- the service requester is a web browser operated by the user, and the service provider is a web server connected to the service requester via the Internet.
- Item 13 6. The combination system according to 6.
- (138) The combination system according to item 1336, wherein the service requester provides the request to the service provider in a format described in XML.
- a plurality of service providers each of the plurality of service providers being configured to be able to access a database storing at least one digital cell, wherein each of the at least one digital cell is
- Each of the at least one experimental data is represented by a cell parameter specifying the cell and culturing the cell specified by the cell parameter.
- An environmental parameter for specifying an environment, a stimulus parameter for specifying a stimulus given to the cell specified by the cell parameter, and the cell specified by the cell parameter under the environment specified by the environmental parameter According to the above stimulus parameters
- a plurality of service providers including a stimulus response result indicating a result of responding to the identified stimulus;
- a service registry that has registered at least one service that can be provided by the plurality of service providers.
- the stimulus response result is provided to the service requester. Means to do;
- the service requester is a Web server connected to a Web browser operated by the user via the Internet, and each of the plurality of service providers is connected to the service requester via the Internet.
- 140. The computer system according to item 140, wherein the computer system is a web server connected to the computer.
- 142 The combination system according to item 140, wherein the service requester provides the request to the service provider in a format described in XML.
- a method for generating profile data on cell information comprising: a) fixing and arranging cells on a support;
- the cell is fixed to the support by a composition comprising: a) a complex of a positively charged substance and a negatively charged substance; and b) a salt, and the actin acting substance is 144.
- a composition comprising: a) a complex of a positively charged substance and a negatively charged substance; and b) a salt, and the actin acting substance is 144.
- the biological factor includes a nucleic acid molecule encoding a gene.
- the method according to 144 is a nucleic acid molecule encoding a gene.
- the above-mentioned profile includes a profile of intermolecular interaction.
- the profile is in the form of a cell, and the method comprises applying a stimulus to the cell, the stimulus being selected from the group consisting of overexpression, underexpression or knockdown of a gene, addition of an exogenous factor, and environmental change.
- a stimulus being selected from the group consisting of overexpression, underexpression or knockdown of a gene, addition of an exogenous factor, and environmental change.
- the profile includes a profile of an interaction between molecules existing in the cell, and the method includes a step of performing observation using a technique selected from the group consisting of a two-hybrid method, FRET, and BRET.
- a technique selected from the group consisting of a two-hybrid method, FRET, and BRET.
- a method according to item 144 comprising a step of comparing phases of the profiles. (189) The method according to item 144, comprising a step of taking a difference between the cell profile and the control profile.
- the present invention provides continuous information (profile) data on the state of cells.
- Information and data (especially continuous information and continuous profiles) on the state of cells under the same environmental conditions can be obtained with good reproducibility.
- the present invention provides a method and system for accurately presenting such data.
- the surprising effect of the system and method for presenting information at the cellular level under the same environmental conditions directly or directly from the viewpoint of a complex system and such data and data arrangement technology itself has been provided. It is.
- the present invention has the further advantage of providing a digital cell based on actual raw data and a method of using the same, which was previously impossible.
- the present invention has made it possible to determine, test and study the state of cells by observing surprisingly few factors. Such a judgment makes it possible to apply it to diagnosis, prevention, and treatment, and its application extends not only to medicine but also to various fields such as food, cosmetics, agriculture, and the environment. In addition, the ability to reproduce live experiments on a computer has the effect of enabling education and research in biotechnology.
- FIG. 1 shows an example of the results of using various actin-acting substances and gelatin as a control when HEK293 cells were used. 9 shows the effect of attached molecules on transfection efficiency.
- PEGFP-N1 was transfected into HEKK293 cells by using Effecttene reagent.
- FIG. 2 shows an example of the results of transfection efficiency using a fibronectin fragment.
- FIG. 3 shows an example of the results of transfection efficiency using a fibronectin fragment.
- FIG. 4 shows an example of the results of transfection efficiency when the fibronectin fragments summarized from FIGS. 2 and 3 are used.
- FIG. 5 shows an example of the results of examining the transfusion efficiency in various cells.
- FIG. 6 shows an example of the results showing the state of the transfer extrusion when various plates were used.
- Figure 7 shows that transfection was performed on various plates with fipronectin concentrations of 0, 0.27, 0.53, 0.8, 1.07 and 1.33 ( ⁇ gZ x L each). The result of the case shown below is shown.
- FIG. 8 shows an example of a photograph showing a cell adhesion profile with and without fibronectin.
- FIG. 9 shows an example of a section photograph showing a cell adhesion profile with and without fibronectin.
- hMSC human mesenchymal stem cells
- Figure 10 shows the evolution of the surface area of the nucleus. Section of confocal laser scanning microscope image. Relative nuclear surface area determined by observation. Human mesenchymal stem cells were fixed by incubating several times with 4% PFA.
- FIG. 11 shows an example of the result of a transfection experiment when constructed as a transfection array chip.
- FIG. 12 is an example showing the state of contamination between spots on the array.
- FIG. 13 is a diagram showing the incorporation of spatially separated DNA into cells by the solid-phase transfection of the present invention in Example 4.
- FIG. 13A is a diagram schematically showing a method for producing a solid-phase transfection array (SPTA). This figure illustrates the methodology of solid phase transfection.
- FIG. 13B shows the results of the solid-phase transfer extrusion. The results of producing SPTA using the HEK293 cell line are shown. The green part indicates the transfected adherent cells. From this result, the method of the present invention made it possible to prepare a spatially separated population of cells transfected by different genes.
- FIG. 13 AB shows the scheme of the transaction (SPTA) as a whole.
- FIG. 13A shows the outline of SPTA determination
- FIG. 13B shows the results of SPTA in the HEK 293 cell line. Par is 3 mm.
- FIG. 13C shows a solid-state transfection methodology.
- FIGS. 14A and 14B show the results of comparison between liquid phase transfection and SPTA.
- FIG. 14A shows the results of measuring the GFP intensity / mm 2 for the five cell lines used in the experiment.
- FIG. 14A shows a method of determining the transfection efficiency as the total fluorescence intensity per unit area.
- FIG. 14B is a fluorescence image of a cell expressing EGFP corresponding to the data shown in FIG. 14A.
- a region indicated by a white circle indicates a region in which the plasmid DNA is immobilized.
- EGFP-expressing cells were not observed even though the cells were immobilized on the solid phase.
- White bars indicate 500 ⁇ .
- the measured fluorescence / mm 2 corresponds to FIG. 14A for the 5 cell lines.
- 4 is a fluorescence photograph of EGFP-expressing cells.
- the white circle corresponds to the plasmid DNA print region. Cells in the region outside this region express EGFP, but cells also adhere to regions other than the print region.
- FIG. 14C shows an example of the transfer extrusion method of the present invention.
- FIG. 14D shows an example of the transfection method of the present invention.
- FIG. 15 shows the result of reducing the cross contamination by coating the chip.
- Figure 15 shows a liquid phase transfer method using HEK293 cells, HeLa cells, NIT3T3 cells (shown as “3T3”), HepG2 cells, and! IMSC. The result of performing A is shown. Transfection efficiency is indicated by GFP intensity.
- FIG. 15A The transfection efficiency of hMSC according to the N / P ratio used is shown in Figure 15A.
- FIG. 15B In the case of conventional liquid phase transfection (FIG. 15B, upper), hM SC cells are killed, and in the case of SPTA, the cell line pair is normal (FIG. 15B, lower).
- FIG. 16 is a diagram showing a situation regarding mutual contamination between spots. A nucleic acid mixture containing a predetermined concentration of fibronectin was immobilized on a chip coated with APS or PLL (poly-L-lysine), and cell transfection was performed using the immobilized chip. Was not observed (top and interrupted). In contrast, when the chip was not coated, significant mutual contamination of the immobilized nucleic acid was observed (lower).
- pEGFP—N1 and pDs Red 2—N1 are printed in a checkered pattern and HMSC (panel A) or HEK293 (panel B).
- FIG. 16C shows the correlation between the type of substance used in the mixture used in the immobilization of nucleic acids and the rate of cell adhesion.
- This graph shows the increase in the percentage of adherent cells over time. A gradual slope of the graph indicates that more time is required for cell adhesion than a steeper graph.
- FIG. 16D is an enlarged view of the graph in FIG. 16C.
- FIG. 17 shows a configuration example when the method of the present invention is executed on a computer.
- FIG. 18A shows an example of the mathematical analysis method of the present invention. Measure the time course of the fluorescence intensity of the promoter file as shown in Figure 18A (mean pNEFAT-d2 EGF PZ negative control) and Figure 18B (mean pERE-d2EGF P / negative control). To get by. This profile has been normalized using the autofluorescence of the cells or medium. Thereafter, in order to compare the amplitudes of the reporter expression fluctuations, the expression fluctuations with an amplitude width of 5 or more (TH ⁇ 5) were judged to have changed state. In addition, the sections were divided into early (0-17.5 hours) and late (17.5-31.5 hours) and total (0-31.5 hours) sections of differentiation induction.
- FIG. 18B shows another example of the mathematical analysis method of the present invention.
- a + B + ... n When extracting an arbitrary reporter (A + B + ... n), n waveforms are integrated, and this is divided by n to create an average waveform, and fluctuations above the threshold are regarded as changes.
- Figure 18B Left, two The reporter profile is integrated and the average waveform is drawn with a red line (black square). Changes in the average profile of 5 or more were evaluated as expression changes. Then, as shown in the table below, you can evaluate the fluctuation of the two extracted reporters.
- FIG. 19 shows an example of the promoter-containing plasmid used in the present invention and an example of the prayer of the present invention.
- the 17 types of transcription factors shown on the left in Fig. 19 were used as repo overnight, and their expression profiles were obtained over time (Fig. 19 right).
- An arbitrary number of profiles were extracted from these 17 types of profiles, and evaluated by the method described above (FIG. 18) based on the fluctuation width of the response profile of each transcription factor.
- FIG. 20 shows an example of the result of mathematical analysis in the early stage of differentiation induction.
- the results as shown in FIG. 20 were obtained.
- the number of extractions an arbitrary number of repos were extracted from each of the 17 reporter groups, the average profile was calculated by the method shown in Fig. 18, and those with a fluctuation range ⁇ 5 were derived. The results were evaluated in the sections from 0 to 31.5, 0 to 17.5, and 17.5 to 31.5 hours from the start.
- the number of extractions is 17 ways. However, the number of extractions 17 is one.
- the ratios of some combinations determined to have fluctuation are shown in the table in FIG. 20 and in the graph of FIG.
- FIG. 21 shows an example of the results of mathematical analysis in maintaining undifferentiation. As in Fig. 20, when any combination was extracted under the undifferentiated maintenance condition, the results shown in the graph were obtained. Compared to the results of differentiation induction described above in FIG. From this comparison, is the cell heading for differentiation induction? W
- FIG. 22 shows a schematic diagram of the cocktail party process.
- FIG. 23 shows an example of construction of a gene transcription switch reporter (transfection plasmid used in the present invention).
- FIG. 24 shows an example of construction of a transcription factor reporter set.
- FIG. 25 shows an example of the transcription factor reporter.
- FIG. 26 shows an example of time-series measurement of transcription factor activity in the process of bone differentiation.
- FIG. 27 shows an example of oscillation phenomenon and phase analysis of transcription factor activity.
- FIG. 28 shows the protocol of the siRNA experiment.
- FIG. 29A shows the results of a si RNA experiment.
- the top shows the results for hMS C, and the bottom shows the results for HeLa cells.
- the numbers indicate the concentration of siRNA ( ⁇ g / ⁇ L).
- the results with anti-GFP siRNA are shown on the left, and the results with scrambled siRNA are shown on the right.
- FIG. 29B shows the effect of si RNA when solid phase transfection (PC 12) was performed on collagen IV coating.
- FIG. 29B (A) shows PC12 cells cotransfected with EGFP vector and anti-EGF Psi RNA. As shown, it was found that only He Red developed color, and the green signal derived from pEGFP-Nl was suppressed.
- FIG. 29B (B) shows an example using scrambled siRNA. As shown, green fluorescence was observed, confirming that the effect in FIG. 29B (A) was due to RNAi.
- FIG. 29B (C) shows the relative intensities of the fluorescence in FIGS. 29B (A) and 29B (B). The y-axis is shown by relative luminance. It can be seen that the effect of EGFP is almost completely suppressed.
- Fig. 29C shows a summary graph of the results.
- the left panel is a photograph comparing EGFP RNAi with scrambled (Mock) RNAi when the ratio of RNAi to pDNA was varied.
- E GFP RNAi showed an inhibitory effect whereas scrambled did not.
- D s Red 2 in the right panel.
- the experimental conditions were as described above. As a result, red (signal derived from DsRed) and green (signal derived from EGFP) were shown in proportion to the effect of RNAi.
- FIG. 29D shows a schematic diagram of a chip using the RNAi reporter.
- RNAi When RNAi is used as an input signal, when a gene product capable of transmitting a signal such as EGF and a nucleic acid encoding a target gene (including a promoter) are introduced together as the output, the signal is transmitted as an output. By observing, it is possible to extract cell information.
- Figure 29E shows the different reporters (pAPl-EGFP, pAPl (PMA) pMyc-EGFP, pNFAT-EGFP, pNFkB-EGFP, pRARE-EGFP, pRb-EGFP, pSTST3-EGFP, pSRE-EGFP, pTRE-EGFP, pp53-EGFP, pCREB-sensor, plkB-sensor, pp53-sensor, pCasapase3- sensor; cis-element system [J is purchased from Clontech. Plasmid vector created by recombination of fluorescent protein gene].
- FIG. 30 shows the appearance of changes when using a tetracycline-dependent promoter.
- FIG. 31 is a diagram showing the expression state when a tetracycline-dependent promoter and a tetracycline-independent promoter are used.
- FIG. 31B shows a schematic diagram in which neurons were analyzed for the effects of the tyrosine kinase RN Ai using a transfection microarray.
- FIG. 31C shows the response of retinoic acid (RA) and nerve growth factor (NGF) by various tyrosine kinases. Inhibition with siRNA. /. showed that.
- FIG. 31D shows a schematic diagram of the signal transduction pathway obtained as a result of the analysis.
- Figure 3 IE shows the results obtained from the above analysis. This is a comprehensive analysis of tyrosine kinases responsible for human neuron differentiation. It is classified as a dopaminergic neuron, a cholinergic neuron, or both, or not both. It can be analyzed that those involved in both are likely to be involved in neurite formation.
- FIG. 31F is an example showing real-time monitoring of transcriptional regulation of apoptosis in HeLa cells.
- the left panel shows the results of the analysis over time and the right panel shows the results of the signal transduction pathway analysis obtained based on the analysis.
- Figure 32 shows a system configuration example.
- FIG. 33A is an example of a digital cell of the present invention.
- FIG. 33B is another example of the digital cell of the present invention.
- FIG. 34 shows an example of the method for producing digital cells of the present invention.
- FIG. 35 shows an example of the configuration of a computer system 3501 that provides a service for reproducing experimental results on real cells using digital cells.
- FIG. 36 shows an example of a procedure of a process for providing a service for reproducing an experiment result on a real cell using a digital cell.
- FIG. 37 shows an example of an input screen for inputting a cell parameter, an environmental parameter, and a stimulus parameter to the service requester 3510.
- FIG. 380 shows an example of the configuration of a computer system 3801, which provides a service for reproducing experimental results on real cells using digital cells.
- Fig. 39 shows an example of the procedure for providing a service that reproduces the results of experiments on real cells using digital cells. Description of Sequence Listing
- SEQ ID NO: 1 Fibronectin nucleic acid sequence (human)
- SEQ ID NO: 2 Amino acid sequence of fibronectin (human)
- SEQ ID NO: 3 Nucleic acid sequence of vitronectin (mouse)
- SEQ ID NO: 4 Amino acid sequence of vitronectin (mouse)
- SEQ ID NO: 5 Laminin nucleic acid sequence (mouse ⁇ -chain)
- SEQ ID NO: 6 Amino acid sequence of laminin (mouse ⁇ -chain)
- SEQ ID NO: 7 Laminin nucleic acid sequence (mouse chain)
- SEQ ID NO: 8 amino acid sequence of laminin (mouse chain)
- SEQ ID NO: 9 Nucleic acid sequence of laminin (mouse ⁇ chain)
- SEQ ID NO: 10 Laminin amino acid sequence (mouse ⁇ chain)
- SEQ ID NO: 11 Amino acid sequence of fibronectin
- SEQ ID NO: 12 s i RNA used in Examples
- SEQ ID NO: 13 Nucleic acid of mouse olfactory receptor I7 (heptanal-sensitive) (Genbank accession number (AccessionNumber) AF 106007).
- SEQ ID NO: 14 protein encoded by the nucleic acid set forth in SEQ ID NO: 13
- SEQ ID NO: 15 Nucleic acid of mouse olfactory receptor S 1 (mc 9 / bc 9-equi-sensitev) (Genbank accession number AF121972).
- SEQ ID NO: 16 protein encoded by the nucleic acid set forth in SEQ ID NO: 15
- SEQ ID NO: 17 Nucleic acid of mouse olfactory receptor S50 (c c9-se ns i t i v e) (GenBank accession number AF 121980).
- SEQ ID NO: 18 protein encoded by the nucleic acid set forth in SEQ ID NO: 17
- SEQ ID NO: 19 Nucleic acid of mouse olfactory receptor S19 (mc9 / mh9 / bc9-equi-senisite) (Genbank accession number AF121976).
- SEQ ID NO: 20 protein encoded by the nucleic acid set forth in SEQ ID NO: 19
- SEQ ID NO: 21 Nucleic acid of mouse OR 23 (lyral-sensitive) (Genbank accession number X92969 coding region only).
- SEQ ID NO: 22 protein encoded by the nucleic acid set forth in SEQ ID NO: 21
- SEQ ID NO: 23 mOR for mouse olfactory receptor—nucleic acid of EV (vani 11 inn—senstive) (Genbank accession number AB061)
- SEQ ID NO: 24 protein encoded by the nucleic acid set forth in SEQ ID NO: 23
- SEQ ID NO: 25 mouse. r 37 a nucleic acid (G en b a n k accession number A J 1
- SEQ ID NO: 26 protein encoded by the nucleic acid set forth in SEQ ID NO: 25
- SEQ ID NO: 27 Nucleic acid of mouse olfactory receptor C6 (GenBank accession number AF 102523).
- SEQ ID NO: 28 protein encoded by the nucleic acid set forth in SEQ ID NO: 27
- SEQ ID NO: 29 Nucleic acid of mouse olfactory receptor F5 (Genbank accession number AF102531).
- SEQ ID NO: 30 protein encoded by the nucleic acid set forth in SEQ ID NO: 29
- SEQ ID NO: 31 Nucleic acid of mouse olfactory receptor S6 (Genbank accession number AF121974).
- SEQ ID NO: 32 protein encoded by the nucleic acid set forth in SEQ ID NO: 31
- SEQ ID NO: 33 Nucleic acid of mouse olfactory receptor S18 (GenBank accession number AF121975)
- SEQ ID NO: 34 protein encoded by the nucleic acid set forth in SEQ ID NO: 33
- SEQ ID NO: 35 Nucleic acid of mouse olfactory receptor S25 (GenBank accession number AF121977).
- SEQ ID NO: 36 protein encoded by the nucleic acid set forth in SEQ ID NO: 35
- SEQ ID NO: 37 Nucleic acid of mouse olfactory receptor S46 (GenBank accession number AF121979).
- SEQ ID NO: 38 protein encoded by the nucleic acid set forth in SEQ ID NO: 37
- SEQ ID NO: 39 Nucleic acid of mouse G protein ⁇ subunit (Genba ⁇ k accession number M36778).
- SEQ ID NO: 40 protein encoded by the nucleic acid set forth in SEQ ID NO: 39
- SEQ ID NO: 41 nucleic acid of mouse G protein i3 subunit (GenBank accession number M87286).
- SEQ ID NO: 42 protein encoded by the nucleic acid set forth in SEQ ID NO: 41
- SEQ ID NO: 43 nucleic acid of mouse G protein ⁇ subunit (GenBank accession number U37527)
- SEQ ID NO: 44 protein encoded by the nucleic acid set forth in SEQ ID NO: 43
- SEQ ID NO: 45 Nucleic acid of mouse epidermal growth factor (EGF) receptor (GenBank accession number BC023729).
- SEQ ID NO: 46 protein encoded by the nucleic acid set forth in SEQ ID NO: 45
- SEQ ID NO: 47 Sequence of si RNA used in Example 9.
- SEQ ID NO: 48 Sequence of scrambled RNA used in Example 9.
- cell is defined in the broadest sense as used in the art, and is a constituent unit of a tissue of a multicellular organism, wrapped in a membrane structure that isolates the outside world, An organism that has self-renewal ability and has genetic information and its expression mechanism.
- the cell used in the present specification may be a naturally occurring cell or an artificially modified cell (for example, a fused cell, a genetically modified cell).
- the source of cells can be, for example, a single cell culture or cells from a normally grown transgenic animal embryo, blood, or body tissue, or a normally grown cell line. Cell mixtures such as, but not limited to.
- digital cell refers to at least one set of experimental data for a cell under test. These experimental data relate the experimental conditions and results of experiments performed on real cells. Digital cells are constructed so that given experimental conditions, the experimental results associated with the experimental conditions can be reproduced. Digital cells envisioned herein include all experimental cells. Therefore, it should be understood that all cell descriptions described herein also apply to digital cells where applicable.
- the cells used in the present invention may be cells from any organism (eg, unicellular organisms of any kind (eg, bacteria, yeast) or multicellular organisms (eg, animals (eg, vertebrates, invertebrates), plants) (Eg, monocotyledonous plants, dicotyledonous plants, etc.)).
- organisms eg, unicellular organisms of any kind (eg, bacteria, yeast) or multicellular organisms (eg, animals (eg, vertebrates, invertebrates), plants) (Eg, monocotyledonous plants, dicotyledonous plants, etc.)).
- cells derived from vertebrates are used, and more specifically, mammals (eg, monopores) Species, marsupials, oligodonts, skin wings, winged wings, carnivores, carnivores, longnoses, hoofed horses, artiodactyla, tube teeth,. , Primates, rodents, etc.).
- mammals eg, monopores
- primates eg, chimpanzees, macaques, humans
- the cells used in the present invention may be stem cells or somatic cells. Such cells may also be adherent cells, floating cells, tissue forming cells, and mixtures thereof. Such cells may be those used for transplantation purposes.
- an organ when an organ is a target, such an organ may be any organ, and a tissue or a cell targeted by the present invention may be derived from any organ or organ of an organism. Les ,.
- organ or “organ” is used interchangeably, and a function of a living individual is localized and operated in a specific part of the individual, and the part is morphologically independent. Structure. In general, in multicellular organisms (eg, animals, plants), organs consist of several tissues with a specific spatial arrangement, and tissues consist of many cells. Such organs or organs include those involved in the vasculature.
- the organs targeted by the present invention include skin, blood vessels, cornea, kidney, heart, liver, umbilical cord, intestine, nerve, lung, placenta, kidney, brain, peripheral extremities, retina, and the like.
- the cells separated from the pluripotent cells of the present invention include epidermal cells, knee parenchymal cells, knee duct cells, hepatocytes, blood cells, cardiomyocytes, skeletal muscle cells, Examples include, but are not limited to, osteoblasts, skeletal myoblasts, nerve cells, vascular endothelial cells, pigment cells, smooth muscle cells, adipocytes, bone cells, chondrocytes, and the like.
- tissue refers to a cell population having substantially the same function and / or morphology in a multicellular organism.
- tissues can be referred to as tissues, even if they are cell populations having the same origin, but different origins, if they have the same function and / or morphology. Therefore, when regenerating a tissue using the stem cells of the present invention, a cell population having two or more different origins can constitute one tissue.
- tissue forms part of an organ. Animal tissues can be divided into epithelial, connective, muscle, and nervous tissues based on morphological, functional, or developmental evidence. Plants are classified into meristems and permanent tissues according to the stage of development of the constituent cells, and are classified into single tissues and complex tissues according to the type of the constituent cells.
- stem cell refers to a cell that has self-renewal ability and has pluripotency (ie, pluripotency) (“: luripotency”).
- pluripotency ie, pluripotency
- Stem cells can usually regenerate tissue when it is damaged.
- a stem cell can be, but is not limited to, an embryonic stem (ES) cell or a tissue stem cell (also referred to as a tissue stem cell, a tissue-specific stem cell, or a somatic stem cell).
- ES embryonic stem
- tissue stem cell also referred to as a tissue stem cell, a tissue-specific stem cell, or a somatic stem cell.
- artificially produced cells can also be stem cells as long as they have the above-mentioned ability.
- Embryonic stem cells refer to pluripotent stem cells derived from early embryos.
- tissue stem cells unlike embryonic stem cells, are cells in which the direction of differentiation is restricted, exist at specific locations in the tissue, and have an undifferentiated intracellular structure. Thus, tissue stem cells have a low level of pluripotency. Tissue stem cells have a high nuclear / cytoplasmic ratio and poor intracellular organelles. Tissue stem cells are generally pluripotent, have a slow cell cycle, and maintain their proliferative potential over the life of an individual. In this specification If used, the stem cells may be embryonic stem cells or tissue stem cells.
- Tissue stem cells can be classified into, for example, the skin system, digestive system, myeloid system, and nervous system when classified according to the site of origin.
- Skin tissue tissue stem cells include epidermal stem cells and hair follicle stem cells.
- Gastrointestinal tissue stem cells include knee (common) stem cells and hepatic stem cells.
- myeloid stem cells include hematopoietic stem cells and mesenchymal stem cells.
- Tissue stem cells of the nervous system include neural stem cells and retinal stem cells.
- somatic cell refers to a cell other than a germ cell, such as an egg or sperm, and refers to any cell that does not directly transfer its DNA to the next generation. Somatic cells usually have limited or lost pluripotency.
- the somatic cells used in the present specification may be naturally occurring or genetically modified.
- Cells can be classified according to their origin into stem cells derived from ectoderm, mesoderm and endoderm.
- the cells derived from the ectoderm mainly exist in the brain, and include neural stem cells and the like.
- Mesodermal-derived cells are mainly present in bone marrow and include vascular stem cells, hematopoietic stem cells, mesenchymal stem cells, and the like.
- Endoderm-derived cells are mainly present in organs and include hepatic stem cells, knee stem cells, and the like.
- somatic cells can be from any germ layer.
- lymphocytes, spleen cells or testis-derived cells can be used. .
- isolated refers to a substance that is naturally associated with the substance in a normal environment is at least reduced, and preferably substantially free.
- an isolated cell refers to a cell that is substantially free from other attendant materials (eg, other cells, proteins, nucleic acid molecules, etc.) in its natural environment.
- isolated refers to substantially isolating cellular material or culture medium, for example, when made by recombinant DNA technology.
- a nucleic acid molecule or polypeptide that is substantially free of precursor or other chemicals when chemically synthesized.
- the isolated nucleic acid molecule is preferably a sequence that naturally flanks the nucleic acid molecule in the organism from which the nucleic acid molecule is derived (ie, located at the 5, and 3, terminal ends of the nucleic acid). Array) is not included.
- an “established” or “established” cell is one that maintains a particular property (eg, pluripotency) and that can continue to proliferate stably under culture conditions. It becomes the state that became. Therefore, the established stem cells maintain pluripotency.
- a particular property eg, pluripotency
- differentiated cells refer to cells with specialized functions and morphologies (eg, muscle cells, nerve cells, etc.) and, unlike stem cells, have little or no pluripotency .
- the differentiated cells include, for example, epidermal cells, ⁇ parenchymal cells, covering cells, hepatocytes, blood cells, cardiomyocytes, skeletal muscle cells, osteoblasts, skeletal myoblasts, nerve cells, vascular endothelial cells, pigment cells , Smooth muscle cells, fat cells, bone cells, chondrocytes and the like.
- the term “state” of a cell refers to a condition relating to various parameters of the cell (eg, cell cycle, response to an external factor, signal transduction, gene expression, gene transcription, etc.). Examples of such a state include, but are not limited to, a differentiated state, an undifferentiated state, a cell response to an exogenous factor, a cell cycle, a proliferative state, and the like.
- differentiation refers to two morphologically and / or functionally qualitative differences in a daughter cell population derived from the division of one cell. This refers to the phenomenon in which the above types of cells occur. Therefore, cell differentiation (cell lineage) derived from cells for which no special characteristics can be detected originally is also included in differentiation. At present, it is common to consider cell differentiation as a state in which specific genes in the genome are expressed. Yes, cell differentiation can be identified by searching for intracellular or extracellular factors or conditions that lead to such gene expression states. The result of cell differentiation is stable in principle, and especially in animal cells, differentiation to another type of cell occurs only exceptionally.
- pluripotency or “pluripotency” is used interchangeably and refers to the property of a cell, and refers to the ability to differentiate into one or more, preferably two or more various tissues or organs. . Accordingly, “pluripotency” and “pluripotency” are used interchangeably herein with “undifferentiated” unless otherwise specified. Normally, the pluripotency of cells is limited as they develop, and in adults, the constituent cells of one tissue or organ rarely change into cells of another. Thus, pluripotency is usually lost. In particular, epithelial cells are unlikely to change into other epithelial cells. When this happens, it is usually a pathological condition and is called metaplasia (metaplasia).
- Embryonic stem cells have pluripotency.
- Tissue stem cells have pluripotency.
- pluripotency the ability to differentiate into all kinds of cells constituting a living body such as a fertilized egg is called totipotency, and pluripotency can encompass the concept of totipotency.
- pluripotency includes, but is not limited to, formation of embryoid bodies (EmbryodBody) in an in vitro culture system, culture under differentiation-inducing conditions, and the like.
- Assays for the presence or absence of pluripotency using living organisms include the formation of teratomas by transplantation into immunodeficient mice, the formation of chimeric embryos by injection into blastocysts, and the Examples include, but are not limited to, transplantation and proliferation by infusion into ascites.
- the pluripotency the ability to differentiate into all kinds of cells constituting a living body such as a fertilized egg is called “totipotency”, and the pluripotency includes the concept of totipotency. I can do it. The ability to differentiate in only one direction is also called unipotency.
- the term “agent” refers to any substance or other element (eg, energy such as light, radioactivity, heat, electricity, etc.) as long as the intended purpose can be achieved. Is also good.
- substances include, for example, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (eg, DNA such as cDNA, genomic DNA, niRNA).
- RNA Ribonucleic acid
- polysaccharides oligosaccharides
- lipids small organic molecules (eg, hormones, ligands, signal transducers, small organic molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals)
- Molecules eg, small molecule ligands, etc.
- conjugated molecules but are not limited thereto.
- a polynucleotide typically, a polynucleotide having a certain degree of sequence homology (for example, 70% or more sequence identity) and complementarity to the sequence of the polynucleotide is used.
- polypeptides such as transcription factors that bind to the promoter region, but are not limited thereto.
- the factor specific to a polypeptide includes an antibody specifically directed against the polypeptide, a derivative thereof, or an analog thereof (eg, a single-chain antibody), and the polypeptide.
- the receptor or the ligand is a specific ligand or receptor, or when the polypeptide is an enzyme, examples thereof include, but are not limited to, a substrate thereof.
- biological factor refers to a factor associated with a living organism (eg, a cell).
- biological factors present in cells under normal conditions are referred to as biological factors. Examples of such biological factors include, but are not limited to, nucleic acid molecules, proteins, sugars, fats, metabolites, small molecules, complexes thereof, and factors having a temporal element. Not done.
- biological factors include current, potential (for example, membrane potential, etc.), pH, osmotic pressure, etc., as encompassed by the present invention.
- Biological factors useful herein include, for example, transcription control sequences (eg, promoters), structural genes, or Nucleic acid molecules to be loaded. “Aggregate” of “biological factors” as used herein refers to more than one biological factor (same or different). Preferably, it refers to a biological agent with which it works.
- the term “gene” refers to a factor that defines a transgenic trait. Usually arranged in a certain order on the chromosome. Those that define the primary structure of a protein are called structural genes, and those that control its expression are called regulatory genes (eg, promoters). As used herein, a gene includes a structural gene and a regulatory gene unless otherwise specified. In recent years, the genome has been analyzed and the entire sequence itself has been revealed. Its function is not always known, but there are sequences that encode neither protein nor RNA. It is well understood that such sequences also have an effect on transgenes, and thus such sequences are also understood to fall within the broadest definition of the gene herein. Is done.
- a cyclin gene usually includes both a cyclin structural gene and a cyclin promoter.
- gene refers to “polynucleotide”, “oligonucleotide”, “nucleic acid molecule” and “nucleic acid” and Z or “protein”, “polypeptide”, “oligopeptide J and“ Peptide ".
- gene product also refers to “polynucleotide”, “oligonucleotide”, “nucleic acid molecule” and “nucleic acid” expressed by a gene, and / or “protein”, “polypeptide”, “Oligopeptide” and “peptide” are included. Those skilled in the art can understand what a gene product is, depending on the situation.
- homologous refers to the degree of identity between two or more gene sequences. Thus, the higher the homology between two genes, the higher the identity or similarity between their sequences. Whether the two genes are homologous is determined by direct sequence comparison or, in the case of nucleic acids, by hybridization under stringent conditions. I can be. When two gene sequences are compared directly, the DNA sequences between the gene sequences are typically at least 50% identical, preferably at least 70% identical, more preferably at least 8% identical. If 0%, 90%, 95%, 96%, 97%, 98% or 99% are identical, then the genes are homologous.
- similarity refers to the homology of two or more gene sequences when conservative substitutions are regarded as positive (identical) in the above homology. Refers to the degree of identity to each other. Thus, if there are conservative substitutions, identity and similarity will vary depending on the presence of the conservative substitution. In the absence of conservative substitutions, identity and similarity indicate the same value.
- the comparison of similarity, identity and homology between an amino acid sequence and a base sequence is calculated using FASTA, a sequence analysis tool, using default parameters.
- protein As used herein, the terms “protein,” “polypeptide,” “oligopeptide,” and “peptide” are used interchangeably herein and refer to a polymer of amino acids of any length. This polymer may be linear, branched, or cyclic. Amino acids may be natural or non-natural, and may be modified amino acids. The term may also include those assembled into a complex of multiple polypeptide chains. The term also includes naturally or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lysification, acetylation, phosphorylation, or any other manipulation or modification (eg, conjugation with a labeling component).
- modifications include, for example, disulfide bond formation, glycosylation, lysification, acetylation, phosphorylation, or any other manipulation or modification (eg, conjugation with a labeling component).
- This definition also includes, for example, polypeptides containing one or more analogs of an amino acid (eg, including unnatural amino acids, etc.), peptide-like compounds (eg, peptoids), and others known in the art. Modifications are included. Transmission of extracellular matrix proteins such as fibronectin The progeny product usually takes the form of a polypeptide.
- polynucleotide As used herein, the terms “polynucleotide”, “oligonucleotide”, “nucleic acid molecule” and “nucleic acid” are used interchangeably herein and refer to a polymer of nucleotides of any length. The term also includes “derivative oligonucleotides” or “derivative polynucleotides.” “Derivative oligonucleotide” or “derivative polynucleotide” refers to an oligonucleotide or polynucleotide containing a derivative of a nucleotide or having an unusual linkage between nucleotides, and is used interchangeably.
- oligonucleotides include, for example, 2,1-O-methyl-1-ribonucleotide, a derivative oligonucleotide in which a phosphodiester bond in an oligonucleotide is converted to a phosphorothioate bond, and a derivative in an oligonucleotide.
- Derivative oligonucleotide in which phosphodiester bond is converted to N3'-P5 'phosphoramidate bond Derivative oligonucleotide in which ribose and phosphodiester bond in oligonucleotide are converted to peptide nucleic acid bond
- derivative oligonucleotide in which peracyl in oligonucleotide is substituted with C-5 thiazole peracyl cytosine in oligonucleotide is C-5 propynyl cytosine
- cytosine in oligonucleotide is C-5 propynyl cytosine
- Substituted derivative oligonucleotide Derivative oligonucleotide in which cytosine in the oligonucleotide is substituted
- a particular nucleic acid sequence may also be conservatively modified (eg, degenerate codon substitutions) and its conservative variants, as are the explicitly indicated sequences. And capture sequences.
- the degenerate codon substitution is This can be achieved by creating a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or deoxyinosine residue (Batzer et al., Nuc. 1 eic Ac id Re s.
- Probes 8 91-98 (1 994)). Genes such as extracellular matrix proteins such as fibronectin usually take this polynucleotide form.
- the molecules to be transfused are also the polynucleotides.
- corresponding amino acid or nucleic acid refers to an amino acid or nucleic acid having the same action as a given amino acid in a polypeptide or polynucleotide as a reference for comparison in a certain polypeptide molecule or polynucleotide molecule, respectively.
- a transcription control sequence of a polynucleotide may be a similar portion in an ortholog corresponding to a specific portion of the transcription control sequence.
- a “corresponding” gene (eg, a polypeptide or nucleic acid molecule) is defined as having, in a certain species, an effect similar to, or predicted to have, the same effect as a given gene in a species that serves as a reference for comparison. Genes that have the same origin in evolution when there are multiple genes having such an action. Thus, the corresponding gene for a gene may be an ortholog or species homolog of that gene. Therefore, a gene corresponding to the mouse cyclin gene can be found in other animals. Such corresponding genes can be identified using techniques well known in the art. Thus, for example, the corresponding gene in an animal can be used as a reference for the corresponding gene. It can be found by searching the sequence database of the animal (eg, human, rat) using the sequence of the gene (eg, mouse cyclin gene) as a query sequence.
- fragment refers to a polypeptide or polynucleotide having a sequence length of l to n-1 with respect to a full-length polypeptide or polynucleotide (length is n).
- the length of the fragment can be appropriately changed depending on the purpose.
- the lower limit of the length is 3, 4, 5, 6, 7, 8, 9 for polypeptides.
- 10, 15, 20, 25, 30, 40, 50 and more amino acids and the length represented by an integer not specifically listed herein (for example, 1 1) may also be appropriate as a lower limit.
- the lengths of the polypeptide and the polynucleotide can be represented by the number of amino acids or nucleic acids, respectively, as described above.
- the above numbers are not absolute, and the upper limit is limited as long as they have the same function.
- the above-mentioned number as an addition or subtraction is intended to include a few above and below (or, for example, 10% above and below) the number.
- "about” may be used before a number. It should be understood, however, that the presence or absence of “about” does not affect the interpretation of the numerical value herein.
- biological activity refers to an activity that a certain factor (eg, a polypeptide or a nucleic acid molecule) can have in a living body, and exerts various functions (eg, a transcription promoting activity). Activity is included. For example, when a factor is an antisense molecule, its biological activity includes binding to a target nucleic acid molecule, thereby suppressing expression. For example, a factor is an enzyme If so, the biological activity includes the enzymatic activity. In another example, where an agent is a ligand, the ligand involves binding to the corresponding receptor. When the biological activity is a transcription regulatory activity, it refers to an activity that regulates the transcription level or its fluctuation. Such biological activity can be measured by techniques known in the art.
- polynucleotide that hybridizes under stringent conditions refers to well-known conditions commonly used in the art.
- a polynucleotide selected from the polynucleotides of the present invention as a probe, a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like is used. Such a polynucleotide can be obtained.
- hybridization was performed at 65 ° C in the presence of 0.7 to 1.0 M NaCl using a filter on which DNA derived from colonies or plaques was immobilized, and then Filter at 65 ° C using a 1- to 2-fold concentration of 33 C (saline—sodium citrate) solution (the composition of the 1-fold concentration SSC solution is 150 mM sodium chloride and 15 mM sodium citrate).
- SSC solution is 150 mM sodium chloride and 15 mM sodium citrate.
- Hybridization is performed as follows: Mo 1 ecu 1 ar C loning 2 nde d., Current Protocolsin Mo lecular Biology, Suppl ement 1 to 38, DNA Cloning 1: Core T c hniques, AP ractical It can be carried out according to a method described in an experiment book such as Ap proach, Eco Edition, Ox ford Uiversity Press (1995).
- sequences that hybridize under stringent conditions preferably exclude sequences containing only the A sequence or only the T sequence.
- “Hybridizable polynucleotide” means to hybridize to another polynucleotide under the above hybridization conditions.
- polynucleotide that can be Specific examples of the hybridizable polynucleotide include polynucleotides having at least 60% homology with the nucleotide sequence of DNA encoding the polypeptide having the amino acid sequence specifically shown in the present invention.
- a polynucleotide having a homology of 80% or more, more preferably a polynucleotide having a homology of 95% or more can be mentioned.
- salt is used in the same meaning as the broadest meaning usually used in the art, and includes both inorganic salts and organic salts. Salts are usually formed by the neutralization reaction of acids and bases. In addition to those, such as N a C 1, K 2 S0 4 produced in the neutralization reaction in salt, there is Pb so 4, various types such as Z nC 1 2 formed by the reaction between the metal and acid, they However, even if it is not directly produced by the neutralization reaction, it can be regarded as produced by the neutralization reaction between the acid and the base.
- salt normal salts (which OH of H and base of an acid is not included in the salts, e.g., Na C 1, NH 4 C 1, CH 3 COONa, Na 2 C0 3), acid salts (acid those H is Zantsu salts, e.g., NaHC0 3, KHS0 4, C aHP0 4), basic salts (which OH salt groups remaining in the salt, for example, Mg C 1 (OH) , CuCl (OH)), etc., but those classifications are not so important in the present invention.
- acid salts acid those H is Zantsu salts, e.g., NaHC0 3, KHS0 4, C aHP0 4
- basic salts which OH salt groups remaining in the salt, for example, Mg C 1 (OH) , CuCl (OH)), etc., but those classifications are not so important in the present invention.
- Preferred salts include those constituting the culture medium (eg, calcium chloride, sodium hydrogen phosphate, sodium hydrogen carbonate, sodium pyruvate, HEPES, calcium chloride, sodium chloride, potassium chloride, magnesium sulfide, iron nitrate, amino acid) , Vitamins, and salts that constitute a buffer solution (for example, calcium chloride, magnesium chloride, sodium hydrogen phosphate, and sodium salt) are preferred because they have a higher effect of maintaining or improving affinity for cells.
- These salts may be used alone or in combination of two or more.Preferably, a plurality of salts are used because the affinity for cells tends to be high.
- Salts contained in the medium eg, calcium chloride
- Magnesium chloride, sodium hydrogen phosphate, and sodium chloride e.g., sodium chloride
- glucose may be added.
- the term "substance” is included in the broadest sense used in the art and includes those that can be positively or negatively charged.
- the term “positively charged substance” includes all substances having a positive charge. Such substances include, but are not limited to, for example, cationic substances such as cationic polymers and cationic lipids. Preferably, such a positively charged substance is advantageously a substance capable of forming a complex. Positively charged substances capable of forming such a complex include, for example, a substance having a certain molecular weight such as a cationic polymer, or a specific solvent such as a cationic lipid (for example, water). , Aqueous solutions, etc.), but are not limited thereto. Such preferred positively charged substances include, but are not limited to, for example, polyethyleneimine, poly-L-lysine, synthetic polypeptides or derivatives thereof.
- positively charged substances include, but are not limited to, biomolecules such as histones, synthetic polypeptides, and the like.
- the type of such preferred positively charged substance will vary depending on the type of negatively charged substance that is the partner that forms the complex. Selecting a preferred complex-forming partner is straightforward to one of skill in the art, and such selection can be made using techniques well known in the art.
- Various parameters can be considered in the selection of such preferred complexing partners. Such parameters include, for example, charge, molecular weight, hydrophobicity, hydrophilicity, nature of substituents, pH, temperature, salt concentration, pressure, etc. Physical parameters, chemical parameters, etc., but are not limited thereto.
- cationic polymer has a cationic functional group.
- a polymer refers to, for example, but is not limited to, polyethyleneimine, poly L-lysine, synthetic polypeptide or a derivative thereof. .
- cationic lipid refers to a lipid having a cationic functional group, and includes, but is not limited to, phosphatidylcholine, phosphatidinoleethanolamine, phosphatidylserine, and derivatives thereof. Les ,.
- examples of the cationic functional group include, but are not limited to, primary amine, secondary amine, and tertiary amine.
- negatively charged substance includes all substances having a negative charge.
- Such substances include, but are not limited to, for example, biomolecular polymers such as DNA and anionic substances such as anionic lipids.
- a negatively charged substance is advantageously a substance capable of forming a complex.
- the negatively charged substance capable of forming such a complex is, for example, a substance having a certain molecular weight such as an anionic polymer such as DNA, or a specific substance such as an aionic lipid.
- a solvent eg, water, aqueous solution, etc.
- Such preferred negatively charged substances include, but are not limited to, for example, DNA, RNA, PNA, polypeptides, compounds, and complexes thereof.
- negatively charged substances include, but are not limited to, biological agents or biomolecules such as DNA, RNA, PNA, polypeptides, compounds, and complexes thereof, and the like.
- the type of such preferred negatively charged substances will vary depending on the type of positively charged substance that is the partner that forms the complex. It is easy for a person skilled in the art to select a preferred complex-forming partner, and such The choice can be made using techniques well known in the art.
- Various parameters can be considered in selecting such a preferred complexing partner. Such parameters also include a variety of parameters, as well as the parameters to be considered for positively charged materials described above.
- anionic polymer refers to a polymer having an anionic functional group, and includes, but is not limited to, DNA, RNA, PNA, polypeptides, compounds, and complexes thereof. .
- anionic lipid refers to a lipid having an anionic functional group, and includes, but is not limited to, for example, phosphatidic acid and phosphatidylserine.
- examples of the anionic functional group include, but are not limited to, a carboxyl group and a phosphate group.
- complex refers to a substance in which two or more substances directly or indirectly interact with each other so that the total of those substances behaves as one substance.
- complex partner when referring to one member that forms a complex, refers to another member that interacts directly or indirectly with that member.
- the conditions for forming a complex herein vary depending on the type of complex partner. Such conditions can be readily understood by those skilled in the art, and any complex partner (eg, positively charged Substances and negatively charged substances) can form a complex.
- any complex partner eg, positively charged Substances and negatively charged substances
- immobilized when used with respect to a solid support, refers to a state in which a substance of interest (eg, a biomolecule) is retained on the support for at least a certain period of time, or the like. It means to be in a state. Therefore, if conditions change (for example, immersion in another solvent) after the substance is immobilized on the solid support, the immobilized state may be released.
- a substance of interest eg, a biomolecule
- cell affinity means that a substance interacts with a cell (eg, a bacterial cell, an animal cell, a yeast, a plant cell, etc.) or an object containing a cell (eg, a tissue, an organ, a living body, etc.). A property that does not have a harmful effect on the cell or the object containing the cell when placed in a state where it can act.
- the substance having cell affinity may be, but is not limited to, a substance with which cells interact preferentially.
- the substance to be immobilized eg, a positively charged substance and a Z or negatively charged substance
- the substance to be immobilized preferably has, but is not limited to, cell affinity.
- the substance to be immobilized has a cell affinity
- the cell affinity is retained or improved when the substance is immobilized according to the invention.
- the effect of the present invention is immeasurable considering that cell affinity is not always maintained.
- probe refers to a substance to be searched for, which is used in biological experiments such as screening in vitro and Z or in vivo, and includes, for example, a nucleic acid molecule containing a specific base sequence or a specific substance. But not limited thereto.
- Nucleic acid molecules that are usually used as probes include nucleic acids that are at least 8 contiguous nucleotides in length that are homologous or complementary to the nucleic acid sequence of the gene of interest. And those having a sequence. Such nucleic acid sequences are preferably at least 9 contiguous nucleotides in length, more preferably 10 contiguous nucleotides in length, even more preferably 11 contiguous nucleotides in length, 12 contiguous nucleotides in length.
- the nucleic acid sequence may be 40 contiguous nucleotides in length, 50 contiguous nucleotides in length.
- Nucleic acid sequences used as probes include nucleic acid sequences that are at least 70% homologous to the above sequences, more preferably at least 80% homologous, even more preferably 90% homologous, and 95% homologous. It is.
- search refers to the use of a nucleobase sequence electronically, biologically, or by another method to search for another nucleobase sequence having a specific function and Z or property.
- BLAST A 1 tschu 1 etal., J. MoI. Biol. 2 15: 403—410 (1 990)
- FASTA Pearson & Lipman Sad., USA 85: 2444—2448 (1988)
- Sm ithand Wa ter man method Sm ithand Wa ter man, J. Mol. 48: 1995-197 (1981)
- Need 1 emanand Wunsch method Need 1 emanand Wunsch, J. Mol. Biol. 48: 443— 4
- Bio searches include stringent hybridization, a macroarray in which genomic DNA is attached to a nylon membrane, a microarray in which the DNA is attached to a glass plate (microarray assay), PCR, Examples include, but are not limited to, insitu hybridization.
- the term “primer” refers to a synthesis in a polymer synthase reaction. Refers to a substance necessary for initiating the reaction of the polymer compound.
- a nucleic acid molecule synthesis reaction eg, a nucleic acid molecule
- DNA or RNA can be used.
- Nucleic acid molecules that are usually used as primers include those having a nucleic acid sequence that is complementary to the nucleic acid sequence of the gene of interest and has at least eight consecutive nucleotides in length. Such nucleic acid sequences are preferably at least 9 contiguous nucleotides in length, more preferably 10 contiguous nucleotides in length, and even more preferably 12 contiguous nucleotides in length, 12 contiguous nucleotides in length.
- nucleic acid sequences used as probes may be at least 70% homologous, more preferably at least 80% homologous, even more preferably 90% homologous, 95% homologous nucleic acid sequences to the above sequences. Is included.
- a suitable sequence as a primer may vary depending on the nature of the sequence to be synthesized (amplified), but those skilled in the art can appropriately design a primer according to the intended sequence.
- the design of such primers is well known in the art, and may be performed manually or using a computer program (for example, LASERGENE, Primer Se1 ect, DNAStat).
- epitopes refers to antigenic determinants whose structure is apparent.
- the epitopes must contain a set of amino acid residues involved in recognition by a particular immunoglobulin, or, in the case of T cells, recognition by T cell receptor proteins and / or major histocompatibility complex (MHC) receptors. A key set of amino acid residues is included.
- MHC major histocompatibility complex
- the term is also used interchangeably with "antigenic determinant” or "antigenic determinant site.”
- epitopes are molecular features (eg, primary, secondary or tertiary peptide structures and charges) that are recognized by immunoglobulins, T cell receptors or HLA molecules. To be formed.
- Epitopes containing peptides may contain more than two amino acids in a spatial conformation unique to the epitope.
- an epitope is composed of at least five such amino acids, and typically is composed of at least six, seven, eight, nine, or ten such amino acids.
- Longer epitopes are generally preferred because longer lengths resemble the antigenicity of the original peptide, but may not always be so in view of conformation.
- Methods for determining the spatial conformation of amino acids are known in the art and include, for example, X-ray crystallography, and two-dimensional nuclear magnetic resonance spectroscopy.
- identification of the epitope in a given protein is readily accomplished using techniques well known in the art. See, for example, Geysen et al.
- epitopes including peptides
- methods for determining epitopes are well known in the art, and such epitopes, once provided with the primary sequence of nucleic acids or amino acids, will be readily apparent to those of skill in the art using such well known techniques.
- At least 3 A sequence of amino acid length is required, and preferably this sequence has at least 4 amino acids, more preferably 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 15 amino acids, 2 amino acids A sequence of 0 amino acids, 25 amino acids in length may be required.
- an agent that specifically binds to a nucleic acid molecule or polypeptide means that the level of binding of the agent to the nucleic acid molecule or polypeptide is a nucleic acid molecule or polypeptide outside the nucleic acid molecule or polypeptide. A factor whose level is equal to or higher than the level of binding of the factor to the peptide.
- Such factors include, for example, when the target is a nucleic acid molecule, a nucleic acid molecule having a sequence complementary to the target nucleic acid molecule, a polynucleotide binding to the target nucleic acid sequence,
- the target is a polypeptide
- examples include, but are not limited to, one of an antibody, a single-chain antibody, a receptor-ligand pair, and an enzyme-substrate. .
- antibody refers to polyclonal, monoclonal, human, humanized, multispecific, chimeric, and anti-idiotype antibodies, and fragments thereof, e.g., F (ab '). 2 and Fab fragments, as well as other recombinantly produced conjugates. Further, such an antibody may be covalently linked to an enzyme, such as alkaline phosphatase, horseradish peroxidase, ⁇ -galactosidase, or the like, or may be recombinantly fused.
- an enzyme such as alkaline phosphatase, horseradish peroxidase, ⁇ -galactosidase, or the like, or may be recombinantly fused.
- the term "monoclonal antibody” as used herein refers to an antibody composition having a homogeneous antibody population. This term is not limited by the manner in which it is made. The term includes whole immunoglobulin molecules as well as Fab molecules, F (ab ') 2 fragments, Fv fragments, and other molecules that exhibit the immunological binding properties of the original monoclonal antibody molecule. Methods for making polyclonal and monoclonal antibodies are known in the art, and are described more fully below. be written.
- Monoclonal antibodies can be obtained using standard techniques well known in the art (eg, Kohler and Milstein, Nature (1975) 256: 495) or modifications thereof (eg, Buck et al., '982) In V itro 18: 3 77).
- a mouse or rat is immunized with a protein conjugated to a protein carrier, boosted, and the spleen (and several large lymph nodes, if necessary) is removed, and the single cell is harvested. Dissociate.
- the spleen cells can be screened after removal of non-specifically adherent cells by applying the cell suspension to a plate or well coated with antigen.
- B cells expressing immunoglobulin specific for the antigen bind to the plate and are not rinsed off of the suspension residue. Then, the obtained B cells (ie, all detached spleen cells) are fused with myeloma cells to obtain a hybridoma, and the monoclonal antibody is produced using the hybridoma.
- the term “antigen” refers to any substrate that can be specifically bound by an antibody molecule.
- the term “immunogen” refers to an antigen that can initiate lymphocyte activation that produces an antigen-specific immune response.
- certain amino acids in the sequence are replaced by other amino acids in the protein structure, such as in the cationic region or the binding site of a substrate molecule, without appreciable loss or loss of interaction binding capacity. obtain. It is the interaction capacity and nature of a protein that defines the biological function of a protein. Thus, certain amino acid substitutions can be made in the amino acid sequence, or at the level of its DNA coding sequence, resulting in a protein that retains its original properties after the substitution. Thus, without obvious loss of biological utility, various modifications can be made to the peptides disclosed herein or the entirety thereof. It can be performed on the corresponding DNA encoding the peptide.
- the hydropathic index of amino acids can be considered.
- the importance of the hydrophobic amino acid index in conferring interactive biological functions on proteins is generally recognized in the art (Kyte. J and Doo 1 itt 1 e, RF J. Mo. 1. Biol. 157 (1): 105-132, 1982).
- the hydrophobic nature of amino acids contributes to the secondary structure of the resulting protein, which in turn defines the interaction of that protein with other molecules (eg, enzymes, substrates, receptors, DNA, antibodies, antigens, etc.).
- Each amino acid is assigned a hydrophobicity index based on its hydrophobicity and charge properties.
- one amino acid can be replaced by another amino acid having a similar hydrophobicity index and still yield a protein having a similar biological function (eg, a protein equivalent in enzymatic activity). It is.
- the hydrophobicity index is preferably within ⁇ 2, more preferably within ⁇ 1, and even more preferably within ⁇ 0.5. It is understood in the art that such amino acid substitutions based on hydrophobicity are efficient.
- hydrophilicity index can also be considered in conservative substitutions. As described in US Pat. No. 4,554,101, the following hydrophilicity indices have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); Aspartic acid (+ 3.0 ⁇ 1); gnoletamic acid (+ 3.0 ⁇ 1); serine (+0.3); asparagine (+0.2); gnoletamine (+0.2); glycine (0); Threonine (10.4); Proline (10.5 ⁇ 1); Alanine (10.5); Histidine (10.5); Cystine (11.0); Methionine (1-1.3); Palin (1-1.5); Leucine (1-1.8); Isoleucine (1-1.8); Tyrosine (1-2.3); Phenylalanine (1-2.5); and Tributophane (_3.4) .
- an amino acid can have a similar hydrophilicity index and be replaced with another that can still provide a bioisostere.
- the hydrophilicity index is preferably within soil 2, more preferably within ⁇ 1, and even more preferably within ⁇ 0.5.
- a “profile” for a cell refers to a collection of measurements of the biological state of the cell.
- a profile can be a set or continuous measurement of the levels of "cell components" that are quantitatively measured.
- Cell components include gene expression levels in biological systems, transcript levels (activity levels of transcription control sequences), abundance of mRNA encoding a particular gene, and protein expression levels.
- the levels of various cellular components, such as mRNA and / or protein expression levels encoding genes, can change in response to treatment with drugs or perturbation or vibration of other cell biological states. are known.
- this profile is increasingly used in cell analysis and in-depth analysis, as the measurement of multiple such "cell components" contains a wealth of information on the effects of the stimulus on the biological state of the cell It is important.
- Individual cell profiles are usually complex. The profile of a given state of a biological system is often measured after the biological system has been subjected to a stimulus.
- Such stimuli include biology Experimental or environmental conditions associated with the biological system, such as exposure of biological systems to drug candidates, introduction of exogenous genes, passage of time, deletion of genes from the system, or culture conditions And changes. Extensive measurement of cellular components, or cell profiling.
- the profile of gene replication or transcription in cells and the expression of proteins and their response to stimuli can be compared to drug effects in addition to examining the cells themselves. It has a wide range of benefits, including studies, disease diagnosis, and optimization of patient medication. In addition, they are useful in basic life science research.
- Such profiles are generated and presented as data in various forms. Such forms include, but are not limited to, the form of a function of numbers and time, a graph form, an image form, and the like. Therefore, data relating to a profile is sometimes referred to herein as "profile data". Such data generation can be easily achieved by computer. Coding of appropriate programs may also be performed by techniques well known in the art.
- various detection methods and detection means can be used as long as information attributable to cells or substances interacting with the cells can be detected.
- detection methods and means include, for example, visual, optical microscopy, fluorescence microscopy, readers using laser light sources, surface plasmon resonance (SPR) imaging, electrical signals, chemical or biochemical marker One or more of them may be used, and methods and means may be used, but the present invention is not limited thereto.
- time profile when a term “time profile” is specifically referred to with respect to a specific cell, it refers to a profile showing a change over time of a parameter relating to the cell.
- Such temporal profiles include, but are not limited to, the temporal profile of the transcriptional state, the temporal profile of the expression state (translational state), the temporal profile of the signal transduction, and the temporal profile of the nerve potential.
- a certain parameter for example, a signal resulting from a marker relating to a transcription state
- the “time-dependent profile” herein may sometimes be referred to as a continuous profile.
- the term “information” of a cell refers to a substance that functions to link a number of elements present in a cell to direct a target as a whole. It can be said that a collection of information constitutes a digital cell.
- the term “state” of a cell, an organism, and the like refers to a state relating to various parameters of a cell (eg, cell cycle, response to an external factor, signal transduction, gene expression, transcription of a gene, and the like). Examples of such a state include, but are not limited to, a differentiated state, an undifferentiated state, a cell response to an exogenous factor, a cell cycle, a proliferative state, and the like.
- the environment of the organism of interest eg, temperature, humidity (eg, absolute humidity, relative humidity, etc.), pH, salt concentration (eg, total salt concentration or specific salt concentration), Nutrition (eg, carbohydrate content), metals (eg, total metal or specific metal (eg, heavy metal) concentration), gases (eg, total gas or specific gas volume), organic solvents (Eg, the total amount of organic solvent or the amount of a particular organic solvent (eg, ethanol)), pressure (eg, local or total pressure), pressure, viscosity, flow rate (eg, If present, the flow rate of the medium, etc.), luminosity (such as the amount of light of a specific wavelength), light wavelength (for example, may include ultraviolet light, infrared light as well as visible light), electromagnetic wave, radiation, gravity, tension, soundFor parameters such as other organisms that are different from the elephant (eg, parasites, pathogens), chemicals (eg, pharmaceuticals), antibiotics, natural products, mental stress, physical stress, etc.
- Nutrition eg, carb
- environmental factors inorganic environment
- Abiotic environmental factors inorganic environment
- the effects of these various environmental factors on living organisms are not always performed independently, but are often related to each other. Therefore, in this specification, the environment may be observed for each factor, or may be recognized as a total of environmental factors (a total of various parameters). It has traditionally been considered difficult to maintain such an environment.
- the same environment means that the cells have substantially the same environment. Therefore, as long as cells can proliferate, differentiate, etc., such an environment can be said to be the same environment.
- the same environment means that other parameters are the same except for a specific stimulus (for example, an external stimulus).
- Factors that take such an environment into account include, for example, temperature, humidity, pH, salt concentration, nutrition, metals, gases, organic solvents, pressure, pressure, viscosity, flow velocity, luminosity, light wavelength, electromagnetic waves, radiation, At least one selected from the group consisting of gravity, tension, sound waves, other organisms different from the organism of interest (eg, parasites), chemicals, antibiotics, natural products, chemical stress and physical stress Two factors are included as parameters.
- the temperature for example, a wide range such as a high temperature, a low temperature, an ultra-high temperature (for example, at 95), an ultra-low temperature (for example, 180 ° C.), Temperature is not limited to these.
- the humidity include, but are not limited to, any point between 0 and 100%, such as 100% relative humidity and 0% relative humidity.
- pH examples include, but are not limited to, arbitrary points of 0 to 14.
- salt concentration examples include, but are not limited to, any one of NaC1 concentration (3% or the like) and salt concentrations of other salts of 0 to 100%.
- Nutrition includes, but is not limited to, for example, protein, glucose, lipids, vitamins, inorganic salts, and the like.
- metals include, but are not limited to, heavy metals (eg, mercury, cadmium, etc.), lead, gold, uranium, and silver.
- gas examples include, but are not limited to, oxygen, nitrogen, carbon dioxide, carbon monoxide, nitric oxide, and a mixture thereof.
- organic solvent examples include, but are not limited to, ethanol, methanol, xylene, and propanol.
- Examples of the pressure include, but are not limited to, any point of 0 to 10 tons / cm 2.
- the atmospheric pressure includes, for example, any point of 0 to 100 atmospheric pressure, but is not limited thereto.
- Viscosity includes, but is not limited to, viscosity in any fluid, such as water, glycerol, or mixtures thereof.
- Examples of the flow velocity include, but are not limited to, any point from 0 to the speed of light. ,
- the luminous intensity includes, for example, one point between darkness and sunlight, but is not limited thereto.
- the light wavelength includes, for example, any wavelength such as visible light, ultraviolet light (UV-A, UV-B, UV-C, etc.), and infrared light (far-infrared light, near-infrared light, etc.). Not limited to them.
- the electromagnetic wave may have any wavelength.
- Radiation may be of any intensity.
- Gravity can include, but is not limited to, any point between any gravity on earth or zero gravity to gravity on earth, or any point greater than or equal to gravity on earth, and tension of any strength Things.
- organisms different from the target organism include, but are not limited to, for example, parasites, pathogens, insects, and nematodes.
- Examples of the chemical include, but are not limited to, hydrochloric acid, sulfuric acid, and caustic soda.
- antibiotics examples include, but are not limited to, penicillin, kanamycin, streptomycin, quinoline and the like.
- Examples of natural products include, but are not limited to, blowfish poison, snake venom, and alkyloid.
- Examples of physical stress include, but are not limited to, vibration, noise, electricity, and impact.
- the environment is referred to as “environmental parameters.”
- Environmental parameters media (type, composition), H, temperature, humidity, C 0 2 concentration, 0 2 concentration, presence of antibiotics, although including etc. presence or absence of a particular nutrient but not limited to.
- the term “stimulus” refers to an agent that induces the expression or enhancement of a specific life activity given to a cell from the outside.
- Stimuli include, but are not limited to, physical stimuli, chemical stimuli, biological stimuli, and biochemical stimuli.
- the physical stimulus include, but are not limited to, light, radio waves, electric current, pressure, and sound (vibration).
- chemical stimuli Examples include, but are not limited to, chemical stimuli, including, but not limited to, antibiotics, nutrients, vitamins, metals, ions, acids, alkalis, salts, buffers, and the like.
- Biological stimuli include, but are not limited to, for example, the presence of other organisms (eg, presence of parasites, density of cell populations, etc.).
- Biochemical stimuli include, but are not limited to, the presence of cell signaling factors.
- the stimulus is presented as a "stimulus parameter".
- the stimulus parameters parameters corresponding to any of the above-described stimuli can be used.
- the stimulation parameters include factors for transmitting the stimulus (eg, a reporter).
- reporters include, but are not limited to, for example, on / off for antibiotics, transcription control sequences, radioactivity, fluorescent materials, and the like.
- response to a stimulus means any response the cell has to a stimulus (eg, changes in cell shape, metabolic changes, other behavior changes, changes in signal transmission, etc.). .
- the results of a digital cell experiment in the present invention can be recorded as cell kinetic data.
- such a stimulus response result may be raw data of the reporter or data obtained by converting the data of the reporter.
- transcription control sequence refers to a sequence that can regulate the transcription level of a gene. Such a sequence is at least two nucleotides in length. Such sequences typically include promoters, enhancers, silencers, terminators, flanking sequences of structural genes in other genomic structures and genomic sequences other than exon, and sequences in exon. But not limited to them.
- the transcription control sequence used in the present invention is It is not about certain types. Rather, the important information as a transcription control sequence is its temporal variation. Such a change is also called a (cell state change) process. Therefore, in the present invention, such a transcription control sequence can be arbitrarily selected. Such transcription control sequences may include those not conventionally used as markers. Preferably, the transcription control sequence has the ability to bind to a transcription factor.
- transcription factor refers to a factor that regulates the process of gene transcription. Transcription factors mainly refer to factors that regulate the transcription initiation reaction. A group of basic transcription factors necessary for placing RNA polymerase in a promoter region on DNA, and various types of factors that regulate the frequency of initiation of RNA synthesis by binding to cis-acting elements upstream and downstream of the transcription region It is roughly divided into transcription regulatory factors.
- TATA binding proteins are said to be common to all transcription systems.
- transcription factors There are many types of transcription factors, but they usually consist of a structurally necessary part for DNA binding and a part required for transcriptional activation or repression. Factors that have a DNA binding site and can bind to a cis-acting element are collectively called trans-acting factors.
- Transcriptional regulators are classified into several groups or families based on the structural characteristics of these parts, and many factors have important roles in development or cell differentiation.
- Such transcription factors include, for example, STAT1, STAT2, STAT3, GAS, NFAT, Myc, AP1, CREB, NF / cB, E2F, Rb, p53, RUNX1 ⁇ RUNX 2, RUNX 3, Nk x _2, CF 2-II, S kn-1, SRY, HFH-2, Oct-1, Oct 3 Sox-5, HNF-3b, P PAR ⁇ , etc. But not limited to them.
- the term "terminator” refers to a sequence that is usually located downstream of a region encoding a protein of a gene and that is involved in termination of transcription when DNA is transcribed into mRNA and addition of a poly A sequence. Say. It is known that the terminator is involved in mRNA stability and affects the gene expression level.
- promoter refers to a region on DNA that determines the transcription initiation site of a gene and that directly regulates the frequency of transcription, and usually initiates transcription by binding to RNA polymerase. It is a nucleotide sequence. Therefore, in the present specification, a portion of a certain gene that functions as a promoter is referred to as “promoter portion”. Since the promoter region is usually a region within about 2 kbp upstream of the first exon of the putative protein coding region, if the DNA coding software is used to predict the protein coding region in the genomic nucleotide sequence, However, the promoter region can be estimated.
- the putative promoter region varies for each structural gene, but is usually, but not limited to, upstream of the structural gene, and may be located downstream of the structural inheritance “?. Preferably, the putative promoter region is a first exon.
- the promoter is located within about 2 kbp upstream from the translation initiation point Examples of the promoter include, but are not limited to, a constitutive promoter, a specific promoter, and an inducible promoter.
- enhancer refers to a sequence used to enhance the expression efficiency of a target gene. Such enhancers are well known in the art. A plurality of enhancers can be used, but one or no enhancer may be used.
- silencer refers to a sequence having a function of suppressing gene expression and quiescent.
- any type of silencer may be used as long as it has the function, and a silencer may not be used.
- operably linked refers to a transcription / translation regulatory sequence (eg, promoter, enhancer, sile) having the expression (activity) of a desired sequence. Or under the control of a translation regulatory sequence.
- a promoter to be operably linked to a gene, the promoter will usually be located immediately upstream of the gene, but need not be.
- flanking sequences of structural genes in other genomic structures and genomic sequences other than exons, and sequences in exons may also be important.
- flanking sequences of structural genes other than the sequences with the specific names described above are also expected to be related to transcriptional regulation from the viewpoint of “process”. Accordingly, such flanking sequences are also included herein as transcription control sequences.
- Genomic sequences other than exons and sequences in exons are also expected to be related to transcriptional regulation in terms of “process”.
- genomic sequences other than exons and sequences in exons are also included herein as transcription control sequences.
- RNAi is an abbreviation for RNA interference.
- a factor that causes RNAi such as double-stranded RNA (also referred to as dsRNA)
- dsRNA double-stranded RNA
- homologous mRNA can be specifically identified. It refers to the phenomenon of degradation and suppression of the synthesis of transgene products and the technology used for it.
- RNAi may also be used synonymously with factors that cause RNAi in some cases.
- factor that causes RNAi refers to any factor that can cause RNAi.
- factor causing RNAi refers to causing RNAi relating to the gene and achieving the effect of RNAi (for example, suppressing the expression of the gene).
- Factors that cause such RNAi include, for example, at least a sequence having at least about 70% homology to a portion of the nucleic acid sequence of the target gene or a sequence that hybridizes under stringent conditions, RNA containing a double-stranded portion 10 nucleotides in length or a variant thereof But not limited to it.
- the factor preferably contains 3, a protruding end, and more preferably, the 3, protruding end may be a DNA having a length of 2 nucleotides or more (eg, DNA having a length of 2 to 4 nucleotides.
- RNAi RNAi works is that when a molecule that causes RNAi, such as dsRNA, is introduced into a cell, it is relatively long (eg, 40 base pairs or more).
- a dicer D icer
- a helicase domain excises the molecule in the presence of ATP, and cuts out about 20 base pairs at each end from a short dsRNA (also called siRNA).
- siRNA short dsRNA
- siRNA is an abbreviation for short interfering RNA, which is artificially or biochemically synthesized, synthesized in a living organism, or Double-stranded RNA of 40 bases or more A short double-stranded RNA of 10 base pairs or more, which is formed by degrading RNA in the body, and usually has the structure of 5, monophosphate and 3, 1OH. And 3. The end protrudes about 2 bases.
- a specific protein binds to this siRNA and forms RISC (RNA-induced RNAi-com1ex). This complex recognizes and binds mRNA having the same sequence as the siRNA, and cleaves the mRNA at the center of the siRNA by RNase III-like enzyme activity.
- the relationship between the sequence of the siRNA and the sequence of the mRNA to be cleaved as a target is preferably 100%.
- mutation of a base at a position off the center of the siRNA does not completely eliminate the RNAi cleavage activity, but retains partial activity.
- mutation of the base at the center of the siRNA has a large effect, and the activity of cleaving mRNA by RNAi is extremely reduced. Utilizing such properties, for mRNAs with mutations, only those mRNAs containing mutations can be specifically degraded by synthesizing siRNAs with the mutations in the center and introducing them into cells. . Therefore, in the present invention, the siRNA itself is used to induce RNAi.
- a factor that produces siRNA eg, dsRNA typically having about 40 bases or more
- siRNAs bind the antisense strand of the siRNA to mRNA and act as a primer for RNA-dependent RNA polymerase (RdRP), ds It is also contemplated that RNA is synthesized and this dsRNA becomes a substrate for the dicer, generating new siRNA and amplifying the effect. Therefore, in the present invention, a factor that produces si RNA itself or si RNA is also useful. In fact, in insects, for example, 35 dsRNA molecules almost completely degrade more than 1,000 copies of intracellular mRNAs, and as a result, the siRNA itself and factors that generate the siRNA are useful. It is understood that
- RNA having a length of about 20 bases (eg, typically about 21 to 23 bases) or less, which is referred to as siRNA, can be used.
- S RNA suppresses gene expression by expressing it in cells and suppresses expression of pathogenic genes targeted by the siRNA, so it should be used for treatment, prevention, prognosis, etc. of disease. Can be.
- the siRNA used in the present invention may take any form as long as it can cause RNAi.
- the factor that causes RNAi of the present invention may be a short hairpin structure (shRNA; shorthairin RNA) having an overhang at the 3 ′ end.
- shRNA refers to a single-stranded, partially palindromic base sequence that takes on a double-stranded structure within the molecule, resulting in a hairpin-like structure of about 20 bases. Refers to a pair or more molecules.
- shRNAs are artificially chemically synthesized.
- such a shRNA is a hairpin D-link that connects the sense and antisense strand DNA sequences in reverse orientation.
- NA can be produced by in vitro synthesis of RNA with T7 RNA polymerase.
- shRNAs can be as long as about 20 bases (typically, for example, 21 bases, 22 bases, 23 bases) in a cell after introduction into the cell. It should be understood that they are degraded and cause RNAi in the same manner as siRNA, and have the therapeutic effect of the present invention. It is to be understood that such effects are exerted on a wide variety of organisms, including insects, plants, animals (including mammals). As described above, shRNA causes RNAi similarly to siRNA, and thus can be used as an active ingredient of the present invention. The shRNA may also preferably have 3, overhanging ends.
- the length of the double-stranded portion is not particularly limited, but may be preferably about 10 nucleotides or more, more preferably about 20 nucleotides or more.
- the 3 'protruding end may be preferably DNA, more preferably DNA having at least 2 nucleotides or more, and further preferably DNA having 2 to 4 nucleotides in length.
- RNAi used in the present invention can be either artificially synthesized (for example, chemically or biochemically) or a naturally occurring factor. There is no essential difference. Chemically synthesized products are preferably purified by liquid chromatography or the like.
- RNAI antisense or sense RNA from type I DNA.
- T7 RNA polymerase and T7 promoter are used to synthesize antisense or sense RNA from type I DNA.
- RNAI is induced through the mechanism described above, and the effects of the present invention are achieved.
- RNA can be introduced into cells by the calcium phosphate method.
- Factors that cause RNAi according to the present invention also include factors such as single-stranded, or all similar analogs of nucleic acids, that are capable of hybridizing to mRNA. Such factors are also useful in the treatment methods and compositions of the present invention.
- “temporal” refers to associating some action or phenomenon with the passage of time.
- the term “monitor” refers to observing the state of a cell using at least one parameter (for example, a label signal resulting from transcription) as an index.
- the monitoring is performed using an instrument such as a detection instrument or a measurement instrument. More preferably, such equipment is connected to a computer for recording and Z-processing of data.
- a monitor can include the step of obtaining image data of a solid support (eg, arrays, plates, etc.).
- the term “real time” means that a certain state is displayed substantially simultaneously in another form (for example, as an image on a display or as a graph processed with data).
- the real-time has a time lag corresponding to the time required for data processing, and such a time lag is included in the real-time if it can be substantially ignored.
- Such a time lag is usually within 10 seconds, and preferably can be within 1 second, but is not limited thereto. In some applications, a time lag of more than 10 seconds may also be referred to as real time.
- “determination” of the state of a cell can be performed using various methods. Such methods include, but are not limited to, mathematical processing (eg, signal processing, multivariate analysis, etc.), empirical processing, phase changes, and the like.
- the term “difference” refers to a mathematical process for presenting a certain profile by subtracting the value of a control profile (for example, when there is no stimulus).
- phase refers to a profile, which refers to determining whether the profile has increased or decreased from a reference point (usually 0) and expressed as + or 1 respectively. And analysis by it.
- correlation between a profile (eg, a time-lapse profile) and a state of a cell refers to associating a profile (eg, a time-lapse profile) or specific information of the change with the state of the cell. Such a relationship is called a correlation.
- a profile eg, a temporal profile
- correlating refers to associating at least one profile or variation thereof with a change in a state of a cell, tissue, organ or organism (eg, affinity, drug resistance), eg, a profile or a variation thereof. This can be done by a quantitative or constant 1 "biological association of the variation with at least one parameter of the state of the cell.
- the number of at least one profile used in the correlation is determined by the correlation
- the number may be as small as possible, and is usually, but not limited to, at least one, preferably at least two, more preferably at least three.In the present invention, at least two, preferably at least three Identify almost all cells by identifying at least one profile Such an effect, which was unpredictable with conventional profiling or Atsui's point of view, was a special effect first brought about by the present invention.
- at least one profile for example, a temporal profile
- mathematical processing using a determinant may be performed.
- At least one form used to correlate It may be advantageous for the number of profiles (eg, profiles for promoters) to be at least eight.
- Specific methods for correlation include, but are not limited to, signal processing methods (eg, using wavelets) and multivariate analysis (eg, cluster analysis).
- the correlation may be performed in advance, or may be performed using a control for each cell determination.
- exogenous factor when referring to a certain cell refers to a factor (eg, substance, energy, etc.) that is not normally present inside the cell.
- factor may be any substance or other element (eg, energy such as ionizing radiation, radiation, light, sound waves, etc.) as long as the intended purpose can be achieved.
- substances include, for example, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (eg, DNA such as cDNA, genomic DNA, etc.).
- RNA including mRNA, RNAi), polysaccharides, oligosaccharides, lipids, small organic molecules (eg, hormones, ligands, messengers, small organic molecules, synthesized in combinatorial chemistry) Molecules, small molecules that can be used as pharmaceuticals (eg, small molecule ligands, etc.), and composite molecules thereof, but are not limited thereto.
- One external factor is used However, a combination of two or more may be used.
- the extrinsic factors include temperature change, humidity change, electromagnetic wave, potential difference, visible light, infrared ray, ultraviolet ray, X-ray, chemical substance, pressure, gravity change, gas partial pressure and osmotic pressure. But not limited to them.
- the exogenous factor can be a biomolecule or a chemical compound.
- biomolecule refers to a molecule associated with a living organism.
- organ refers to a biological organism, including, but not limited to, animals, plants, fungi, viruses, and the like. Therefore, in the present specification, a biomolecule includes, but is not limited to, a molecule extracted from a living body, and is defined as a biomolecule as long as it is a molecule that can affect a living body. Therefore, molecules synthesized in combinatorial chemistry and small molecules that can be used as pharmaceuticals (for example, small molecule ligands) are also included in the definition of biomolecules as long as their effects on living organisms can be intended.
- biomolecules include proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (eg, DNA such as cDNA, genomic DNA, mRNA, etc.).
- RNA DNA such as cDNA, genomic DNA, mRNA, etc.
- polysaccharides, oligosaccharides, lipids, small molecules for example, hormones, ligands, signaling substances, small organic molecules, etc.
- complex molecules glycolipids, glycoproteins, lipoproteins
- Biomolecules can also include cells themselves, or portions of tissues, as long as introduction into the cell is contemplated.
- biomolecules can be nucleic acids, proteins, lipids, sugars, proteolipids, lipoproteins, glycoproteins, proteoglycans, and the like.
- the biomolecules include nucleic acids (DNA or RNA) or proteins.
- the biomolecule is a nucleic acid (eg, genomic or cDNA, or DNA synthesized by PCR or the like).
- the biomolecule can be a protein.
- such a biomolecule may be a hormone or a cytokine.
- the term "chemical compound” refers to any substance that can be synthesized using ordinary chemical techniques. Such synthesis techniques are well known in the art, and those skilled in the art will be able to combine such techniques as appropriate to produce chemical compounds.
- cytoforce in is defined in the broadest sense as used in the art and refers to a bioactive substance produced from a cell and acting on the same or different cells. Cytokines are generally proteins or polypeptides that regulate immune responses, regulate the endocrine system, regulate the nervous system, antitumor, antiviral, regulate cell growth, regulate cell differentiation, etc. Have. As used herein, cytoforce may normally be in the protein form, although it may be in a protein or nucleic acid form or other form. As used herein, “growth factor” refers to a substance that promotes or controls cell growth. Growth factors are also called growth factors or growth factors. Growth factors can be added to the medium in cell or tissue culture to replace the action of serum macromolecules.
- Cytokines typically include interleukins, chemokines, hematopoietic factors such as colony stimulators, tumor necrosis factors, and interferins.
- Typical growth factors are platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) )).
- PDGF platelet-derived growth factor
- EGF epidermal growth factor
- FGF fibroblast growth factor
- HGF hepatocyte growth factor
- VEGF vascular endothelial growth factor
- hormone is used in the broadest sense commonly used in the art, and refers to an organ that is produced by a specific organ or cell of animals and plants and is separated from the site where it is produced. A physiological organic compound that exhibits its specific physiological action. Such hormones include growth hormone, sex Hormones, thyroid hormones, and the like, but are not limited thereto. Such hormones may, in part, overlap with the cytokines described above.
- actin acting substance refers to a substance having a function of directly or indirectly interacting with actin in a cell to change the form or state of actin.
- substances include, but are not limited to, for example, extracellular matrix proteins (eg, fibronectin, vitronectin, laminin, etc.).
- actin agonists include those identified by Atsushi as follows. In the present specification, the interaction with actin is evaluated by visualizing the actin with a actin staining reagent (Molecular Probes, Tes Red—Xpha 11 oidin), etc., and then microscopically examining the actin aggregation.
- actin acting substance used in the present invention is derived from a living body, the substance may be of any origin, and examples thereof include human, mouse, and mammal species such as mammals.
- cell adhesion factor or “cell adhesion molecule” (Cell adhesion molecule) or “adhesion factor” or “adhesion molecule” is used interchangeably and refers to the approach of two or more cells to each other.
- Cell adhesion or A molecule that mediates adhesion between a substrate and a cell.
- cell-cell adhesion a molecule involved in cell-cell adhesion (cell-cell adhesion)
- cell-substrate adhesion cell — S ub strateadhesion mo lecu 1 e.
- the cell adhesion molecule This includes proteins on the substrate side during cell-substrate adhesion, but in the present specification, proteins on the cell side (eg, integrins) are also included, and even if it is a molecule other than a protein, it mediates cell adhesion. As far as it goes, the concept of the cell adhesion molecule or cell adhesion molecule herein is included.
- cadherin a number of molecules belonging to the immunoglobulin superfamily (NCAM, L1, ICAM, facyclin II, III, etc.), selectins, etc., are known, each of which has a unique molecular reaction to regulate cell membranes. It is also known to combine.
- the major cell adhesion molecule that works for cell-substrate adhesion is integrin, which recognizes and binds to various proteins contained in the extracellular matrix. All of these cell adhesion molecules are on the cell membrane surface and can be considered as a type of receptor (cell adhesion receptor). Accordingly, such receptors located on the cell membrane can also be used in the tissue pieces of the present invention. Such receptors include, but are not limited to, for example, alpha integrins, integrins, CD44, syndecan, and aggrecan. Techniques for cell adhesion are also well known in addition to those described above, and are described, for example, in Extracellular Matrix-Clinical Application-Medical Review.
- Whether a molecule is a cell adhesion molecule is determined by biochemical quantification (SDS-PAG method, labeled collagen method), immunological quantification (enzyme antibody method, fluorescent antibody method, immunohistological examination) PDR method, hybridization It can be determined by deciding that it is positive in Atsushi, such as the dimensionation method.
- cell adhesion molecules include collagen, integrins, fibronectin, laminin, vitronectin, fiprinogen, immunoglobulin superfamily (eg, CD2, CD4, CD8, ICM1, ICAM2, VCAM1), selenium Examples include, but are not limited to, cutin, cadherin, and the like. Many of these cell adhesion molecules are activated by cell-cell interaction simultaneously with cell adhesion.
- auxiliary signals Transmits auxiliary signals into cells. Whether such capture signals can be transmitted into cells can be determined by biochemical quantification (303-? 0 method, labeled collagen method), immunological quantification (enzyme antibody method, fluorescent antibody method, (Immunohistological examination) The determination can be made by determining that the antibody is positive in the PDR method or the hybridization method.
- Cell adhesion molecules include, for example, cadherin, immunoglobulin superfamily single molecule (CD2, LFA-3, ICAM-1, CD2, CD4, CD8, ICM1, ICAM2, VCAM1, etc.); Integrin family Molecules (LFA-l, Maccl, gpIIbIIIa, pl50, 95, VLA1, VLA2, VLA3, VLA4, VLA5, VLA6, etc.); Selectin family molecules (L-selectin, E-selectin) , P-selectin, etc.), but are not limited thereto.
- extracellular matrix protein refers to a protein that is a protein in “extracellular matrix”.
- ECM extracellular matrix
- extracellular matrix is also referred to as “extracellular matrix” and is used in the same sense as commonly used in the art. (som aticcell).
- the extracellular matrix is involved not only in the support of the ligament, but also in the composition of the internal environment necessary for the survival of all somatic cells.
- Extracellular matrices are generally produced by connective tissue cells, but some are also secreted by cells that themselves possess basement membranes, such as epithelial and endothelial cells.
- the fiber component is roughly divided into the matrix and the matrix that fills the space.
- the fiber component includes collagen fibers and elastic fibers.
- the basic constituent of the substrate is glycosaminodalican (acid mucopolysaccharide), most of which binds to non-collagenous proteins to form a macromolecule of proteodalican (acid mucopolysaccharide-protein complex).
- the substrate also includes glycoproteins such as laminin in basement membrane, microfibrils around elastic fibers, fibers, and fibronectin on cell surfaces. Specially differentiated pairs
- the basic structure is the same in weaving, for example, chondroblasts produce characteristically large amounts of proteodalican-containing cartilage matrix in hyaline cartilage, and osteoblasts produce a bone matrix in which calcification occurs in bone. .
- examples of the extracellular matrix used in the present invention include, but are not limited to, collagen, elastin, proteodalican, glycosaminodalican, fibronectin, vitronectin, laminin, elastic fiber, collagen fiber, and the like.
- receptor refers to a molecule that is present on a cell or in the nucleus, has a binding ability to a factor from the outside or a factor in a cell, and transmits a signal by binding.
- Receptors usually take the form of proteins.
- the binding partner of the receptor is usually called a ligand.
- agonist refers to a factor that binds to a receptor of a certain biologically active substance (ligand) and exerts the same (or similar) action as that of the substance.
- antagonist refers to a factor that acts antagonistically on the binding of a certain biologically active substance (ligand) to a receptor and does not itself exert a physiological action via the receptor. Antagonists, blockers (blockers), inhibitors (inhibitors), etc. are also included in this antagonist.
- the term “device” refers to a part that can constitute part or all of an apparatus, and includes a support (preferably a solid support) and a target substance to be carried on the support. Be composed.
- Such devices include, but are not limited to, chips, arrays, microtiter plates, cell culture plates, petri dishes, finolems, beads, and the like.
- support refers to a material (materia 1) that can immobilize a substance such as a biomolecule.
- the material of the support may be either a covalent bond or a non-covalent bond. Any solid material that has the property of binding to a substance such as or that can be derivatized to have such property is included.
- any material capable of forming a solid surface may be used, for example, glass, silica, silicon, ceramic, silicon dioxide, plastic, metal (alloy) ), And naturally occurring synthetic polymers (eg, polystyrene, cellulose, chitosan, dextran, and nylon), and the like.
- the support may be formed from a plurality of different material layers.
- inorganic insulating materials such as glass, quartz glass, alumina, sapphire, forsterite, silicon oxide, silicon carbide, and silicon nitride can be used.
- Polyethylene Polyethylene, ethylene, polypropylene, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetate, acryl resin, polyacrylonitrile, Polystyrene, acetal resin, polycarbonate, polyamide, phenolic resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, atarilonitrile butadiene styrene copolymer, silicone resin, polyphenylene Organic materials such as oxide and polysulfone can be used.
- a membrane used for blotting such as a nitrocellulose membrane, a nylon membrane, or a PVDF membrane
- a membrane used for blotting such as a nitrocellulose membrane, a nylon membrane, or a PVDF membrane
- the material constituting the support is a solid phase, it is referred to herein as a “solid support”.
- it may take the form of a plate, a microwell plate, a chip, a slide glass, a film, a bead, a metal (surface), or the like.
- the support may be coated or uncoated.
- liquid phase is used in the same meaning as usually used in the art, and usually means a state in a solution.
- solid phase has the same meaning as used in the art. Used for taste and usually refers to the solid state. In this specification, liquids and solids are sometimes collectively referred to as fluids.
- substrate refers to the material (preferably solid) from which the chip or array of the invention is constructed. Thus, the substrate is included in the concept of a plate.
- the material of the substrate can be any covalent or non-covalent bond that has the property of binding to the biomolecule used in the present invention or that can be derivatized to have such property.
- Solid materials are mentioned.
- any material capable of forming a solid surface may be used, for example, glass, silica, silicon, ceramic, silicon dioxide, plastic, metal (alloy) ), Natural and synthetic polymers (eg, polystyrene, cellulose, chitosan, dextran, and nylon).
- the substrate may be formed from multiple layers of different materials.
- inorganic insulating materials such as glass, quartz glass, alumina, sapphire, forsterite, silicon carbide, silicon oxide, and silicon nitride can be used.
- Organic materials such as polyphenylene oxide and polysulfone can be used.
- a preferable material for the substrate varies depending on various parameters such as a measuring instrument, and a person skilled in the art can appropriately select an appropriate material from the various materials described above.
- slide glasses are preferred.
- a substrate can be coated.
- coating when used with respect to a solid support or substrate refers to forming a film of a substance on the surface of the solid support or substrate and to such a film. Coating may be performed for various purposes, for example, to improve the quality of the solid support and the substrate (for example, to improve the service life, to improve environmental resistance such as acid resistance), to be bonded to the solid support or the substrate. Often aimed at improving the affinity of substances.
- various materials can be used, and in addition to the materials used for the solid support and the substrate itself, biological materials such as DNA, RNA, protein, and lipid, Polymers (eg, poly-L-lysine, MAS (Matsunami Glass, Kishiwada, available from Japan), water-repellent fluoroplastics), silanes (APS (eg, ⁇ -aminopropyl silane)), metals (Eg, gold) can be used, but is not limited to such.
- biological materials such as DNA, RNA, protein, and lipid
- Polymers eg, poly-L-lysine, MAS (Matsunami Glass, Kishiwada, available from Japan), water-repellent fluoroplastics), silanes (APS (eg, ⁇ -aminopropyl silane)), metals (Eg, gold)
- APS eg, ⁇ -aminopropyl silane
- metals eg, gold
- such a coating comprises poly-L-lysine, silane, (eg, epoxysilane or mercaptosilane, APS ( ⁇ -aminopropyl silane)), MAS, hydrophobic fluorine.
- silane eg, epoxysilane or mercaptosilane, APS ( ⁇ -aminopropyl silane)
- MAS ⁇ -aminopropyl silane
- hydrophobic fluorine It may be advantageous to use resins, metals such as gold.
- resins eg. epoxysilane or mercaptosilane, APS ( ⁇ -aminopropyl silane)
- MAS ⁇ -aminopropyl silane
- chip or “microchip” is used interchangeably, refers to a microminiature integrated circuit that has various functions and becomes part of a system. Examples of the chip include, but are not limited to, a DNA chip and a protein chip.
- the term “array” refers to a pattern in which threads (eg, DNA, protein, and transfect mixture) containing one or more (eg, 1000 or more) target substances are arranged. Or a substrate having a pattern (for example, a chip) itself. In the array, on a small substrate (eg, 10 x 10 mm The above patterned one is called a microarray, but in the present specification, the microarray and the array are used interchangeably. Thus, those patterned into larger substrates than those described above are sometimes referred to as microarrays. For example, an array is comprised of a set of desired transfatate mixtures that are themselves immobilized on a solid surface or membrane.
- Array preferably comprises at least 0 two identical or different that antibody, more preferably at least 1 0 3, and at least 0 4 preferably in the further, even more preferably at least 1 0 5 a.
- These antibodies are preferably located on a surface of 125 x 80 mm, more preferably 10 x 10 mm.
- a microtiter plate such as a 96-well microtiter plate, a 384-well microtiter plate, etc., and a size similar to a slide glass are contemplated.
- the composition containing the target substance to be immobilized may be one kind or a plurality of kinds. The number of such types can be any number from 1 to the number of spots. For example, a composition containing about 100 kinds, about 100 kinds, about 500 kinds, about 100 kinds of target substances can be immobilized.
- a solid phase surface or film such as a substrate
- the size of the substrate is preferably smaller.
- the spot size of a composition containing a target substance can be as small as the size of a single biomolecule (which can be on the order of 1-2 nm) .
- the minimum substrate area is determined in some cases by the number of biomolecules on the substrate.
- a target to be introduced into a cell The composition containing the quality is usually 0.01 mir! Arrays with covalent bonds in the form of spots of ⁇ 10 mm are fixed in sequence by physical interaction.
- spots of biomolecules may be arranged.
- spot refers to a certain set of compositions containing a target substance.
- spotting refers to producing spots of a fibrous material containing a certain target substance on a certain substrate or plate. Spotting can be performed in any manner, for example, by pipetting, or by automated equipment, and such methods are well known in the art.
- the term “address” refers to a unique location on a substrate that may be distinguishable from other unique locations. The address is suitable for association with the spot with that address, and has any shape so that the entity at every address can be distinguished from the entity at the other address (eg, optically). Can be taken.
- the shape defining the dress may be, for example, circular, oval, square, rectangular, or irregular. Therefore, “address” indicates an abstract concept, and “spot” may be used to indicate a specific concept. However, if there is no need to distinguish between the two, the term “address” will be used herein. "Address” and “spot” can be used interchangeably.
- the size defining each address depends, among other things, on the size of the substrate, the number of addresses on a particular substrate, the amount and / or available reagents of the composition containing the target substance, the size of the microparticles and the array thereof. Depends on the degree of resolution required for any method used.
- the size can be, for example, in the range of 1-2 nm to several cm, but can be any size consistent with the application of the array.
- the spatial configuration defining the address is designed to suit the particular application for which the microarray is used.
- the addresses can be densely arranged and widely dispersed, or can be subgrouped into desired patterns appropriate for a particular type of analyte.
- Microarrays are widely reviewed in the Protocols for Genome Function Research (Experimental Medicine Separate Volume, Experimental Lectures in the Post-Genome Era 1), Genomics Medical Science and Future Genomics Medicine (Experimental Medical Science Extra Edition).
- a cell or a substance interacting therewith Various detection methods and detection means can be used as long as the information resulting from the detection can be detected.
- detection methods and means include, for example, visual inspection, optical microscopy, confocal microscopy, fluorescence microscopy, readers using laser light sources, surface plasmon resonance (SPR) imaging, electrical signals, chemical or biochemical Examples include, but are not limited to, a method using one or more target markers.
- Such detectors also include, but are not limited to, fluorescence analyzers, spectrophotometers, scintillation counters, CCDs, luminometers, and any other means that can detect biomolecules. May be something.
- markers refers to a biological factor that reflects the level or frequency of a substance or state of interest.
- markers include, but are not limited to, for example, nucleic acids encoding genes, gene products, metabolites, receptors, ligands, antibodies, and the like.
- the marker relating to the state of a cell includes, in addition to a transcription control factor, an intracellular factor (for example, a nucleic acid encoding a gene, a gene product (eg, an mRNA, a protein) , Post-translationally modified proteins), metabolites, receptors and the like (eg, ligands, antibodies, complementary nucleic acids), and the like, but are not limited thereto.
- the present invention includes generating and analyzing a temporal profile for such a marker.
- Such a marker may advantageously advantageously interact specifically with the factor of interest.
- specificity for example, refers to the property of having a significantly higher degree of interaction with a molecule of interest than similar molecules.
- such a marker is preferably present inside a cell, but may be extracellular.
- label refers to an entity (eg, a substance, energy, an electromagnetic wave, or the like) for distinguishing a target molecule or substance from others.
- the labeling method include an RI (radioisotope) method, a fluorescence method, a biotin method, and a chemiluminescence method.
- the labeling is performed with fluorescent substances having different fluorescence emission maximum wavelengths. The difference between the fluorescence emission maximum wavelengths is preferably 10 nm or more.
- a base portion of the nucleic acid e.g., C y D y e TM Series C y 3, C y 5, etc.
- Rhodamine 6 G It is preferable to use reagents such as N-acetoxy N2-acetylaminofluorene (AA F), AA IF (iodine derivative of AAF) and the like.
- Examples of the fluorescent substance having a difference in the maximum fluorescence emission wavelength of 1 O nm or more include, for example, a combination of Cy5 and rhodamine 6G reagent, a combination of Cy3 and fluorescein, and a combination of rhodamine 6G reagent and fluorescein. Combinations and the like can be given.
- the target can be modified using such a label so that it can be detected by the detection means used. Such modifications are known in the art and those skilled in the art can implement such methods as appropriate depending on the label and the intended target.
- the term "interaction” includes hydrophobic interaction, hydrophilic interaction, hydrogen bonding, van der Waals force, ionic interaction, nonionic interaction, electrostatic interaction, and the like. But not limited to them.
- the level of such an interaction can be measured by methods well known in the art. Such methods include, for example, measuring the number of cells that have actually interacted and are in a fixed state, directly or indirectly (for example, by using reflected light, Intensity) counting, staining with a cell-specific marker, antibody, fluorescent label, etc., and measuring the intensity, but are not limited thereto. These levels are It can be displayed directly from the marker or indirectly via a label. From such measured values, for example, the number or frequency of genes actually transcribed or expressed in a certain spot can be calculated.
- display and “presentation” are used interchangeably and refer to a profile obtained according to the method of the present invention or information derived therefrom, directly or indirectly, or in the form of information processing. To embody. There are various types of such display modes, such as graphs, photographs, tables, and animations, and there is no limitation. Such techniques include, for example, MET HOD SIN CELL BI OLOGY, VOL. 56, ed.
- Real-time display and presentation can also be performed using techniques well known in the art. For example, after all the images have been acquired and stored in semi-permanent memory, or substantially simultaneously with the acquisition of the images, they can be processed with appropriate application software to obtain the processed data. For example, the method of processing the acquired data is to play back an uninterrupted sequence of images or to display in real time, as a continuous change in the focal plane, or as a ⁇ movie '' showing the illuminated light can do.
- the measurement and display application includes software for setting the conditions for normal stimulation and the recording conditions for the obtained detection signal.
- the computer not only constitutes means for applying stimulation to cells and means for processing signals detected from cells, but also optical observation means (SIT camera and image file device) and / or Alternatively, control of the cell culture means can be performed.
- desired complex stimulus conditions can be set by inputting stimulus conditions on the screen using a keyboard, touch panel or mouse.
- various conditions such as cell culture temperature and pH can be set using a keyboard, a mouse, and the like.
- the profile detected from the cells or information derived therefrom is displayed in real time or after recording.
- another recorded profile or information derived therefrom can be superimposed and displayed on a microscopic image of a cell.
- measurement parameters during recording stimulation conditions, recording conditions, display conditions, processing conditions, cell conditions, temperature, pH, etc.
- a warning function may be provided when the temperature or pH is out of the allowable range.
- a temporary profile display function and a topography display function may be provided. These analysis results can be displayed over the microscope image stored in the recording medium.
- the technique for introducing a nucleic acid molecule into a cell may be any technique, for example, transformation, transduction, transfection, and the like.
- transfusion is preferred.
- transfection refers to the cultivation or suspension of cells in the form of gene DNA, plasmid DNA, viral DNA, viral RNA, etc., in a nearly naked state that does not take the form of virus particles.
- gene transfer or infection is performed by being taken up by cells.
- a gene introduced by transfection is transiently expressed in cells, but may be permanently taken up.
- vector refers to a vector capable of transferring a target polynucleotide sequence into a target cell.
- vectors include those capable of autonomous replication in host cells such as prokaryotic cells, yeast, animal cells, plant cells, insect cells, animal individuals and plant individuals, or capable of integration into chromosomes, Those containing a promoter at a position suitable for polynucleotide transcription are exemplified.
- the vector suitable for crawling is called “crawling vector”.
- Such cloning vectors usually contain multiple cloning sites which contain more than one restriction enzyme site.
- expression vector refers to a nucleic acid sequence in which various regulatory elements in addition to a structural gene and a promoter that regulates its expression are operably linked in a host cell. Regulatory elements may preferably include terminators, selectable markers such as drug resistance genes, and enhancers. It is well known to those skilled in the art that the type of expression vector of an organism (eg, an animal) and the type of regulatory element used can vary depending on the host cell.
- Recombinant vectors for prokaryotic cells include pcDNA3 (10), pB1uescript-SK (+ / _), pGEM-T, pEF-BOS, pEGFP, pHAT, pUC18, pFT-DEST TM 42 GATEWAY (Invitrogen) and the like.
- recombinant vectors for animal cells include pcDNAIZAmp, pcDNAI, pCDM8 (all commercially available from Funakoshi), AGE107 [Japanese Unexamined Patent Publication (Kokai) No. 3-229 (Inv itrogen), pAGE103 [J. Biochem. , 101, 1307 (1987)], pAMo, pAMoA [J. Biol. Chem., 268, 22782-22787 (1993)], murine stem cell virus (Murine Stem Cell Virus) ( MS CV) based retroviral expression vectors, pEF-BOS, .pEGFP, and the like.
- Recombinant vectors for plant cells include, but are not limited to, pPCV ICEn 4HPT, pCGN1548, pCGN1549, pBI221, pBI121, and the like.
- any method as described above for introducing DNA into cells can be used.
- transfection, Transfection, transformation, etc. eg, calcium phosphate method, ribosome method, DEAE dextran method, electroporation method, particle gun (gene gun) method, etc.
- ribofluxion method e.g., calcium phosphate method, ribosome method, DEAE dextran method, electroporation method, particle gun (gene gun) method, etc.
- ribofluxion method eg. calcium phosphate method, ribosome method, DEAE dextran method, electroporation method, particle gun (gene gun) method, etc.
- ribofluxion method eg., ribofluxion method
- spheroplast method Pro N at 1 USA, 84, 1929 (1 978)
- lithium acetate method J. B acterio 1 153, 163 (1 983)
- Proc. Natl. Ac ad. USA 75, 1929 (1978
- operably linked refers to a transcription / translation control sequence (eg, promoter, enhancer, etc.) or a translation control sequence that has expression (activity) of a desired sequence.
- a transcription / translation control sequence eg, promoter, enhancer, etc.
- a translation control sequence that has expression (activity) of a desired sequence.
- Means to be placed in In order for a promoter to be operably linked to a gene, the promoter will usually be located immediately upstream of the gene, but need not be.
- the term “gene introduction reagent” refers to a reagent used to promote the efficiency of introduction in a gene introduction method.
- a gene transfer reagent include, but are not limited to, cationic polymers, cationic lipids, polyamine-based reagents, polyimine-based reagents, calcium phosphate, and the like.
- Specific examples of the reagents used in transfection include commercially available reagents from various sources.
- Effectene Transfection Reagent (cat.no.301425, Qiagen, CA), Trans Fast TM Tranfection Reagent (E 2431, Prome ga, WI), T fx TM—20 R eagent (E 2391, Prome ga, WI), Super Fect Tr ansfeetion Re agent (301305, Q iagen, CA), Po 1 y Fect Transfection Re agent (301 105, Q iagen, CA), Lipofect AM I NE 2000 Re agent (1 1668—01 9, Invitrogencorporation, (CA), J et PE I (X 4) con e. (101-30, Po 1 y 1 s ⁇ transfection, France) and Ex Gen 500 (R 0 511, Fermenta ntas I n c. , MD), but are not limited to these.
- “detection” or “quantification” of gene expression can be achieved using an appropriate method including, for example, mRNA measurement and immunological measurement.
- the molecular biological measurement method include a Northern plot method, a dot plot method, and a PCR method.
- the immunological measurement method include, for example, an ELISA method using a microtiter plate, an RIA method, a fluorescent antibody method, a Western blot method, and an immunohistological staining method.
- Examples of the quantification method include the ELISA method and the RIA method. It can also be performed by a gene analysis method using an array (for example, a DNA array or a protein array).
- DNA arrays are widely reviewed in (Shujunsha eds., Cell Engineering Separate Volume “DNA Microarrays and the Latest PCR Method”).
- the protein array is described in detail in Nat Genet. 2002 Dec; 32 Suppl: 526-32.
- Methods for analyzing gene expression include, but are not limited to, RT-PCR, RACE, SSCP, immunoprecipitation, two_hybrid system, in vitro translation, and the like, in addition to the above.
- Such a further analysis method is described in, for example, Genome Analysis Experimental Method, Yusuke Nakamura Lab. Manual, edited by Yusuke Nakamura, Yodosha (2002), and all the descriptions in this specification are incorporated herein by reference. Incorporated.
- “Expression level” refers to the level at which a polypeptide or mRNA is expressed in a target cell or the like. Such an expression level can be determined using the antibody of the present invention by any appropriate method including immunological measurement methods such as ELISA, RIA, fluorescent antibody, Western blot, and immunohistological staining.
- Invented poly (II) The polypeptide of the present invention evaluated by the expression level of the peptide at the protein level, or by any appropriate method including a molecular biological measurement method such as the Northern plot method, the dot plot method, and the PCR method. At the mRNA level.
- “Change in expression level” refers to expression of the polypeptide of the present invention at the protein level or mRNA level, which is evaluated by any appropriate method including the above-described immunological measurement method or molecular biological measurement method. It means that the amount increases or decreases.
- screening refers to selecting a target such as an organism or a substance having a specific property of interest from a population including a large number by a specific operation Z evaluation method.
- a target such as an organism or a substance having a specific property of interest from a population including a large number by a specific operation Z evaluation method.
- the method or system of the present invention can be used.
- the antibodies or single-chain antibodies of the invention can be used for immunophenotyping cell lines and biological samples.
- Transcripts and translation products of the gene of the present invention are useful as cell-specific markers, or more specifically, as cell markers that are differentially expressed at various stages of differentiation and / or maturation of a particular cell type. It is.
- Monoclonal antibodies directed against specific epitopes or combinations of epitopes allow for the screening of cell populations expressing the marker.
- a variety of techniques can be employed using monoclonal antibodies to screen cell populations expressing the marker, including magnetic separation using magnetic beads coated with the antibody, solid matrices (ie, And “panning” using antibodies attached to plates, and flow cytometry (eg, US Pat. No. 5,985,660; and Morrison et al., Cell 1, 96). : 737-49 (1999)). These techniques are used to grow cells as can be found in human cord blood. Or used to screen for a cell population that contains undifferentiated cells (eg, embryonic stem cells, tissue stem cells, etc.), such as a cell population that has undergone differentiation or has been modified to an undifferentiated state. Can be done.
- undifferentiated cells eg, embryonic stem cells, tissue stem cells, etc.
- diagnosis refers to identifying various parameters related to a disease, disorder, or condition in a subject, and determining the current state of such disease, disorder, or condition.
- the sugar chain structure can be analyzed and the drug resistance level can be correlated, and such information can be used to treat a disease, disorder, condition, or treatment to be administered to a subject.
- various parameters can be selected, such as formulations or methods for prevention.
- the diagnostic method of the present invention is industrially useful because, in principle, it can be used from the body, and can be carried out without the hands of medical staff such as doctors.
- the term “treatment” refers to preventing the worsening of a disease or disorder when such a condition occurs, preferably maintaining the status quo, more preferably reducing, It preferably refers to fluctuating.
- the term “subject” refers to an organism to which the treatment of the present invention is applied, and is also referred to as “patient J. The patient or the subject may preferably be human.
- pathogenesis refers to a factor associated with a disease, disorder or condition (generally referred to as “lesion” or “disease” in plants) of a subject, such as a causative agent (pathogenic agent). ), Pathogens, diseased cells, pathogenic viruses, and the like.
- the “disease” targeted by the present invention can be any disease associated with a pathogenic gene.
- diseases include cancer, viral or bacterial infections, allergies, hypertension, hyperlipidemia, diabetes, heart disease, cerebral infarction, dementia, obesity, arteriosclerosis Sexual disorders, infertility, neuropsychiatric disorders, cataracts, progeria, UV radiation sensitivity, and the like.
- the “disorder” targeted by the present invention can be any disorder associated with a pathogenic gene.
- cardiovascular diseases eg, anemia (eg, aplastic anemia (particularly severe aplastic anemia)), renal anemia, cancer anemia, secondary Anemia, refractory anemia, etc.), cancer or tumors (eg, leukemia, multiple myeloma), etc .
- cardiovascular diseases eg, anemia (eg, aplastic anemia (particularly severe aplastic anemia)), renal anemia, cancer anemia, secondary Anemia, refractory anemia, etc.
- cancer or tumors eg, leukemia, multiple myeloma
- nervous system disorders e., stroke and sequelae, brain tumors, spinal cord injury, etc.
- immune system disorders etc.
- T-cell deficiency leukemia, etc .
- locomotor and skeletal diseases fractures, osteoporosis, joint prolapse, subluxation, sprains, ligament damage, osteoarthritis, osteosarcoma, Ewing sarcoma, osteogenesis imperfecta, Osteochondrodysplasia, etc.
- dermatological diseases alopecia, melanoma, cutaneous malignant lymphoma, angiosarcoma, histiocytosis, bullous disease, pustulosis, dermatitis, eczema, etc.
- endocrine diseases hypothalamics ⁇ Pituitary disease, thyroid disease, vice Thyroid (parathyroid) disease, adrenal cortex, medulla disease, abnormal glucose metabolism, abnormal lipid metabolism, abnormal protein metabolism, abnormal nucleic acid metabolism, abnormal congenital metabolism (phenylketonuria, galatatosemia, homocystinuria) Disease, maple syrup urine disease), analbumin
- cancer or “cancer” is used interchangeably, is atypical, proliferates faster than normal cells, and can destructively invade surrounding tissues or can cause malignancy or metastasis thereof. It refers to a state in which such a malignant tumor exists.
- cancers include, but are not limited to, solid cancers and hematopoietic tumors.
- solid cancer refers to a cancer having a solid form, and is a concept that is opposed to hematopoietic tumors such as leukemia.
- Such solid cancers include, for example, breast cancer, liver cancer, stomach cancer, lung cancer, head and neck cancer, cervical cancer, prostate cancer, retinoblastoma, malignant lymphoma, esophageal cancer, brain tumor , But not limited to, bone tumors.
- cancer treatment includes surgical treatment performed by administering an anticancer agent (eg, a chemotherapeutic agent, radiation therapy, or the like), or performing surgical removal. .
- an anticancer agent eg, a chemotherapeutic agent, radiation therapy, or the like
- chemotherapeutic agent used in the present specification is well known in the art, and is an anticancer agent Mayual 2nd edition, edited by Shigeru Tsukagoshi et al. Chugai Medical Co .; Pharmacoco 1 ogy; Lippincott Williams & Wi 1 kins , Inc.
- chemotherapeutic agents include, for example, But not limited to: 1) Alkylating agents (alkylate cellular components such as DNA and proteins and exhibit cytotoxicity.
- Examples include, but are not limited to, cyclophosphamide, busulfan, thiotepa, and dacarbazine) 2) antimetabolites (agents that mainly inhibit the synthesis of nucleic acids (eg, methotrexate as an antifolate, 6-mercaptopurine as an antimetaboline, fluorouracil (5-FU) as an antimetabolite, etc.) 3) DNA topoisomerase inhibitors (eg, camptothecin and etoposide (which inhibit topoisomerase I and II, respectively)); 4) Tubulin agonists (inhibit microtubule formation and suppress cell division.
- nucleic acids eg, methotrexate as an antifolate, 6-mercaptopurine as an antimetaboline, fluorouracil (5-FU) as an antimetabolite, etc.
- DNA topoisomerase inhibitors eg, camptothecin and etoposide (which inhibit topoisomerase I and
- Platinum compounds DNA and tamper
- Anticancer antibiotics binding to DNA and inhibiting DNA synthesis and RNA synthesis.
- Adriamycin, daunorubicin, mitomycin C, bleomycin, etc. 7) Hormonal agents (adapted to hormone-dependent cancers such as breast cancer, uterine cancer, and prostate cancer.
- Tamoxifen leuprorelin (LH-RH), etc.); 8) Biological products (asparagine-requiring hematological malignancies) 9) Immunostimulants (enhance immune response and exhibit indirect antitumor activity) Lentinan, a polysaccharide derived from shiitake mushrooms, bestatin, a peptide derived from microorganisms, etc.).
- anticancer agent selectively inhibits the growth of cancer (tumor) cells, and includes both cancer drugs and radiation therapy.
- pile cancer drugs are well known in the art, and are described, for example, in Anticancer Drugs Manual, Second Edition, edited by Shigeru Tsukagoshi et al. Chugai Medical Co .; Ph. Rmacologyi L ippincott Wi 11 i ams & Wilkins, In c.
- Radiation therapy or “radiation therapy” is used interchangeably and refers to the treatment of a disease utilizing ionizing radiation or a radioactive substance.
- Typical Radiation therapy includes, but is not limited to, X-rays, gamma rays, electron beams, proton beams, heavy ion beams, and neutron capture therapy.
- Preferred radiation therapy includes heavy ion beams. The therapy using heavy ions may be large and uncommon.
- Such radiation therapies are well known in the art and include, for example, the fundamentals of radiological examination and treatment; , 6-7 (2002.02).
- the drug resistance identified in the present invention is usually assumed to be chemotherapy, but since the resistance due to radiation therapy is also associated with a profile, radiation therapy falls within the concept of a drug in this specification. .
- pharmaceutically acceptable carrier refers to a substance used in producing agricultural chemicals such as pharmaceuticals or veterinary drugs, which does not adversely affect the active ingredient.
- Such pharmaceutically acceptable carriers include, but are not limited to, for example: antioxidants, preservatives, coloring agents, flavorings, and diluents, emulsifiers, suspending agents Agents, solvents, fillers, bulking agents, buffers, delivery vehicles, diluents, excipients and / or agricultural or pharmaceutical adjuvants.
- the type and amount of the drug used in the treatment method of the present invention are determined based on the information obtained by the method of the present invention (for example, information on drug resistance level), the purpose of use, the target disease (type, severity, etc.). ),
- the patient's age, weight, sex, medical history, and the form or type of the site of the subject to be administered can be easily determined by those skilled in the art.
- the frequency of applying the treatment method of the present invention to a subject (or patient) also depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, sex, medical history, and course of treatment. A person skilled in the art can easily determine the above in consideration.
- the frequency of drug administration or monitoring of drug resistance may be, for example, once every few months (eg, once a week, once a month). It is preferable to administer once a month, once a week, while monitoring the progress. Good.
- the “instruction” describes the tiller-made treatment method of the present invention or the like for a doctor, a patient, or another person who performs administration.
- the instructions include a statement instructing that the medicament or the like of the present invention be administered, for example, immediately after or immediately before radiation treatment (for example, within 24 hours).
- These instructions are prepared in accordance with the format prescribed by the competent authority of the country in which the invention is implemented (for example, the Ministry of Health, Labor and Welfare in Japan and the Food and Drug Administration (FDA) in the United States) and are issued by that competent authority. The approval is clearly stated. Instructions are so-called package inserts, which are usually provided on paper, but are not limited to, e.g., electronic media (e.g., home pages, email provided on the Internet). Such a form can also be provided.
- more than one drug may be used in the treatment of the present invention.
- substances of similar properties or origins may be used, or drugs of different properties or origins may be used.
- Information on drug resistance levels for such methods of administering two or more drugs can also be obtained by the methods of the present invention.
- Gene therapy can also be performed based on the obtained information on drug resistance.
- Gene therapy refers to therapy performed by the administration of an expressed or expressible nucleic acid to a subject.
- the nucleic acids produce their encoded protein, which protein mediates a therapeutic effect.
- the result of analysis of the corresponding profile is correlated with the result of analysis of the corresponding profile.
- an organism of a similar kind for example, mouse to human
- the result of analysis of the corresponding profile is correlated with the result of analysis of the corresponding profile.
- Gene therapy refers to therapy performed by the administration of an expressed or expressible nucleic acid to a subject.
- the nucleic acids produce their encoded protein, which mediates a therapeutic effect.
- Auresube 1 F CurrentPrototocolsinMolleculalarBio1ogy, GleeneP.Associaetes;
- such mathematical processing may be a regression analysis.
- Regression analysis includes, but is not limited to, linear regression (including simple regression analysis, multiple regression analysis, robust estimation, etc.) and nonlinear estimation.
- a and b are model parameters, and is Deviation or error.
- an analysis is usually performed in which a and b are determined so that the average value of the sum of squares of the vertical distance between the data point and the direct point is minimized.
- a value that minimizes the square error can be obtained by performing a partial differentiation, establishing a system of linear equations, and solving this. Such a value is called a least squares estimate.
- the regression direct is calculated for the value obtained by subtracting the average value from each data.
- a regression line As a regression line
- the multiple regression analysis method used in another embodiment considers that y is not a single independent variable, but a function of two or more variables, for example,
- a mouth past estimation method is used.
- the least squares method has no bias in the measured values, the measurement error has a normal distribution, and the model has no approximation error It is based on the assumption that.
- mouth bust estimation since actual measurement errors and simple errors can occur, such unreliable data is detected and removed as outliers from the majority of reliable data, or Statistical processing is called mouth bust estimation.
- Such a mouth bust estimation method can also be used in the present invention.
- Non-linear estimation methods may also be used herein. With such a nonlinear estimation method, it is possible to express a nonlinear model as a vector equation and find a solution.
- principal component analysis method which uses principal component analysis of two-dimensional data, principal component analysis of multidimensional data, singular value decomposition, and generalized inverse matrix.
- canonical correlation analysis, factor analysis, discriminant analysis, cluster analysis, and the like can be used.
- Embodiments for identifying such reference transcription control sequence sets include clustering algorithms (for an overview of clustering algorithms, see, e.g., Fukunaga, 1990, Statistical Pattern Recognition, 2nd ed d). ., Ac ad emic Press, San Diego; An derberg, 1973, Cluster An alysis for Ap lications, Ac ad emic Press: New York; Ev eritt, 1974, Cluster An alysis , London: Hein ema nn E duc. n).
- a transcription control sequence set can also be defined based on a transcription control mechanism.
- Transcription control sequences having transcription factor binding sites of the same or similar sequence in the regulatory region are likely to be co-regulated.
- the regulatory regions of the transcriptional regulatory sequence of interest can be compared using multiplex alignment analysis to decipher possible covalent transcription factor binding sites (Stormo and Hartze 1
- Preferred embodiments for identifying such a basic set of biological factors include a clustering algorithm
- the status of a number of biological agents can be monitored while various stimuli are applied to the biological sample.
- Tables of data containing measurements of the status of biological agents are used for cluster analysis.
- a basic set of biological factors including those that change simultaneously over various conditions, Usually, at least two, preferably at least three, more preferably at least 10, even more preferably more than 50 and most preferably more than 100 stimuli or conditions are used to obtain them.
- the cluster analysis is performed on a table of data having m ⁇ k dimensions, where m is the total number of conditions or stimuli, and k is the number of biological factors to be measured.
- Clustering algorithms use differences or distances between objects when forming clusters.
- the distance used is a Euclidean distance in multidimensional space:
- I (x, y) is the distance between gene X and gene Y (or between any other cellular component (eg, biological factor) X and ⁇ ); ⁇ i is the gene expression response under stimulus i.
- the distance criterion may be the Manhattan distance, for example, between biological factor X and biological factor Y, which is:
- X i and Y i are biological factors or gene expression responses under stimulus i.
- IXI (X ⁇ X) 1/2
- I ⁇ I ( ⁇ ⁇ ⁇ ) Ru 1/2
- distance criteria are used to identify, for example, co-modifying cells or co-regulated cellular components (such as co-modifying or co-regulated biological agents).
- r fret is preferably the formula shown below:
- the correlation coefficient of the above normal weighted inner product is the value + 1 (indicating that the two response vectors are completely correlated and essentially identical) and 1 (the two response vectors are "correlated”). No “or” not facing the same direction “(ie, pointing in the opposite direction).
- These correlation coefficients determine the cellular components (eg, biological factors, transcriptional regulatory sequences) that have a set or cluster that have the same symptom response (eg, biological factors, transcriptional regulatory sequences). Particularly preferred for embodiments of the present invention.
- cellular components that co-regulate or participate in the same biological response or pathway but contain similar and uncorrelated responses (eg, biological factors, transcriptional regulation) It is preferred to identify a set or cluster of In such an embodiment, it is preferable to use the absolute value of either the above-mentioned normalization or the weighted inner product, that is, i r I as the correlation coefficient.
- the relationship between co-regulated and Z or co-changing cellular components is even more complex, and involves multiple biological pathways (eg, In some cases, signal transduction pathways, for example, are clustered into the same cellular component (eg, biological factors, transcriptional regulatory sequences) and produce different results.
- the correlation coefficient specified in the following equation (Equation 5) is particularly useful in such an embodiment:
- Such methods include, for example, the simple association method, the closest point method, and the like, which measure the distance between the two closest objects.
- a fully associative method that can be used in the present invention measures the distance at the maximum distance between two objects in different clusters. This method is particularly useful where genes or other cellular components naturally form distinct "clump" Js.
- the average of the unweighted pair group defines the distance as the average distance between all object pairs in two different clusters.
- This method is also very useful for clustering genes or other cellular components that naturally form distinct “aggregates”.
- a weighted pair group averaging method is also available. This method is the same as the unweighted pair group average method, except that the size of each cluster is used as a weight. This method is particularly useful in embodiments where the size of clusters, such as biological factors, is suspected to be highly variable (Sneath and Sokal, 1973, Nume ricaltaxon omy, San Franci'sco: WH F re ema n & C o.).
- cluster association rules such as unweighted and weighted pair group centroid and quad methods, are also useful in some embodiments of the present invention. See, for example, Ward, 196 3, J. Am. Stats As ssn. 58: 236; Hartigan, 1975, Cluste ergin g algori hthms, New York: Wiley.
- the cluster analysis is based on he1 ust knowledge techniques (eg, "hc1 ust:” from the program S-Plus, Math S oft, Inc., Cambridge, MA). Please refer to the well-known procedure for " It can be performed using:
- Such stimulus conditions include drug treatment at different concentrations, different measurement times after treatment, response to genetic mutations in various genes, combinations of drug treatment and mutation, and changes in growth conditions (temperature , Density, and calcium concentration).
- objective tests can be defined by randomizing the findings of the experiments on the response of each cell component across the set of experiments with Monte Carlo.
- an objective test can be defined in the following way: Let p ki be the response of component k in experiment i. ⁇ Let (i) be a random permutation of the index of the experiment. Then, P kn (i) is set for each of a number (about 100-1000) of different random permutations. For each branch of the original tree, for each sort: (1) Perform hierarchical clustering using the same algorithm used for the unsorted original data (in this case, “hclus tj”) ;
- D k is the square of the distance criterion (average) of component k with respect to the center of the cluster to which it belongs.
- the superscript 1 or 2 indicates whether it relates to the center of the whole branch or to the center of the preferred cluster of the two subclusters.
- D l—r, where r is the phase between the responses of one component across the experimental set, relative to another (or relative to the average cluster response). The relation number.
- objective statistical tests can preferably be used to determine the statistical reliability of any clustering method or algorithm grouping decision.
- similar tests can be used for both hierarchical and non-hierarchical clustering methods.
- the compactness of a cluster is determined, for example, as the average of the squares of the distances of the elements of the cluster from the "mean of the cluster", and more preferably as the reciprocal of the average of the squares of the distances of the elements from the mean of the cluster. , Is defined quantitatively.
- the cluster mean for a particular cluster is generally defined as the mean of the response vectors of all elements of the cluster.
- ie, 1 1 ⁇
- a different definition of compactness of the cluster such as, but not limited to, the mean of the squares of the distances between all pairs of elements in the cluster.
- the compactness of a cluster can be defined to mean the average distance (or more preferably the reciprocal of the average distance) from each element (eg, cell component) of the cluster to other elements of the cluster. it can.
- element eg, cell component
- Other definitions that can be used in the statistical methods used in the present invention will be apparent to those skilled in the art.
- the profiles of the present invention can be analyzed using signal processing techniques.
- signal processing techniques by defining a correlation function, calculating a correlation coefficient, defining an autocorrelation function and a cross-correlation function, and calculating them so that their weights sum to one, A moving average can be obtained.
- Rhythm is often important in the analysis of natural phenomena, especially the dynamic characteristics of life and living organisms.
- a time function f (t) a function that satisfies the following condition is called a periodic function.
- the value of the function at this time is f (0), and after various changes, the time T At that point, it will return to the same value as f (0).
- a function is called a periodic function, and a typical example of such a function is a sine-like wave.
- T is called a period.
- the concept expressed by the number of cycles per unit time is called frequency. If the frequency is expressed as f,
- time and frequency have a front and back relationship.
- time domain When dealing with time, it is called time domain, and when dealing with frequency, it is called frequency domain.
- the frequency can also be expressed electrically.
- a period can be expressed as 360 ° or 2 ⁇ radians, with one period converted to an angle.
- a sine wave can be represented as a time delay of a cosine wave, and this time delay is called a phase (phase).
- phase phase
- the phase is 90 ° in a sine wave.
- the sum of a sine wave and a cosine wave has an amplitude increase of 2 and a phase of ⁇ / 4.
- a Fourier series and frequency analysis technique can be used. It is also possible to use Fourier transform, discrete Fourier transform and power spectrum. In Fourier series expansion, a wavelet transform method or the like can be used. Such techniques are well known in the art, and are described in Mathematics for Life Systems Analysis, Corona, Yuki Shimizu (1992), Wavelet Analysis for Clinical Medicine, Medical Publishing, Yasuhiro Ishikawa Have been.
- the present invention provides a method for presenting a state of a cell.
- Such methods include: a) monitoring the status of cells associated with at least one biological factor selected from the group of biological factors derived from the cells over time and monitoring the profile of the cells; Obtaining said profile; and b) presenting said profile.
- a function of change can be obtained and displayed by performing an interval differentiation on a profile of the signal strength obtained as a result of monitoring.
- such a profile is preferably obtained by taking a difference with respect to a biological factor assumed to be unchanged, for example, a constitutive promoter. , But is not limited thereto.
- any method may be used to display the profile. For example, it may be displayed visually using a display (for example, time on the X-axis and signal strength on the y-axis), or may be displayed as a numerical table using spreadsheet software or the like. .
- the signal intensity can be displayed on the display as some other light intensity.
- the profile may be displayed by audio.
- the cells are monitored while fixed to a support (preferably, a solid support, such as an array, plate, microtiter plate, etc.).
- a support preferably, a solid support, such as an array, plate, microtiter plate, etc.
- Such a fixation method can be performed based on any method known in the art or the method described herein. By fixing the cells, the tests can be performed systematically.
- such a profile can be presented in real time.
- the real-time may have a certain time lag as long as it can be displayed substantially simultaneously.
- the allowed time lag depends on the required real-time concurrency, but can be, for example, up to 10 seconds, and more preferably up to 1 second.
- the present invention provides a method for determining a state of a cell. Since such a determination of the state of a cell involves observing a change in the transcriptional state of a transcription factor as a process, an element that has not been observed at all in the past is added to the determination factor. Therefore, the method for determining a cell state according to the present invention makes it possible to determine various states that could not be observed conventionally.
- Such a method comprises: a) monitoring the transcriptional status associated with at least one biological factor selected from the group of biological factors derived from the cell over time to obtain a profile of the cell; And b) determining the state of the cell from the profile of the transcription state.
- the cells comprise a support (preferably a solid support, eg, an array, Plate, microtiter plate, etc.).
- a fixing method can be performed based on a method known in the art or a method described herein.
- the method for determining a cell state of the present invention may advantageously further include a step of pre-correlating the profile with the state of the cell, or the information of such correlation is provided in advance. Is also good. Such a correlation process may be performed each time a determination is made, or a correlation saved as a database may be used.
- the biological agent used may be a transcription control sequence, such as a promoter, enhancer, silencer, flanking sequence of a structural gene in another genomic structure. And genomic sequences other than exon, but are not limited thereto. Promoters are preferred. This is because the transcriptional state can be measured directly, and the transcriptional state often reflects the state of the cell.
- transcription control sequences may be constitutive promoters, specific promoters, inducible promoters, and the like.
- the biological element (eg, promoter) of the present invention is not particularly limited, and may be any kind.
- a profile can be analyzed from the viewpoint of “process”. Therefore, any biological factor (eg, promoter, structural gene, etc.) or its heterologous or homologous species can be analyzed. It has become possible to determine the state of the cells using the set. Such a determination is impossible with the conventional technology, and the present invention is highly useful in the sense that it has achieved what was not achievable from the conventional technology.
- At least two biological agents to be monitored are used. Observe at least two biological factors This makes it possible to identify a cell state of usually 80% or more (preferably 90% or more, and in some cases almost 100%). More preferably, the biological agent to be monitored comprises at least three biological agents. Observing at least three biological factors, usually identifying more than 90% (preferably more than 95%, and in some cases nearly 100%) of the biological agents This is because it becomes possible. In a most preferred embodiment, the biological agent being monitored comprises at least eight transcription control sequences. Observing at least eight biological factors usually makes it possible to identify all cellular states.
- the biological agent being monitored comprises at least eight transcription control sequences. Observing at least eight biological factors usually makes it possible to identify all cellular states.
- the status of almost all cells can be determined by selecting only a small number as described above and monitoring it. Is much more simple, accurate, and accurate than the traditional method of observing each time point and statistically processing it as a heterogeneous population. Will be provided.
- the monitoring method of the present invention preferably further comprises a step of arbitrarily selecting at least one biological factor from a group of biological factors before monitoring.
- a biological factor that does not show specificity in point-by-point research can be used.
- the data obtained will more accurately reflect the state of the cell of interest. Data of such accuracy cannot be obtained by the conventional technology.
- the profiles obtained in the present invention can be presented in real time.
- data can be obtained in real time.
- “Real time” as used herein means that a certain time lag may occur as long as they can be displayed substantially simultaneously. Forgiveness The time lag accommodated depends on the required real-time concurrency, but can be, for example, up to 10 seconds, more preferably up to 1 second. For example, in treatments that require real-time diagnosis, the real-time capability
- the state determined by the cell state determination method of the present invention includes, for example, a differentiated state, an undifferentiated state, a cell response to an exogenous factor, a cell cycle, and a proliferative state. More specifically, such conditions include, for example, the response of cancer cells to anticancer agents, drug resistance, response to biological time, stem cells (eg, mesenchymal stem cells, neural stem cells, etc.) Differentiation state, or undifferentiated state of purified stem cells (eg, embryonic stem cells), changes in cell morphology, cell migration state, intracellular localization of molecules, secretory substance-producing ability, and the like. Not limited.
- cells used in the present invention include, but are not limited to, stem cells or somatic cells or mixtures thereof.
- stem cells or somatic cells or mixtures thereof may be adherent cells, floating cells, tissue forming cells, and mixtures thereof.
- the cell state determining method of the present invention can be performed on cells fixed on a substrate as a support (preferably a solid support).
- a support preferably a solid support
- the solid support is called a chip, and if the cells are aligned, also called an array.
- the reporter in the method for determining a cell state of the present invention, when the biological factor (for example, a transcription control sequence) to be determined is a nucleic acid molecule, the reporter is operably linked to the nucleic acid molecule.
- the reporter is transfected into the cell of interest in the form of a nucleic acid molecule comprising a single gene sequence.
- the transcription state can be measured as a reporter gene signal.
- transfections can be performed on a solid phase or in a liquid phase.
- a method for increasing the efficiency of introducing a target substance into cells can be used.
- the present invention presents a target substance (for example, DNA, RNA, polypeptide, bran chain, or a complex thereof) that is hardly introduced into a cell under normal conditions to a cell together with an actin acting substance such as fibronectin. (Preferably, contact) by utilizing the effect that the target substance is efficiently introduced into cells.
- this transfection method comprises in random order A) a step of providing a target substance (ie, a DNA containing a transcription control sequence); B) a step of providing an actin agonist (eg, fibronectin). And d) contacting the target substance and the actin acting substance with the cell.
- the target substance and the actin-acting substance may be provided together or separately.
- the actsin-acting substance As the actin-acting substance, the forms described in detail in the above-mentioned thread composition for increasing the efficiency of introduction of the target substance of the present invention into cells can be applied. Those skilled in the art can select and implement an appropriate form based on the description in this specification. Therefore, as the actin acting substance, a person skilled in the art can arbitrarily select a form applied in a composition for increasing the efficiency of introducing the target substance of the present invention into cells, and carry out the present invention. it can.
- the actin agonist may be an extracellular matrix protein (eg, fibronectin, vitronectin, laminin, etc.) or a variant thereof. More preferably, fibronectin or a variant or fragment thereof can be used.
- the sequence when the biological agent used in the present invention is a transcription control sequence, the sequence has the ability to bind to the transcription factor.
- transcription factors include, for example, ISRE, RARE, STAT3, GAS, NFAT, MIC, AP1, SRE, GRE, CRE, NFKB, ERE, TRE, E2F, Rb, p53, etc. But not limited to them. like this As the transcription factors, those commercially available from BD Biosciences Clonetech, CA, USA can be used. Where ISRE is associated with STAT 1Z2 and RARE is associated with retinoic acid.
- STAT 3 is related to differentiation control, and GRE is related to glucose metabolism.
- CRE is related to cAMP and TRE is related to thyroid hormone.
- E2F is associated with the cell cycle, and p53 is associated with the G1 checkpoint. Therefore, it is possible to determine the cell state based on such information.
- the determining step in the present invention includes comparing the phases of the profiles obtained in the present invention.
- the calculation of the phase can be appropriately performed by a person skilled in the art in consideration of the general method described above in the present specification and the method described in the examples.
- the determination step in the present invention includes a step of taking a difference between the open file of the cell and the control profile.
- the calculation of the difference can be appropriately performed by a person skilled in the art in consideration of the general method described above in the present specification and the method described in Examples.
- the judging step in the present invention includes a mathematical process selected from the group consisting of a signal processing method and a multivariate analysis.
- Such mathematical processing is well known to those skilled in the art, and can be easily performed in consideration of the description in this specification.
- the present invention provides a method for correlating an exogenous factor with a response of a cell to the exogenous factor.
- the external factors to be correlated in the present invention may be any. That Such exogenous factors are preferably those that are directly or indirectly applicable to cells.
- the method of exposing a foreign factor is well known in the art, and varies depending on the type of the foreign factor. For a substance, exposure is achieved by dissolving the substance in a solvent and dropping the solution into a medium containing cells.
- the generation of the profile can be performed as described above.
- the correlation between the external factor and the profile in the correlation method of the present invention can be performed by providing various methods. For simplicity, it is possible to pattern the profile when a certain extrinsic factor is dropped, and if the difference from the profile is small, it can be estimated that the exogenous factor has been dropped.
- the cells are monitored while immobilized on a solid support (eg, array, plate, microtiter plate, etc.).
- a solid support eg, array, plate, microtiter plate, etc.
- Such a fixing method can be performed based on a method known in the art or a method described herein.
- the correlation method of the present invention may include a step of using at least two extrinsic factors to obtain a profile for each extrinsic factor. In one embodiment, at least three, or four, and more preferably, at least ten such exogenous factors may be used, but are not limited thereto.
- the correlation method of the present invention comprises a step of categorizing at least two profiles to classify an extrinsic factor corresponding to the profile.
- classification can be easily performed by those skilled in the art in view of the description in this specification. By such a classification, correlation and identification of unknown foreign factors can be achieved using the method of the present invention.
- a transcriptional control sequence may include a promoter, enhancer, silencer, or other gene. It may be, but is not limited to, flanking sequences of structural genes in the nome structure and genomic sequences other than exons. Promoters are preferred. This is because the transcription state can be measured directly.
- transcription control sequences may be constitutive promoters, specific promoters, inducible promoters, and the like.
- the promoter may be any type, and rather, it is characterized by not choosing a type.
- the profile can be analyzed from the viewpoint of “process”, so that the state of the cell can be determined using an arbitrary promoter or a set thereof. . Such a determination is impossible with the conventional technology, and the present invention is highly useful in the sense that the present invention has achieved what was not possible with the conventional technology.
- At least two biological agents to be monitored are used. Observing at least two biological factors usually allows identification of more than 80% (preferably more than 90%, and in some cases nearly 100%) of the cell state. More preferably, the biological agent to be monitored comprises at least three biological agents. Observing at least three biological factors, usually identifying more than 90% (preferably more than 95%, and in some cases nearly 100%) of the biological agents This is because it becomes possible. In a most preferred embodiment, the biological agent being monitored comprises at least eight transcription control sequences. Observing at least eight biological factors usually makes it possible to identify all cellular states. Thus, despite the choice of any biological factor, the status of almost all cells can be determined by selecting only a small number as described above and monitoring it. This is much more simple, accurate, and accurate than the conventional judgment method, which observes each time point and statistically processes it as a heterogeneous population. Will provide a good judgment.
- the biological agent to be monitored comprises at least three biological agents. Observing at least three biological factors, usually identifying more than 90% (preferably more than 9
- the monitoring method of the present invention preferably further comprises a step of arbitrarily selecting at least one biological factor from a group of biological factors before monitoring.
- biological factors that do not show specificity in point-by-point studies can be used.
- such a profile can be presented in real time.
- the real-time may have a certain time lag as long as it can be displayed substantially simultaneously.
- the allowed time lag depends on the required real-time concurrency, but can be, for example, up to 10 seconds, and more preferably up to 1 second.
- the real-time property may be, for example, up to 1 second or up to 0.1 second.
- a profile corresponding to the data may be presented with a time lag based on the stored data.
- the phase of the profile is used as information of the profile used for correlation with an external factor.
- the phase is represented by two types of signal intensity, plus and minus, in a certain cycle, and cells can be identified or exogenous factors can be identified by using such a simplified method. The precision of the method is demonstrated.
- the cells are advantageously cultured on an array.
- an array By culturing on an array, many cells can be observed at once.
- salts may be used.
- monitoring the state of the cells over time comprises obtaining image data from the array.
- the step of correlating the extrinsic factor with the profile comprises identifying a difference in the phase of the profile.
- the phase is a simple parameter, and its information processing is simple. It is possible to sufficiently identify cells only by the simple information processing.
- the extrinsic factors to be identified in the method of the present invention include: temperature change, humidity change, electromagnetic wave, potential difference, visible light, infrared light, ultraviolet light, X-ray, chemical substance, pressure, gravity change, gas partial pressure. And osmotic pressure, but are not limited thereto. Such factors could not be sufficiently identified by conventional methods, but by using the process-oriented cell determination method of the present invention, it is possible to sufficiently investigate the effects of the factors on cells. It is now possible.
- the exogenous factor to be identified in the method of the invention is a chemical, such as a biomolecule, a chemical compound or a medium.
- biomolecules include, but are not limited to, for example, nucleic acids, proteins, lipids, sugars, proteolipids, lipoproteins, glycoproteins, and proteodaricans. Such biomolecules are known to have an effect on living organisms, or even if they are unknown, they are sufficiently likely to be important, so they are considered to be important targets for investigation.
- hormones are used as biomolecules to be investigated.
- the present invention provides a method for estimating an unidentified exogenous factor given to a cell from the profile of the cell.
- the method of the present invention comprises: a) exposing the cell to a plurality of known exogenous factors; b) an organism present in the cell. Monitoring the transcriptional status associated with at least one biological factor selected from a group of biological factors over time to obtain a profile of the cell for each of the known exogenous factors; c) Correlating each of the exogenous factors with each of the profiles; d) exposing the cells to an unidentified exogenous factor; e) estimating a state of the cells that are related to the selected biological factor over time.
- exposing the exogenous agent can be performed as described herein above and as exemplified in the Examples.
- Generating a profile can also be performed as described herein above and illustrated in the examples.
- Correlation can also be performed as described herein above and as exemplified in the examples.
- the same monitoring is performed for the unidentified foreign factor, and they are compared to determine whether the unidentified foreign factor is known.
- the profiles are exactly the same, it is naturally possible to judge that they are the same, but if they are substantially the same, it is also judged as a known extrinsic factor It is possible to do.
- Such a determination depends on the quantity and quality of the information on the known extrinsic factor. Such determination is easy for those skilled in the art, and can be determined in consideration of various factors.
- the present invention provides a method for estimating an unidentified exogenous factor given to a cell from the profile of the cell.
- a method for estimating an unidentified exogenous factor given to a cell from the profile of the cell comprises the steps of: a) for at least one promoter selected from the group of promoters present in the cell, a known exogenous factor, Providing data on the correlation with the profile; b) exposing the cells to an unidentified foreign agent; C) monitoring the status of the cells associated with the selected biological agent over time. Obtaining a profile of the cell; d) determining a profile corresponding to the profile obtained in the step (c) from the profiles provided in the step (a); and e. ) Determining that the unidentified extrinsic factor is the known exogenous factor corresponding to the determined profile;
- the exogenous gene exposure, profile generation, correlation, and the like can use the techniques described above in the present specification and exemplified in the examples.
- the present invention provides a system for presenting a state of a cell.
- Such a system comprises: a) means for monitoring the transcriptional status associated with at least one biological factor selected from the group of biological factors derived from the cell over time to obtain a profile of the cell; And b) provide means for presenting the above profile.
- An example of the system configuration is shown in Figure 32.
- FIG. 17 shows a configuration example of 500 of a computer that executes the cell state presentation method of the present invention.
- An example of the system configuration is shown in Figure 32.
- the computer 500 includes an input unit 501, a CPU 502, an output unit 503, a memory 504, and a bus 505.
- the input unit 501, the CPU 502, the output unit 503, and the memory 504 are interconnected by a bus 505.
- the input section 501 and the output section 503 are connected to an input / output device 506. The outline of the process of presenting the cell state executed by the computer 500 will be described.
- a program for executing the cell state presentation method (hereinafter referred to as a cell state presentation program) is stored in the memory 502, for example.
- the display programs can be recorded independently or together on any type of recording medium such as a floppy disk, M0, CD-ROM, CD-R, or DVD-ROM. Alternatively, it may be stored in the application server.
- the cell state presentation program recorded on such a recording medium is loaded into the memory 504 of the computer 500 via the input / output device 506 (for example, a disk drive, a network (for example, the Internet)).
- the CPU 502 executes the cell state presentation program
- the computer 500 functions as a device that executes the processing of the cell state presentation method of the present invention.
- the input unit 501 Through the input unit 501, information on cells and the like is input.
- the measured profile data is also input.
- Information about known information may also be entered as needed.
- the CPU 502 generates display data from profile data and cell information based on the information input at the input unit 501, and stores the display data in the memory 504. Thereafter, CPU 502 may store such information in memory 504. After that, the output unit 503 outputs the state of the cell selected by the CPU 502 as display data. In another aspect, the output data can be output from the input / output device 506.
- the present invention provides a system for determining a state of a cell. Such a system comprises: a) means for monitoring a transcriptional state associated with at least one biological factor selected from the group of biological factors derived from the cell over time to obtain a profile of the cell; and b) means for determining the state of the cell from the profile of the transcription state. An example of the system configuration is shown in FIG.
- FIG. 17 shows a configuration example of a computer 500 that executes the cell state determination method of the present invention.
- the computer 500 includes an input unit 501, a CPU 502, an output unit 503, a memory 504, and a path 505.
- the input unit 501, the CPU 502, the output unit 503, and the memory 504 are interconnected by a bus 505.
- the input unit 501 and the output unit 503 are connected to an input / output device 506. The outline of the cell state determination process executed by the computer 500 will be described.
- a program for executing the cell state determination method (hereinafter, referred to as a cell state determination program) is stored in the memory 502, for example.
- the cell state determination programs can be recorded on any type of recording medium such as a floppy disk, M0, CD-ROM, CD-R, DVD-ROM, independently or together. Alternatively, it may be stored on the application server.
- the cell state determination program recorded on such a recording medium is loaded into the memory 504 of the computer 500 via the input / output device 506 (for example, a disk drive, a network (for example, the Internet)).
- the CPU 502 executes the cell state determination program
- the computer 500 functions as a device that executes the processing of the cell state determination method of the present invention.
- the input unit 501 Through the input unit 501, information on cells and the like is input.
- the measured profile data is also input.
- Information about known information may also be entered as needed.
- the CPU 502 determines the state of the cell from the profile data and the cell information based on the information input at the input unit 501, generates the result as the determination result data, and stores the determination result in the memory 504. Store the data. Thereafter, CPU 502 may store such information in memory 504. After that, the output unit 503 outputs the state of the cell selected by the CPU 502 as determination result data.
- the output data can be output from the input / output device 506.
- the present invention provides a system for correlating an exogenous factor with a response of a cell to the exogenous factor.
- the system comprises: a) means for exposing the cell to an exogenous factor; b) monitoring over time the transcriptional state associated with at least one promoter selected from the group of promoters present in the cell. Means for obtaining a profile; and c) means for correlating the extrinsic factor with the profile.
- a system can also be realized by using a computer similarly to the above-described system. An example of the system configuration is shown in Figure 32.
- the present invention provides a system for estimating an unidentified exogenous factor given to a cell from a profile of the cell.
- a system for estimating an unidentified exogenous factor given to a cell from a profile of the cell.
- Such a system comprises: a) means for exposing the cell to a plurality of known exogenous factors; b) relating to at least one biological factor selected from the group of biological factors present in the cell. Means for monitoring the state of the cells over time to obtain a profile of the cell for each of the known exogenous factors; c) means for correlating each of the known exogenous factors with each of the profiles; d E) means for exposing the cell to an unidentified exogenous factor; e) means for monitoring the transcriptional state associated with the selected promoter over time to obtain a profile of the cell for the unidentified exogenous factor.
- F) means for determining a profile corresponding to the profile obtained by means (e) from the profiles obtained by means (b) above; and g) the unidentified foreign factor comprises: Comprising means for determining the upper Symbol a known external factor corresponding to the serial determined in section (f) profile.
- the present invention provides a system for estimating an unidentified exogenous factor given to a cell from a profile of the cell.
- a system includes: a) at least one selected from the group of biological factors present in the cells; With respect to the promoter, means for providing data on the correlation between a known exogenous factor and the profile of the cell corresponding to the known exogenous factor; Means for monitoring the state of the cell relating to the selected biological factor over time to obtain a profile of the cell; d) from among the profiles provided in the means (a), Means for determining a profile corresponding to the profile obtained in the means (c); and e) Means for determining that the unidentified foreign factor is the known foreign factor corresponding to the determined profile.
- Such a system can also be realized using a computer as in the above-described system. An example of the system configuration is shown in Figure 32.
- each component may be implemented using the same detailed or preferred embodiment as the present invention is provided as a method. Selection of such a preferred embodiment is easy for a person skilled in the art, and a person skilled in the art can easily make a preferred embodiment of such a system in consideration of the description in the present specification.
- An example of the system configuration is shown in Figure 32.
- the present invention provides a computer-readable recording medium recording a program for causing a computer to execute a process of presenting a state of a cell.
- the recording medium includes at least: a) a transcription state associated with at least one biological factor selected from a group of biological factors derived from the cells, which is monitored over time, B) a procedure for obtaining the profile; and b) a procedure for presenting the profile, and a program for executing the procedure is recorded.
- the present invention provides a computer-readable recording medium recording a program for causing a computer to execute a process of determining a state of a cell.
- Such recording media include at least a) Monitoring the transcriptional status associated with at least one biological factor selected from the group of biological factors over time to obtain a profile of the cell; and b) determining the status of the cell from the profile of the transcriptional status.
- the present invention provides a computer-readable recording medium recording a program for causing a computer to execute a process for correlating an exogenous factor with a response of a cell to the exogenous factor.
- the recording medium includes at least a) a procedure of exposing the cells to an exogenous factor; b) a method of monitoring a transcriptional state associated with at least one promoter selected from a group of promoters present in the cells over time, A program for performing a procedure for obtaining a cell profile; and c) a procedure for correlating the extrinsic factor with the profile is recorded.
- the present invention provides a computer-readable recording medium recording a program for causing a computer to execute a process for estimating an unidentified external factor given to a cell from a profile of the cell. I do.
- the recording medium includes at least a) a procedure for exposing the cells to a plurality of known exogenous factors; b) a transcriptional state associated with at least one promoter selected from a group of promoters present in the cells over time.
- step (b) Monitoring to obtain a profile of the cell for each of the known exogenous factors; c) correlating each of the known extrinsic factors with each of the profiles; d) exogenous factor whose cell has not been identified E) monitoring the transcriptional status associated with the selected promoter over time to obtain a profile of the cell for an unidentified exogenous factor; f) obtaining the profile from step (b) above. Determining a profile corresponding to the profile obtained in step (e) from the obtained profiles; and g) the unidentified foreign factor Performing the step of determining that the known external factor corresponds to the profile determined in step (f). The program to make it work is recorded.
- the present invention provides a computer-readable recording medium recording a program for causing a computer to execute a process for estimating an unidentified external factor given to a cell from a profile of the cell. I do.
- this recording medium at least a) a correlation between a known exogenous factor and a file of the cell corresponding to the known exogenous factor with respect to at least one promoter selected from a group of promoters present in the cell.
- step (b) exposing the cells to an unidentified exogenous factor; c) monitoring the transcriptional status associated with the selected promoter over time to obtain a profile of the cells; d) a step of determining a profile corresponding to the profile obtained in step (c) from the profiles provided in step (a) above; and e ) the unidentified extrinsic factor is Determining a known external factor corresponding to the determined profile. Beam is recorded.
- each component may be implemented using the same detailed or preferred embodiments as the present invention is provided as a method.
- the selection of such a preferred embodiment is easy for those skilled in the art, and the skilled person can easily perform such a preferred embodiment of the recording medium in consideration of the description in the present specification. .
- the present invention provides a program for causing a computer to execute a process of presenting a state of a cell.
- the program includes at least a) a profile of the cells related to at least one biological factor selected from the group of biological factors derived from the cells over time, and a profile of the cells. And b) perform the steps of presenting the above profile.
- the present invention provides a computer, comprising: Provide a program to execute
- the program comprises at least a) monitoring the transcriptional status associated with at least one biological factor selected from the group of biological factors derived from the cell over time to profile the cell; A) obtaining an isle; and b) determining a state of the cell from the profile of the transcription state.
- the present invention provides a program for causing a computer to execute a process for correlating an exogenous factor and a response of a cell to the exogenous factor.
- the program comprises: 'at least a) a step of exposing the cells to an exogenous factor; b) monitoring over time the transcriptional state associated with at least one promoter selected from the group of promoters present in the cells, Performing a step of obtaining a profile of cells; and c) a step of correlating the external factor with the profile.
- Techniques for executing such a procedure are well known in the relevant field, and those skilled in the art can create an appropriate program according to the purpose.
- the present invention provides a program for causing a computer to execute a process for estimating an unidentified foreign factor given to a cell from a profile of the cell.
- the program includes at least a) exposing the cells to a plurality of known exogenous factors; b) a condition of the cells associated with at least one biological factor selected from a group of biological factors present in the cells.
- the present invention provides a program for causing a computer to execute a process for estimating an unidentified external factor given to a cell from a profile of the cell.
- the program includes at least a) a known extrinsic factor and a profile of the cell corresponding to the known exogenous factor for at least one biological factor selected from a group of biological factors present in the cell.
- each configuration requirement can be implemented by applying the same detailed or preferred embodiment as that in which the present invention is provided as a method.
- the selection of such a preferred embodiment is easy for those skilled in the art, and the skilled person can easily perform such a preferred embodiment of the program in consideration of the description in the present specification. .
- the description format of such a program is well known to those skilled in the art, and for example, C + language can be applied.
- the present invention provides a method and system for diagnosing a subject.
- This diagnostic method comprises the steps of: a) monitoring the state of a cell associated with at least one biological factor selected from a group of biological factors derived from the cell of the subject over time to obtain a profile of the cell; B) determining the state of the cell from the profile of the state; and c) detecting the test from the state of the cell. Determining the condition, disorder or disease of the body.
- the system of the present invention includes: a) a cell associated with at least one biological factor selected from the group of biological factors derived from the cells of the subject.
- the present invention can be applied to tailor-made diagnosis and treatment, such as various cell states, survival, differentiation, drug resistance, selection of an appropriate anticancer agent, and selection of an appropriate transplant cell.
- the diagnostic method of the present invention is provided as a therapeutic or prophylactic method including a step of subjecting a subject to a treatment or prophylaxis selected in accordance with a diagnostic result.
- the diagnostic system of the present invention is provided as a treatment or prevention system comprising means for providing a selected treatment or prevention in response to a diagnosis. An example of the system configuration is shown in Figure 32.
- FIG. 17 shows a configuration example of 500 of a computer that executes the diagnosis method of the present invention.
- An example of the system configuration is shown in Figure 32.
- the computer 504 includes an input unit 501, a CPU 502, an output unit 503, a memory 504, and a path 505.
- the input section 501, the CPU 502, the output section 503, and the memory 504 are interconnected by a path 505.
- the input section 501 and the output section 503 are connected to an input / output device 506.
- a program for executing the correlation method and / or the selection of the treatment or the prevention (hereinafter referred to as a correlation program and a selection program, respectively) is stored in the memory 502, for example.
- the correlating program and the selecting program can be used independently or together with a floppy disk, It can be recorded on any type of recording medium such as MO, CD-ROM, CD-R, DVD-ROM. Alternatively, it may be stored on the application server.
- the correlation program and / or the selection program recorded on such a recording medium are loaded into the memory 504 of the computer 500 via the input / output device 506 (for example, a disk drive, a network (for example, the Internet)).
- the CPU 502 executes the correlation program and the Z or selection program
- the computer 500 functions as a device that executes the processing of the correlation method and / or the selection method of the present invention.
- the result of profile analysis for example, phase
- information on the state of cells are input.
- additional information such as status, disability or disease, treatment and / or prevention information that may be correlated with the profile may also be entered.
- the CPU 502 correlates information on the profile with information on the state of the cell or the state of the subject, information on the disorder or disease, and, if necessary, a preventive or therapeutic method based on the information input at the input unit 501. Then, the correlation data is stored in the memory 504. Thereafter, the CPU 502 may store the information in the memory 504. Thereafter, the output unit 503 outputs, as diagnostic information, information on the state of the cell selected by the CPU 502, information on the state of the subject, information on a disorder or disease, and a prevention or treatment method as necessary. The output data can be output from the input / output device 506.
- the present invention provides a method for generating profile data on cell information.
- the method comprises the steps of: a) fixing and arranging the cell on a support; and b) monitoring the biological factor or the aggregate thereof on or in the cell over time to determine the profile of the cell. Generating data.
- a) fixing and arranging the cell on a support a support
- b) monitoring the biological factor or the aggregate thereof on or in the cell over time to determine the profile of the cell. Generating data.
- One of the important features of the present invention in this aspect is that For example, cells can be fixed to substantially the same location on a support so that the same information can be obtained (for example, over time). This has made it possible to monitor biological factors of cells and their aggregates over time. The ability to monitor over time has made it possible to obtain cell profiles and to construct digital cells.
- the present invention may use a fixing agent such as a salt in the support.
- Cells can be immobilized on a support by a combination of a salt, a complex of a positively charged substance and a negatively charged substance, and cells.
- a salt can be used, for example, calcium chloride, sodium hydrogen phosphate, sodium hydrogen carbonate, sodium pyruvate, HEPES, calcium chloride, sodium chloride, potassium chloride, Magnesium sulfide, iron nitrate, amino acids and vitamins may be used, but are not limited thereto.
- a combination of a positively charged substance and a negatively charged substance includes, for example, a negatively selected substance selected from the group consisting of DNA, RNA, PNA, polypeptide, chemical compound, and a complex thereof.
- a complex of a charged substance and a positively charged substance selected from the group consisting of a cationic polymer, a cationic lipid, a cationic polyamino acid and a complex thereof can be used, but is not limited thereto.
- the biological agent of interest can be a nucleic acid molecule or a molecule derived from the nucleic acid molecule. Nucleic acid molecules are often responsible for genetic information, and cell information can be obtained for such transmission information.
- the present invention provides a method comprising: a) fixing and arranging a cell on a support; and b) monitoring a biological factor or an aggregate thereof on or in the cell over time. Generating data of the cell profile. Such data is data obtained by methods that did not exist before, and is new in itself. Therefore, the present invention provides a recording medium including such data.
- the present invention relates to a method for generating profile data relating to information on cells (preferably, a plurality of cells) in the same environment. The method comprises the steps of: a) arranging a plurality of cells on a support capable of maintaining the same environment; and b) transferring a biological agent or an aggregate thereof on or in the cells over time. Monitoring to generate profile data of the cell.
- the present invention can use an immobilizing agent such as a salt in the support.
- Cells can be immobilized on a support by a combination of a salt, a complex of a positively charged substance and a negatively charged substance, and a cell.
- any such salts can be used, for example, calcium chloride, sodium hydrogen phosphate, sodium bicarbonate, sodium pyruvate, HEPES, calcium chloride, sodium chloride, potassium chloride, magnesium sulfide , Iron nitrate, amino acids and vitamins and the like may be utilized, but are not limited thereto.
- the combination of such a positively charged substance and a negatively charged substance is, for example, selected from the group consisting of DNA, RNA, PNA, polypeptide, chemical compound, and a complex thereof.
- the biological agent of interest can be a nucleic acid molecule or a molecule derived from the nucleic acid molecule. Nucleic acid molecules often control genetic information, and cell information can be obtained from such genetic information.
- the target cell is preferably provided with an actin agonist.
- Actin acting substances act on intracellular actin, transforming the internal skeleton of cells, and introducing foreign factors from outside. This has the effect that it becomes easier. The presence of such an actin acting substance makes it possible to examine the effects of a target foreign factor in cells.
- the biological factor of interest in the present invention comprises at least one factor selected from the group consisting of a nucleic acid, a protein, a sugar chain, a lipid, a small molecule, and a combination thereof. .
- the cells of interest are preferably cultured without stimulation for some period of time before monitoring. This is to synchronize the target cells.
- a period required for synchronization for example, it may be advantageous to culture for at least 1 day, more preferably for at least 2 days, still more preferably for at least 3 days, and more preferably for at least 5 days.
- the longer the culture the greater the need to maintain culture conditions.
- the synchronization is preferably carried out in the same medium supplied to each cell, it is preferable that the medium during the culture is always the same force, or at least similarly changed. Therefore, preferably, a means for convection of the culture medium may be provided and used for this purpose.
- the biological agent provided to the cell in the present invention may include a nucleic acid molecule encoding a gene.
- the nucleic acid molecule encoding the gene is preferably transfused into the cell.
- a biological factor can be provided together with a transfusion reagent (gene transfer reagent).
- the nucleic acid molecule encoding the gene can be provided to the cell along with the gene transfer reagent and the actin agonist.
- the cell is preferably provided with a complex of a salt, a positively charged substance and a negatively charged substance (here, a nucleic acid molecule and a gene transfer reagent).
- Such a standardized form may be in a form readable by a normal computer (eg, one equipped with a commonly available OS such as Windows, Mac, UNIX, LINUX, etc.).
- the data generated in the present invention may include generated cell profile data, information on experimental conditions used for generation, information on cells, information on environment, and the like.
- the profile targeted by the present invention is labeled with a gene expression profile, an apoptosis signal profile, a stress signal profile, a molecule (preferably, a fluorescent, phosphorescent, radioactive substance or a combination thereof). ) Localization profile, cell morphology changes, Mouth motor profiles, specific drug (eg, antibiotics, ligands, toxins, nutrients, vitamins, hormones, cytokins, etc.) dependent promoter profiles, molecular interaction profiles, and the like.
- the subject of the present invention includes a profile of a specific drug-dependent promoter
- the present invention may preferably further include a step of administering the specific drug.
- the present invention may further comprise the step of providing a foreign stimulus to the cell.
- a foreign stimulus may or may not be a biological factor.
- the extrinsic factor can be any factor, including, but not limited to, a substance or other element (eg, energy such as ionizing radiation, radiation, light, sound waves, etc.).
- the exogenous factor used in the present invention may include RNAi. Since RNAi can be used to examine the suppression of virtually any gene, it is also possible to prepare RNANAi for the existing gene and examine its effect. RNAi can be prepared by methods well known in the art.
- the foreign factor used in the present invention may include a chemical substance that is not present in a living body.
- a chemical substance that does not exist in a living body to cells as described above, various information can be collected.
- once collected data can be reused, even if chemical substances that do not exist in living organisms are hardly available, they can be obtained if data can be obtained in the present invention. Research can be continued without regard to gender.
- an exogenous factor to which the present invention can be directed may include a ligand for a receptor of a cell.
- a ligand for a receptor of a cell By analyzing ligands, it is possible to investigate various signaling pathways. Therefore, in such a case, the profile obtained by the present invention includes a profile of the receptor ligand interaction.
- the profile obtained by the present invention is cell morphology, wherein the method of the present invention comprises the group consisting of gene overexpression, underexpression or knockdown, addition of exogenous factors and environmental changes.
- the method may further include the step of providing the cell with a stimulus that can be selected.
- the profile obtained according to the invention comprises a profile of the interactions between molecules present in the cell.
- the profile of such an interaction between molecules include, for example, an interaction between molecules appearing in a signal transduction pathway, an interaction between a receptor and a ligand, an interaction between a transcription factor and a transcription factor sequence, and the like.
- Profiles include, but are not limited to.
- the profile obtained by the present invention comprises a profile of an interaction between molecules present in the cell, wherein the present invention is selected from the group consisting of a two-hybrid method, FRET and BRET.
- the method further includes a step of performing observation using a technique.
- the two-hybrid method detects intermolecular interactions in cells. The details are described in Protein-Protein Interactions, A MOLECULAR CLONING MANUAL, Edited by Erica Golemis, Cold Spring Habor Laboratory Press, Cold Spring Harbor, New York (this document also describes FRET).
- FRET is a technology that detects the resonance energy transfer between molecules and within a molecule as a change in the fluorescence wavelength.
- the cells of interest are preferably arranged in an array on a support.
- the plurality of cells of interest in the present invention each have a maximum of 10 cm, more preferably a maximum of lcm, more preferably a maximum of lmm, most preferably a maximum of 0.1 interval.
- the profile obtained by the present invention may be obtained in real time, but may not be obtained. It may be advantageous to get in real time. In situations where synchronization is important, such real-timeness is important. Or, if the purpose is to store, real-time property is not always necessary.
- the present invention further comprises the step of immobilizing the cells on a solid support.
- the fixation to the solid support may be possible with salts, complexes, actin-substances and the like.
- the data generated by the present invention includes information about the profile.
- the data generated by the present invention may further include information on conditions in the monitor, information on the state of cells, information on exogenous factors, information on the environment, and the like.
- the biological agent monitored in the present invention comprises at least two biological agents, more preferably comprises at least three biological agents, even more preferably at least Contains and obtains eight biological factors.
- the present invention may further comprise the step of optionally selecting such a biological agent.
- the cells targeted by the present invention can be selected from the group consisting of stem cells and somatic cells.
- the support used in the present invention is preferably a solid support. This is because it is easy to fix.
- a solid support any substance known in the art can be used as a material.
- the support may take the form of a base.
- the biological agent is a nucleic acid
- the cell is advantageously transfected with the nucleic acid.
- transfecting with a nucleic acid in this way, it becomes possible to collect a profile of the effect of the nucleic acid on a cell in real time or in a defined storable format. This was not possible with the prior art.
- the transfer may be performed on a solid phase or in a liquid phase. More preferably, the transphosphate is advantageously performed on a solid phase. This is because data collection and standardization are easier.
- the processing of the profile can be processed by a process selected from the group consisting of phase comparison, difference calculation with a control file, signal processing, and multivariate analysis. Data so processed is also within the scope of the present invention.
- the present invention provides a method for presenting profile data relating to information on cells (preferably a plurality of cells) in the same environment.
- the method comprises the steps of: a) placing a plurality of cells on a support capable of maintaining the same environment; b) monitoring a biological factor or an aggregate thereof on or in the cells over time. Generating data of the profile of the cell by; and c) presenting the data.
- the support capable of maintaining a plurality of cells in the same environment can be implemented as described in detail in the present specification.
- the step of generating data can also be performed as described elsewhere herein.
- the step of presenting the data can also be performed as detailed elsewhere herein.
- Examples of such a presentation method include, but are not limited to, a method using various sensory means such as visual, auditory, olfactory, tactile, and taste.
- a visual presentation means is used.
- a display of a computer or the like can be exemplified.
- the presentation can be performed in real time.
- the stored data may be recalled and presented later. If the presentation is to be made in real time, the data signal can be sent directly to, for example, a display.
- the present invention provides a method for determining the state of cells in the same environment.
- the method comprises the steps of: a) arranging a plurality of cells on a support capable of maintaining the same environment; b) aging a biological agent or an aggregate thereof on or in the cells over time. Generating the profile data of the cell by monitoring the cell; and c) determining the state of the cell from the data.
- a support capable of maintaining a plurality of cells in the same environment can be carried out as described in detail in this specification.
- the step of generating data may also be performed as described in detail elsewhere herein.
- the determination can be made by comparing the generated data with information on the cell, or by comparing the data with standard data. In this case, statistical processing may be performed.
- the present invention further comprises a step of pre-correlating the cell file obtained by the present invention with the state of the cell.
- the target cell in the present invention has an already-existing state.
- the biological agent may be of a heterogeneous category (eg, proteins and nucleic acids, etc.) or may be of the same category.
- the present invention further comprises the step of optionally selecting a biological agent.
- a biological agent Regardless of the biological factor chosen, the state of the cell can be somewhat distinguished and, in some cases, identifiable, so the present invention is imagined from the prior art. It can be said that the effect is effective.
- the data is generated in real time. This is because, by generating data in real time, an unknown substance or a cell whose state is unknown can be determined in real time.
- the state of the target cell includes, but is not limited to, a differentiated state, an undifferentiated state, a cell response to an exogenous factor, a cell cycle, and a cultured state. .
- the cells targeted in the present invention may be stem cells or somatic cells.
- the somatic cell may be any cell. Those skilled in the art can appropriately select cells depending on the purpose of using the cells.
- the solid support used in the determination method of the present invention includes a substrate.
- the present invention makes it possible to use the substrate as a part of a computer system and automatically make a determination.
- An example of the system configuration is shown in Figure 32.
- the method according to claim 52 wherein the biological factor is a nucleic acid molecule, and the cell is transfected with the nucleic acid molecule.
- the transfection technology no matter what Good, but preferably, it is advantageous to use a gene transfer reagent. More preferably, the transfection is carried out on a solid support using a salt, an actin acting substance or the like. The transfection may be performed on a solid phase or in a liquid phase, but advantageously it may be advantageously performed on a solid phase.
- the target biological factor may have the ability to bind to another biological factor.
- the network mechanism in cells can be elucidated.
- the determination step may include performing a mathematical process selected from the group consisting of comparing a phase of a profile, collecting a difference from a control profile, a signal processing method, and a multivariate analysis.
- a mathematical process selected from the group consisting of comparing a phase of a profile, collecting a difference from a control profile, a signal processing method, and a multivariate analysis.
- the present invention provides a method for correlating an exogenous factor with a response of a cell to the exogenous factor.
- the method comprises the steps of: a) exposing the cells to exogenous factors on a support capable of maintaining a plurality of cells in the same environment; b) biological factors on or in the cells. Or monitoring the aggregate over time to generate profile data for the cell; and c) correlating the exogenous factor with the profile.
- the exposure to the exogenous factor is achieved by placing the cell and the exogenous factor in an environment in which the exogenous factor comes into contact with the cell.
- exposure can be achieved by adding the exogenous agent on the support.
- Methods for generating and correlating data are also well known in the art, and such methods of generating and correlating can be employed using conventional data processing or a combination thereof. It is preferable to perform statistical processing to generate statistically significant data and information.
- the cells are placed on a support. It may be fixed. By being fixed, data standardization becomes easier and data processing becomes much more efficient.
- the correlation method of the present invention may further include a step of using at least two of the extrinsic factors to obtain a profile for each extrinsic factor. Techniques for obtaining such a profile are fully described herein.
- the correlation may further comprise the step of categorizing at least two profiles to classify the extrinsic factor corresponding to the profile. By classifying, more standardized data processing becomes possible.
- the profiles obtained in the present invention are presented in real time, but need not be in real time especially for the purpose of storing data.
- the cells used in the present invention can be cultured on an array. Therefore, in such a case, the cells are preferably covered with the medium.
- the medium any medium that is usually used for cells can be used.
- monitoring the profile comprises obtaining image data from the array.
- the profile is visual information (for example, emission of fluorescence due to gene expression), it is possible to obtain the profile by obtaining image data.
- the step of correlating the external factor and the profile may include a step of identifying a difference in the phase of the profile.
- the determination of the phase change is a feature achieved by the present invention providing the profile for the first time over time and in the same environment.
- the external factors targeted by the present invention include temperature change, humidity change, electromagnetic wave, potential difference, and It can be selected from the group consisting of visual rays, infrared rays, ultraviolet rays, X-rays, chemicals, pressure, gravity changes, gas partial pressures and osmotic pressures.
- the chemical can be a biomolecule, a chemical compound or a medium.
- biomolecules include, but are not limited to, for example, nucleic acid molecules, proteins, lipids, sugars, proteolipids, lipoproteins, glycoproteins and proteodaricans. Biomolecules can also be, for example, hormones, cytokins, cell adhesion factors and extracellular matrices.
- the chemical may be an agonist or antagonist of the receptor.
- the present invention relates to a method for identifying an unidentified exogenous factor given to a cell from a profile of the cell.
- the method comprises the steps of: a) exposing a cell to a plurality of known exogenous factors on a support capable of maintaining the same environment; b) a biological factor or a collection thereof on or in the cell. Monitoring the body over time and obtaining a profile of the cell for each of the known exogenous factors to generate data of the cell profile; c) each of the known exogenous factors and each of the profiles; D) exposing the cell to an unidentified exogenous factor; e) monitoring a biological factor or an aggregate thereof on or in the cell exposed to the exogenous factor over time.
- the present invention relates to a method comprising: Methods are provided for identifying unidentified foreign factors.
- the method comprises the steps of: a) relating to a biological factor or a collection thereof on or in the cell, data on a correlation between a known foreign factor and a profile of the cell corresponding to the known foreign factor.
- exogenous factor exposure, data generation, correlation, unidentified exogenous factor exposure, etc. are described in detail elsewhere in the present specification, and those skilled in the art can refer to these descriptions to An appropriate form can be appropriately selected according to the purpose.
- the present invention provides a method for obtaining a profile relating to information on cells (preferably a plurality of cells) in the same environment.
- the method comprises the steps of: a) arranging a plurality of cells on a support capable of maintaining the same environment; and b) monitoring a biological factor or an aggregate thereof on or in the cells over time.
- exogenous factor exposure, data generation, correlation, exposure of unidentified exogenous factor, etc. are described in detail elsewhere in this specification, and those skilled in the art can refer to these descriptions.
- An appropriate form can be appropriately selected according to the purpose.
- the present invention relates to a recording medium storing data generated by the method for generating cell profile data according to the present invention.
- the storage format may be any, and the recording medium may be any medium.
- such recording media include CD_ROM, flexible disk, CD-R, CD-RW, MO, minidisc, DVD_ROM, DVD _ R, memory stick, hard disk, etc., but not limited to them.
- the present invention also relates to a transmission medium in which data generated by the method for generating cell profile data of the present invention is stored. Examples of the transmission medium include, but are not limited to, networks such as an intranet and the Internet.
- the recording medium or the transmission medium according to the present invention is related to at least one information selected from the group consisting of information on conditions in the monitor, information on the profile, information on the state of the cell, and information on the biological factor. It may further include data. Data relating to such information may be stored in a linked form. Preferably, these data are advantageously standardized. This is because standardization makes it possible to put them on a certain distribution channel.
- the links may be linked for each cell, or for each biological agent, or both.
- the present invention relates to data generated by the method for generating cell profile data of the present invention. Such data is completely new because it cannot be generated by conventional techniques.
- the present invention provides a system for generating open file data relating to information on a plurality of cells in the same environment.
- the system comprises: a) a support capable of maintaining a plurality of cells in the same environment; b) means for monitoring a biological factor or an aggregate thereof on or in the cells over time; Means for generating profile data of the cell from a signal obtained from the monitoring means.
- Supports that can be kept in the same environment can be implemented by those skilled in the art using the technology first provided by the present invention. Such techniques stem from the ability to immobilize cells and arrange cells without walls.
- a monitoring means for example, a microscope (for example, an optical microscope, a fluorescence microscope, Phase contrast microscope), electron microscope, scanner, naked eye, infrared camera, confocal / non-confocal microscope, CCD camera, etc., but are not limited thereto.
- a microscope for example, an optical microscope, a fluorescence microscope, Phase contrast microscope
- electron microscope scanner
- naked eye infrared camera
- confocal / non-confocal microscope CCD camera
- the system of the present invention need not initially contain cells, but preferably comprises a plurality of cells and is preferably fixed to a support. It is. In such a case, the fixation is preferably standardized. When fixed, the distance between cells may be, for example, 1 mm, but is not limited thereto.
- At least one substance selected from the group consisting of salt-actin acting substances is attached to the support. This is because the attachment of either one or preferably both of the salt-opactin acting substances enhances the effect of immobilization and introduction of Z or cytoplasmic substances.
- Monitoring means that can be used in the system of the present invention include: optical microscopes, fluorescence microscopes, phase microscopes, readers using laser light sources, surface plasmon resonance (SPR) imaging, electrical signals, chemical or biochemical markers. Examples include, but are not limited to, means using one or more species, synchrotron radiation, confocal microscope, non-confocal microscope, differential interference microscope, stereo microscope, video monitor and infrared camera.
- a scanner is used, for example a scanner that scans the substrate surface with a white light source or laser.
- Scanner is preferred because it has the advantages that fluorescence can efficiently transmit excitation energy and that microscopy technology can be easily applied. Furthermore, it has the advantage that measurement can be performed without causing significant damage to cells.
- An example of the system configuration is shown in Figure 32.
- the present invention provides a system for presenting a profile relating to information on cells (preferably a plurality of cells) in the same environment.
- the system comprises: a) a support capable of maintaining a plurality of cells in the same environment; b) a support on or in the cells; Means for monitoring a biological factor or an aggregate thereof in a cell over time; C) means for generating data of a profile of the cell from a signal obtained from the means for monitoring; and
- d) means for presenting the data means for presenting the data.
- the support, the monitoring means, and the data generating means can be implemented as described elsewhere in this specification.
- Means for presenting data can also utilize means well known in the art. Examples of such data presentation means include, but are not limited to, a computer display and a speaker. An example of the system configuration is shown in FIG.
- the presentation system of the present invention preferably further includes a plurality of cells, and the plurality of cells are preferably fixed to the support.
- the support has attached thereto at least one substance selected from the group consisting of salts and actin acting substances. The use of such a substance enhances fixation and / or enhances the introduction of foreign substances into cells.
- the monitoring means can be of any type, for example, optical microscope, fluorescence microscope, phase microscope, reader with laser light source, surface plasmon resonance (SPR) imaging, electrical signal, chemical or biochemical It may be a means using one or more of the target markers.
- the data presentation means may be of any type, and examples include a display and a speaker.
- the present invention provides a system for determining a state of a cell.
- the system comprises: a) a support capable of maintaining a plurality of cells in the same environment; b) means for monitoring a biological factor or an aggregate thereof on or in the cells over time; c) the monitor Means for generating data from a signal obtained from the means; and d) means for detecting the state of the cell from the data.
- the support, the monitor means, and the data generation means are described elsewhere in this specification. W
- Means for detecting the state of cells from data can also be prepared and used using techniques well known in the art. For example, extrapolation can be achieved by comparing the measured data with standard data on known cells, and a device storing a program for such extrapolation or a computer capable of executing it. It can be used as such an external means.
- An example of the system configuration is shown in Figure 32.
- the present invention provides a system for correlating an exogenous factor with a response of a cell to the exogenous factor.
- the system comprises: a) a support capable of maintaining a plurality of cells in the same environment; b) means for exposing an exogenous factor; c) a biological factor or a collection thereof on or in the cell over time. D) generating data of the cell profile from the signal from the monitoring means; and e) means for correlating the extrinsic factor with the profile.
- the support, the monitoring means, and the data generating means can be implemented as described elsewhere herein.
- Means for exposing the exogenous factor can also be appropriately designed and implemented by those skilled in the art depending on the nature of the exogenous factor.
- a recording medium storing a program for the correlation or a computer capable of executing the program can be used.
- the system of the invention comprises a plurality of cells. An example of the system configuration is shown in Figure 32.
- the present invention provides a system for identifying an unidentified foreign factor given to a cell from a profile of the cell.
- the system comprises: a) a support capable of maintaining a plurality of cells in the same environment; b) means for exposing a known exogenous factor; c) a biological factor or an aggregate thereof on or in the cell.
- Means for monitoring over time means for obtaining a profile of the cell for each of the exogenous factors and generating data of the profile of the cell; e) said known exogenous factor Means for correlating each of the profiles with each of the profiles; f) means for exposing the cells to an unidentified exogenous factor; g) a profile of the known exogenous factor obtained in the means (d); Means for comparing a profile of an exogenous factor with a profile of a known exogenous factor, and determining a profile corresponding to a profile of an unknown extrinsic factor from among the open files of known extrinsic factors. Means, the known extrinsic factor corresponding to the determined port file.
- the means for determining the corresponding profile can also be implemented by using a recording medium storing a program for executing such a determination process and a computer for executing the program.
- the system comprises a plurality of cells. An example of the system configuration is shown in Figure 32.
- the present invention provides a system for identifying an unidentified foreign factor given to a cell from a profile of the cell.
- the system comprises: a) data relating to a known foreign factor and a profile of the cell corresponding to the known foreign factor with respect to a biological factor or a collection thereof on or within the cell.
- Means for monitoring a biological factor or an aggregate thereof over time e) means for obtaining a profile of the cell from a signal obtained from the monitoring means; f) said profile stored in said recording medium (a) Means for determining a profile corresponding to a profile obtained for an unknown extrinsic factor, wherein the unidentified extrinsic factor is the known extrinsic factor corresponding to the determined profile.
- the means for determining the corresponding profile can also be implemented by using a recording medium storing a program for executing such a determination process and a computer for executing the program.
- the system comprises a plurality of cells.
- An example of the system configuration is shown in Figure 32.
- the present invention relates to a support capable of maintaining the same environment for a plurality of cells. Such a support was first provided by the present invention. The use of such a support has made it possible to analyze a plurality of cells under the same environment.
- the cells on the support are advantageously arranged in an array. This is because standardized analysis becomes possible.
- Actin activators are preferred, especially in internal analysis, because they increase the efficiency of introducing foreign factors into cells. Therefore, in a preferred embodiment of the present invention, it comprises a salt-opactin acting substance, and more preferably further comprises a complex of a positively charged substance and a negatively charged substance.
- the support of the present invention has a feature that cells can be arranged at intervals of 1 mm. Presenting a wallless environment at such intervals has not been possible in the past. Therefore, the present invention has surprising effects and practicality.
- the support of the present invention is provided further comprising fixed cells. In a more preferred embodiment, the support of the present invention is provided further comprising an immobilized biological agent.
- two or more of the biological factors are fixed.
- biological factors nucleic acid molecules, proteins, sugars, fats, metabolites, small molecules, their complexes, and factors that include physical and / or temporal elements? It may be a factor selected from the group consisting of:
- cells and biological factors are mixed and immobilized on the support of the present invention.
- biological factors and cells can be arranged to interact. Such interactions will vary with biological factors, but the nature of those skilled in the art will understand how they interact and how they are arranged to interact. .
- the support of the present invention comprises a complex of a positively charged substance and a negatively charged substance, and an actin acting substance, comprising a cell or a biological material. Fixed with factors.
- the support of the present invention comprises a salt and a complex of a positively charged substance and a negatively charged substance, and an actin acting substance in an array together with a cell and a biological factor. Fixed in the shape. By adopting such a configuration, a cell chip capable of generating cell profile data is provided.
- the support preferably has a configuration in which salts, gene transfer reagents, actin acting substances, nucleic acid molecules, and cells are immobilized in an array. It is also called "Young Array”.
- the salts used in the support of the present invention include calcium chloride, sodium hydrogen phosphate, sodium hydrogen carbonate, sodium pyruvate, HEPES, calcium chloride, sodium chloride, sodium chloride, magnesium chloride, and magnesium nitrate. Include, but are not limited to, iron, amino acids and vitamins.
- the salt preferably includes, but is not limited to, sodium salt and the like.
- Gene transfer reagents used in the support of the present invention include, but are not limited to, cationic polymers, cationic lipids, polyamine-based reagents, polyimine-based reagents, calcium phosphate, oligofectamine and oligofactors. Not done.
- ribofectamine, Oligofuectamines include, but are not limited to, oligofuectamines.
- Actin acting substances used in the support of the present invention include, but are not limited to, fipronectin, laminin, and vitronectin.
- the actin acting substance preferably includes, but is not limited to, fibronectin.
- the nucleic acid molecule used in the support of the present invention includes a transcription control sequence (eg, a promoter, an enhancer, etc.), a gene coding sequence, a genomic sequence containing an untranslated region, a nucleic acid sequence not encoded by the host genome. (Fluorescent protein gene, Escherichia coli / yeast self-replication origin, GAL4 domain, etc.), but are not limited thereto.
- the nucleic acid molecule preferably includes, but is not limited to, a transcriptional regulatory sequence (eg, promoter, enhancer, etc.), a gene code sequence, and a genomic sequence including an untranslated region.
- Cells used in the support of the present invention include, but are not limited to, stem cells, established cell lines, primary culture cells, insect cells, and bacterial cells.
- the cells preferably include, but are not limited to, stem cells, established cell lines, and primary cultured cells.
- the material of the support used in the support of the present invention includes, but is not limited to, glass, silica, and plastic. This material preferably includes, but is not limited to, the materials described above.
- the present invention provides a method for producing a support comprising a plurality of fixed cells and capable of maintaining the same cell environment.
- the method comprises the steps of: A) providing a support; and B) fixing cells on the support using a salt and a complex of a positively charged substance and a negatively charged substance. .
- Providing the support can be done by obtaining a commercial source or by molding the support material. Thus it can be achieved. When it is necessary to prepare a support material, it can be prepared by mixing raw materials for such a material.
- the fixing step can also be performed using a technique known in the art, and examples of such fixing techniques include, but are not limited to, an ink jet printing method, a pin array method, and a stamp method. Such techniques are well known and can be implemented by those skilled in the art as appropriate.
- the immobilization step in the present invention comprises the steps of: adding the salt; a gene transfer reagent as the positively charged substance; an actin acting substance; a nucleic acid molecule as the negatively charged substance; Fixing the mixture in an array. Such fixation can be achieved using printing techniques.
- the present invention provides an apparatus for producing a support comprising a plurality of fixed cells and capable of maintaining the same environment for the cells.
- the device comprises: A) means for providing a support; and B) means for immobilizing cells on the support using a complex of salt and a positively charged substance and a negatively charged substance.
- the realization of the provision of the support can be achieved using means capable of performing the method described above. Examples of such means include, but are not limited to, a means for molding a support and a means for preparing a material (for example, a mixing means).
- a molding means a technique well-known in the art can be used.
- the fixing means includes a printing means. As such means, a commercially available ink jet printer can be used.
- Digital cell refers to a set of at least one set of experimental data for the cell under study. These experimental data relate the experimental conditions and the experimental results of the experiments performed on real cells. Digital cells are constructed so that given experimental conditions, the experimental results associated with the experimental conditions can be reproduced. When digital cells are used, the results of experiments performed on real cells can be reproduced on a computer system. This will enable research institutions and individuals without experimental facilities to conduct cutting-edge research on cells. As a result, it will be possible to enter this field even from different industries that previously could not enter this field.
- FIG. 33A shows an example of a digital cell data structure.
- the digital cell is represented as a set of three experimental data A1, A2, and A3 for cell A. ,.
- Each of the experimental data A1, A2, and A3 includes a cell parameter, an environmental parameter, and a stimulus parameter as parameters indicating the experimental conditions, and a stimulus response result as an experimental result.
- the cell parameter specifies a cell to be tested.
- the environmental parameter specifies an environment for culturing the cell specified by the cell parameter.
- the stimulus parameter specifies the stimulus given to the cell specified by the cell parameter.
- the stimulus response result indicates the result of the cell specified by the cell parameter responding to the stimulus specified by the stimulus parameter under the environment specified by the environmental parameter.
- Experimental data A 1 is culturing "DMEM” pH “7” in a medium of a temperature “3 7" of the cell A in media conditions that the CO 2 concentration "5"% "T et _OFF C MV EGF It shows that the stimulation response was obtained by applying a stimulus consisting of a reporter called “CMV EGFP” and a chemical stimulus (drug) called “D oxycycene” to cell A. This stimulus response result is represented by “cell kinetic data 1 J” and “reporter measurement data 1”.
- Experimental data A2 is "DMEM” pH “7” in a medium of a temperature “3 7" degree, and culturing the cells A in the culture medium provided that C0 2 concentration "5"%, to as “c one fos” reporter And a stimulus consisting of a chemical stimulus (drug) called "PSC8333" By giving to A, it is shown that a stimulus response result was obtained. This stimulus response result is represented by “cell kinetic data 2” and “reporter measurement data 2”.
- Experimental data A 3 is, p H in a medium called “DMEM”, "5", the temperature “3 9" degree, and culturing the cells A in the culture medium provided that C_ ⁇ 2 concentration "4"%, a reporter called “CREB” It shows that the stimulation response results were obtained by applying a stimulus consisting of a chemical stimulus (drug) called “V indecine” to cell A. The result of this stimulus response is represented by “cell kinetic data 3” and “reporter measurement data 3”.
- the parameters indicating the experimental conditions are associated with the stimulus response results indicating the experimental results. What linked these is called experimental data.
- Digital cells are provided as a set of at least one set of experimental data for the cells under study.
- FIG. 33B shows another example of the data structure of the digital cell.
- the data structure shown in FIG. 33A is hierarchized. By hierarchizing the data structure of the digital cell in this way, it becomes possible to express the same content with a smaller amount of data as compared with the data structure shown in FIG. 33A.
- the parameters indicating the experimental conditions and the experimental results are related by a unidirectional link (arrow in the figure).
- the method of relating them is not limited to this. An arbitrary method can be adopted as a method of relating these.
- Fig. 34 shows an example of the procedure for processing to produce digital cells. This process is performed by any type of computer.
- Step S3401 Cell parameters for specifying cells to be tested are obtained. Acquisition of cell parameters is performed, for example, by a computer receiving cell parameters input by a user. Or an experimental device A computer may automatically acquire or analyze the data output from the computer to acquire the cell parameters.
- Step S3402 Environmental parameters for specifying the environment for culturing the cells specified by the cell parameters are obtained. Acquisition of environmental parameters is performed, for example, by a computer receiving environmental parameters input by a user. Alternatively, environmental parameters may be obtained by a computer automatically collecting or analyzing data output from an experimental apparatus (for example, a sensor for measuring an experimental environment).
- the environmental parameters include, for example, a parameter indicating a medium in which cells are cultured, and a parameter indicating conditions of the medium.
- the conditions of the culture medium e.g., medium p H, temperature, etc. C 0 2 concentration and the like.
- Step S3403 A stimulus parameter for specifying a stimulus to be applied to the cell specified by the cell parameter is obtained.
- the acquisition of the stimulus parameters is performed, for example, by the computer receiving the stimulus parameters input by the user.
- a stimulus parameter may be obtained by automatically collecting or analyzing data output from the experimental apparatus by a computer.
- the stimulus parameters include, for example, a parameter indicating a reporter and a parameter indicating a chemical stimulus.
- Step S3404 A stimulus response result indicating a result of the cell specified by the cell parameter responding to the stimulus specified by the stimulus parameter under the environment specified by the environmental parameter is obtained. Acquisition of the stimulus response result is performed, for example, by a computer automatically collecting or analyzing data output from an experimental device (for example, a monitor device for monitoring the progress of the experiment).
- an experimental device for example, a monitor device for monitoring the progress of the experiment.
- Step S3405 Cell parameters, environmental parameters, stimulus parameters, and stimulus response results are associated. This association allows the target cells to be One experiment data is generated. Such an association is performed using a unidirectional link, for example, as shown in FIG. 33A. However, the method of association does not matter.
- Step S3406 Step S3401 to step S3405 are repeated as necessary. This generates at least one set of experimental data for the cells under test. This at least one set of experimental data is provided as a digital moonpack.
- a computer that executes processing for producing digital cells functions as a device that produces digital cells.
- the digital cells produced are stored, for example, in a database accessible by the consumer.
- FIG. 35 shows an example of the configuration of a computer system 3501 that provides a service for reproducing experimental results on real cells using digital cells.
- the computer system 3501 includes a service requester 35010 for requesting a service desired by a user, and a service provider 3502 for providing a predetermined service in response to the request.
- the computer system 3501 may include a plurality of service requesters 3501.
- Service provider 352 0 stores at least one digital cell It is configured to be able to access database 352 2.
- the data structure of digital cells stored in the database 352 2 is as shown in, for example, FIGS. 33A and 33B.
- the database 352 may be provided inside the service provider 352 or may be provided outside the service provider 352.
- the service provider 352 may be configured to be able to access a plurality of databases each storing at least one digital cell.
- Each of the service requester 355 and the service provider 352 can be any type of computer.
- the service requester 3501 and the service provider 3502 are connected via a network 350.
- the network 354 can be any type of network, but is most preferably the Internet in view of ease of connection and cost.
- the service requester 351 may be a web browser operated by a user, and the service provider 352 may be a service requester via the Internet. It can be a web server connected to 10. With this configuration, users all over the world can easily access the service provider 352.
- FIG. 36 shows an example of a procedure of a process for providing a service for reproducing an experiment result on a real cell using a digital cell. This processing is executed by cooperation between the service requester 350 and the service provider 350.
- Step S3601 The service requester 351 receives the cell parameter, the environmental parameter, and the stimulus parameter, and generates a request including the cell parameter, the environmental parameter, and the stimulus parameter.
- the request is described in XML, for example.
- Step S3602 The service V quester 3501 supports the request. Provide to one screw provider 3 5 2 0.
- Step S3663 The service provider 352020 searches the database 3522 in response to the request, and stores the cell parameter and the environmental parameter included in the request in the database 3522. Determine if there is a stimulus response result associated with the stimulus parameter.
- Step S3664 If it is determined that there is a stimulus response result related to the cell parameter, the environmental parameter and the stimulus parameter included in the request in the database 3522, the service provider 352020 Provides the stimulus-response result to the service requester 3510.
- the stimulus response result is described in, for example, XML.
- Step S3655 The service requester 351 displays the stimulus response results provided by the service provider 352.
- the service provider 352 for example, The result of “not applicable” is provided to the service requester 3510.
- processing shown in FIG. 36 can be performed by a single computer.
- the processes of steps S3601 to S3655 shown in FIG. 36 should be realized by a single program executed by a single computer.
- the single computer functions as a device having both the function of the service requester 350 and the function of the service provider 352.
- FIG. 37 shows an example of an input screen for inputting a cell parameter, an environmental parameter, and a stimulus parameter to the service requester 3510.
- these parameters are entered by the user entering text in a predetermined area of the input screen.
- these parameters may be entered by the user selecting a menu (eg, a pull-down menu, a pop-up menu).
- a menu eg, a pull-down menu, a pop-up menu.
- An arbitrary mode can be adopted as a mode in which the service requester 3510 displays the stimulus response result.
- the service requester 3510 may display the stimulus response result on a display or may output the stimulus response result to a printer.
- the service requester 3510 may display the stimulus response result on a display using a still image or on a display using a moving image.
- the stimulus response result may include, for example, data on the profile of the cell obtained by monitoring a biological agent or a collection thereof on or within the cell over time.
- the data of the profile of the cell as shown in FIG. 19 is displayed by the service requester 3510.
- FIG. 380 shows an example of the configuration of a computer system 3801, which provides a service for reproducing experimental results on real cells using digital cells.
- the computer system 380 1 includes a service requester 380 requesting a service desired by a user, and a plurality of service providers 380 i to 382 providing a predetermined service in response to the request. 0 comprising N and, a service registry 3 8 4 0 a plurality of service provider 3 8 2 0 i ⁇ 3 8 2 0 N has registered at least one service that can be provided.
- N is any integer of 2 or more.
- the computer system 3801 may include a plurality of service requesters 380.
- Service provider 3 8 2 0 i the database 3 8 2 2 which stores at least one digital cell; is configured to allow access.
- the data structure of the digital cells stored in the database 3822i is, for example, as shown in FIGS. 33A and 33B.
- the database 382 2 may be provided inside the service provider 380i or may be provided outside the service provider 380i.
- i l, 2, ⁇ ⁇ ⁇ ⁇ N.
- the service provider 3820 may be configured to be able to access a plurality of databases each storing at least one digital cell.
- Service Registry 3 8 4 0 is configured service provider 3 8 2 0 1 to 3 8 2 0 N allow Akuse scan the database 3 8 4 2 which stores data representing the services that can be provided is.
- the database 3842 may be provided inside the service registry 380, or may be provided outside the service registry 380. By storing data representing a service in the database 384, the service can be registered in the service registry 380. It is preferable that the format of the data stored in the database 3842 is standardized in advance.
- the storage of the data in the database 384 2 may be performed manually by the company that manages the service registry 380 4, or from the service provider 380 2 i to 380 2 N to the network 3 8 3 This may be done by sending data to the service registry 3840 through 0.
- Each of the service requester 3810, the service provider 3SSC ⁇ SSSON, and the service registry 3840 can be any type of computer.
- Each of the service providers 380 i to 380 N has experimental facilities It is preferably operated by a research institution, company or organization studying real cells.
- Each of Service Requester 3810 and Service Registry 3840 is provided by a research institution, company or entity (eg, Digital Cell Promotion Council) that oversees the provision of services that use digital cells to replicate experimental results on real cells. It is preferably operated. Further, in order to guarantee the quality of service registered in the service registry 3 840, it is preferable to require that meet certain criteria to the service provider 3 82 ( ⁇ ⁇ 820 N of operational entities.
- the service requester 3810 and service provider 3820 i ⁇ 3820 N and service registry 3840 are connected via a network 3830.
- Network 3830 can be any type of network, but is most preferably the Internet because of the ease and cost of connectivity.
- the service requester 3810 may be a web server connected to a web browser operated by the user via the Internet, and the service provider 3820 1 to 3820 N Each can be a web server connected to the service requester 3810 via the Internet.
- the service requester 3810 functions as a portal / web site that relays the web browser operated by the user and the web server of the service provider 3820;
- the user is able to easily access the service provider 3820 ⁇ 3820 N worldwide, experimental research institutions and companies around the world for the actual cell using the digital cell It will be possible to participate in businesses that provide services that reproduce the results.
- FIG. 39 shows an example of a procedure of a process for providing a service for reproducing an experiment result on a real cell using a digital cell. This process is performed by the service requester It is performed by 3 8 1 0 and the service provider 3 8 2 0 i ⁇ 3 8 2 0 N cooperate.
- Step S3901 The service requester 38010 receives the cell parameter, the environmental parameter, and the stimulus parameter, and generates a request including the cell parameter, the environmental parameter, and the stimulus parameter.
- the request is described in, for example, XML.
- Step S 3 9 0 2 service requester 3 8 1 0 searches the service registry 3 8 4 0 in response to the request, the request in the service provider 3 8 2 0 E ⁇ 3 8 2 0 N It is determined whether or not the service provider 3820i that can provide the service of the present exists.
- i indicates any of 1 to N.
- any method can be adopted as a method of registering a service that can be provided by the service provider 3 8 2 ( ⁇ 3 8 0 2 0 N in the service registry 3 8 40.
- the service provider 3 8 2 If it is possible to provide a service that reproduces the experimental results for cell A, the cell parameters that specify cell A and the address (for example, URL) that specifies the location of service provider 380 are stored in database 384 may be stored in 2.
- service provider 3 8 2 0 2 cell B and if it is possible to provide a service to reproduce the experimental results for cells C, the cells to identify cell B, and cell C parameters and service prop Ida 3 8 2 0 2 position address identifying (e.g., URL) and may be stored in the database 3 8 4 2.
- the service provider 3 8 2 If 0 3 can provide a service that reproduces the experimental results that satisfy the specific experimental conditions for cell D, the parameters that specify cell D and the parameters that specify the experimental conditions (for example, environmental parameters, stimulation parameters) and service provider 3 8 2 0 3 address identifying the location of (e.g., URL) and may be storing in the database 3 8 4 2.
- Service provider 3 8 2 0; positions may be by connexion identified by referring to the database 3 8 4 2 service registry 3 8 4 0.
- Step S3904 The service provider 38020i searches the database 382022 in response to the request, and stores the cell parameters included in the request in the database 382022i. It is determined whether the stimulus response result related to the environmental parameter and the stimulus parameter exists.
- Step S3905 If it is determined that there is a stimulus response result related to the cell parameter, the environmental parameter and the stimulus parameter included in the request in the database 3822; 0 i provides the stimulus response result to the service requester 3810.
- the stimulus response result is described in, for example, XML.
- Step S3906 The service requester 3810 displays the stimulus response result provided by the service provider 3820i.
- the service provider 380 For example, provide the result "N / A" to the service requester 3810.
- any method can be adopted as a method of inputting the cell parameter, the environmental parameter, and the stimulus parameter to the service requester 380, and the service requester 380 can display any stimulus response result.
- the aspect can be adopted as described above.
- digital cells are used. As a result, it is possible to provide a service that reproduces the results of experiments on real cells. This makes it possible for research institutions and individuals without experimental facilities to conduct cutting-edge research on cells. Further, according to the computer system 3801, by registering a service that can be provided by a plurality of service providers 382 ( ⁇ 380 0 ⁇ ) in the service registry 380, a digital cell can be used. This will give research institutions and companies around the world the opportunity to participate in businesses that provide services that reproduce the results of experiments on real cells.
- the ones prepared in this example are as follows. Various extracellular matritus proteins and their variants or fragments thereof were prepared as actin candidate substances. The components prepared in this example are as follows. Commercially available fibronectin and the like were used, and fragments and modified ones modified by genetic manipulation were used.
Abstract
Description
Claims
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP04746873A EP1640453A4 (en) | 2003-06-25 | 2004-06-25 | DIGITAL CELL |
CA002530350A CA2530350A1 (en) | 2003-06-25 | 2004-06-25 | Digital cell |
US10/562,469 US7747390B2 (en) | 2003-06-25 | 2004-06-25 | Digital cell |
JP2005513404A JPWO2005021744A1 (ja) | 2003-06-25 | 2004-06-25 | デジタル細胞 |
US12/823,620 US20110004415A1 (en) | 2003-06-25 | 2010-06-25 | Digital cell |
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JP2003181915 | 2003-06-25 | ||
JP2003-181915 | 2003-06-25 | ||
JP2003-289469 | 2003-08-07 | ||
JP2003289469 | 2003-08-07 |
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US12/823,620 Continuation US20110004415A1 (en) | 2003-06-25 | 2010-06-25 | Digital cell |
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WO2005021744A1 true WO2005021744A1 (ja) | 2005-03-10 |
Family
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PCT/JP2004/009404 WO2005021744A1 (ja) | 2003-06-25 | 2004-06-25 | デジタル細胞 |
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US (2) | US7747390B2 (ja) |
EP (1) | EP1640453A4 (ja) |
JP (2) | JPWO2005021744A1 (ja) |
CA (1) | CA2530350A1 (ja) |
WO (1) | WO2005021744A1 (ja) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7774134B1 (en) * | 1999-11-15 | 2010-08-10 | Walgreen Co. | Pharmacy network management system and method for searching for a pharmacy store location with a drive through |
WO2023190139A1 (ja) * | 2022-03-30 | 2023-10-05 | 富士フイルム株式会社 | 構成方法、構成装置、及び構成プログラム |
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7747390B2 (en) * | 2003-06-25 | 2010-06-29 | National Institute Of Advanced Industrial Science And Technology | Digital cell |
US20050149546A1 (en) * | 2003-11-03 | 2005-07-07 | Prakash Vipul V. | Methods and apparatuses for determining and designating classifications of electronic documents |
US7519565B2 (en) * | 2003-11-03 | 2009-04-14 | Cloudmark, Inc. | Methods and apparatuses for classifying electronic documents |
US8862582B2 (en) * | 2007-11-15 | 2014-10-14 | At&T Intellectual Property I, L.P. | System and method of organizing images |
US9336302B1 (en) | 2012-07-20 | 2016-05-10 | Zuci Realty Llc | Insight and algorithmic clustering for automated synthesis |
US10083275B2 (en) | 2012-12-13 | 2018-09-25 | International Business Machines Corporation | Stable genes in comparative transcriptomics |
US10542961B2 (en) | 2015-06-15 | 2020-01-28 | The Research Foundation For The State University Of New York | System and method for infrasonic cardiac monitoring |
US11205103B2 (en) | 2016-12-09 | 2021-12-21 | The Research Foundation for the State University | Semisupervised autoencoder for sentiment analysis |
JP2018100903A (ja) * | 2016-12-20 | 2018-06-28 | パナソニックIpマネジメント株式会社 | 樹脂判定方法及び装置 |
WO2023276449A1 (ja) * | 2021-06-29 | 2023-01-05 | 富士フイルム株式会社 | 細胞数理モデルを作成する方法、細胞数理モデル作成プログラム、細胞数理モデル作成装置、細胞数理モデルの判定方法、細胞数理モデル判定プログラム、及び、細胞数理モデル判定装置 |
CN113759196B (zh) * | 2021-09-08 | 2022-09-13 | 山东大学 | 一种高压电气设备实验装置及其实验方法 |
Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998006874A1 (en) * | 1995-08-09 | 1998-02-19 | The Regents Of The University Of California | Systems for generating and analyzing stimulus-response output signal matrices |
WO1998038490A1 (en) | 1997-02-27 | 1998-09-03 | Cellomics, Inc. | A system for cell-based screening |
WO1999060094A2 (de) | 1998-05-20 | 1999-11-25 | Forschungszentrum Jülich GmbH | Verfahren zur durchführung einer messung der interaktion von chemikalien mit zellen |
WO2000003246A2 (en) * | 1998-07-13 | 2000-01-20 | Cellomics, Inc. | A system for cell-based screening |
WO2001063245A2 (en) * | 2000-02-25 | 2001-08-30 | The Technology Partnership Plc | Method and apparatus for high throughput cell - based assays for screening and diagnostics |
WO2002007100A1 (fr) | 2000-07-13 | 2002-01-24 | Takashi Gojobori | Procede, systeme et programme permettant d'indiquer le phenomene d'expression d'un ecosysteme |
US20020072116A1 (en) | 2000-10-12 | 2002-06-13 | Bhatia Sangeeta N. | Nanoporous silicon bioreactor |
US20020197718A1 (en) | 2001-06-06 | 2002-12-26 | Becton, Dickinson And Company | Method of providing a substrate with a ready-to-use, uniformly distributed extracellular matrix |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7293230B2 (en) * | 2000-01-27 | 2007-11-06 | American Express Travel Related Services Company, Inc. | Information architecture for the interactive environment |
US20020055935A1 (en) * | 2000-03-31 | 2002-05-09 | Rosenblum Michael G. | Methods and systems for providing access to one or more databases of information concerning therepeutic and diagnostic agents |
US20030065774A1 (en) * | 2001-05-24 | 2003-04-03 | Donald Steiner | Peer-to-peer based distributed search architecture in a networked environment |
US7747390B2 (en) * | 2003-06-25 | 2010-06-29 | National Institute Of Advanced Industrial Science And Technology | Digital cell |
-
2004
- 2004-06-25 US US10/562,469 patent/US7747390B2/en not_active Expired - Fee Related
- 2004-06-25 CA CA002530350A patent/CA2530350A1/en not_active Abandoned
- 2004-06-25 EP EP04746873A patent/EP1640453A4/en not_active Withdrawn
- 2004-06-25 JP JP2005513404A patent/JPWO2005021744A1/ja active Pending
- 2004-06-25 WO PCT/JP2004/009404 patent/WO2005021744A1/ja active Application Filing
-
2010
- 2010-03-19 JP JP2010065276A patent/JP2010207222A/ja not_active Withdrawn
- 2010-06-25 US US12/823,620 patent/US20110004415A1/en not_active Abandoned
Patent Citations (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998006874A1 (en) * | 1995-08-09 | 1998-02-19 | The Regents Of The University Of California | Systems for generating and analyzing stimulus-response output signal matrices |
US5777888A (en) | 1995-08-09 | 1998-07-07 | Regents Of The University Of California | Systems for generating and analyzing stimulus-response output signal matrices |
WO1998038490A1 (en) | 1997-02-27 | 1998-09-03 | Cellomics, Inc. | A system for cell-based screening |
WO1999060094A2 (de) | 1998-05-20 | 1999-11-25 | Forschungszentrum Jülich GmbH | Verfahren zur durchführung einer messung der interaktion von chemikalien mit zellen |
WO2000003246A2 (en) * | 1998-07-13 | 2000-01-20 | Cellomics, Inc. | A system for cell-based screening |
WO2001063245A2 (en) * | 2000-02-25 | 2001-08-30 | The Technology Partnership Plc | Method and apparatus for high throughput cell - based assays for screening and diagnostics |
WO2002007100A1 (fr) | 2000-07-13 | 2002-01-24 | Takashi Gojobori | Procede, systeme et programme permettant d'indiquer le phenomene d'expression d'un ecosysteme |
EP1302901A1 (en) | 2000-07-13 | 2003-04-16 | Takashi Gojobori | Method, system and program of indicating expression phenomenon of biosystem |
US20020072116A1 (en) | 2000-10-12 | 2002-06-13 | Bhatia Sangeeta N. | Nanoporous silicon bioreactor |
US20020197718A1 (en) | 2001-06-06 | 2002-12-26 | Becton, Dickinson And Company | Method of providing a substrate with a ready-to-use, uniformly distributed extracellular matrix |
Non-Patent Citations (3)
Title |
---|
GRAY ET AL.: "Exploiting chemical libraries, structure, and genomics in the search for kinase inhibitors", SCIENCE, vol. 281, 1998, pages 533 - 538, XP002204861, DOI: doi:10.1126/science.281.5376.533 |
MARTON ET AL.: "Drug target validation and identification of secondary drug target effects using Microarrays", NAT. MED., vol. 4, no. 11, November 1998 (1998-11-01), pages 1293 - 301, XP002940437, DOI: doi:10.1038/3282 |
See also references of EP1640453A4 |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7774134B1 (en) * | 1999-11-15 | 2010-08-10 | Walgreen Co. | Pharmacy network management system and method for searching for a pharmacy store location with a drive through |
WO2023190139A1 (ja) * | 2022-03-30 | 2023-10-05 | 富士フイルム株式会社 | 構成方法、構成装置、及び構成プログラム |
Also Published As
Publication number | Publication date |
---|---|
CA2530350A1 (en) | 2005-03-10 |
EP1640453A4 (en) | 2009-09-02 |
JP2010207222A (ja) | 2010-09-24 |
US20110004415A1 (en) | 2011-01-06 |
JPWO2005021744A1 (ja) | 2006-10-26 |
US20060253258A1 (en) | 2006-11-09 |
US7747390B2 (en) | 2010-06-29 |
EP1640453A1 (en) | 2006-03-29 |
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