JP2006158292A - Method for analyzing promoter - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for more directly comparing and evaluating a polymorphic promoter from a normal promoter to analyze a contribution of mutation or polymorphism to transcription activity, to provide a method for more directly evaluating a difference in the response of a medicine due to the mutation or polymorphism, and to provide a method for screening the medicine. <P>SOLUTION: A mammal cell into which a gene structure having three or more reporter genes under the controls of separated promoters wherein the first reporter gene, the second reporter gene and the remaining reporter gene are placed under the controls of a regular expression promoter, a normal expression promoter and a variant promoter, respectively, are transduced to transiently or stably express, and a method for analyzing the activity of a promoter comprises culturing mammal cells under different culture conditions, measuring the transcription activities of the promoters connected to the reporter gene, and comparing the transcription activities of the promoters between samples. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for directly comparing a plurality of transcriptional activities using a cell sample in which a plurality of reporter genes are incorporated so as to allow expression. More specifically, the present invention relates to a method for analyzing promoter polymorphism by directly comparing transcriptional activities of a normal promoter and a mutant promoter, a responsive promoter and a non-responsive promoter, etc. in the same cell sample.

  Genetic polymorphism includes nucleotide sequence substitution, insertion, and deletion. Among them, in recent years, single nucleotide polymorphism (Single Nucleotide Polymorphism), which is a polymorphism of single nucleotide substitution, has attracted attention and research is progressing. Examples of SNP include rSNP in the gene expression control region, cSNP as a protein coding region, and iSNP in a gene intron (transcribed into mRNA and then removed by splicing and does not encode a protein). Gene polymorphisms including SNPs are considered to be necessary information for medical care tailored to individuality (custom-made medical care). Whether these SNPs are likely to cause diseases such as blood pressure, blood glucose, and allergies, and the effects of drugs. This is because there is an expectation that it can explain at least a part of individual differences.

  Among SNPs, cSNP analysis has attracted attention because it directly contributes to protein function. As an example, it is statistically revealed that flash memory is inferior in the SNPs population present in the neurotrophic factor BDNF, and at the same time, a neurotrophic factor-fluorescent protein is created and the localization of the fusion protein is changed. The difference in the kinetics in the cell was clarified (Non-patent Document 1). On the other hand, the importance of single molecule polymorphism analysis has been pointed out, but a method for analyzing SNP information existing in the gene expression control region has not been well established. Therefore, research on rSNP that may be involved in gene expression control has not progressed, and is far less than cSNP for data accumulation.

  As a technique for analyzing gene expression control analysis, a vector containing a cis-acting base sequence element (a gene expression control sequence (candidate) such as a promoter, enhancer, silencer) linked to a reporter gene) is introduced into an appropriate cell, The activity of the reporter enzyme expressed under certain conditions is measured. Until now, Escherichia coli chloramphenicol acethyltransferase (CAT), human growth hormone, alkaline phosphatase, firefly (Photinus Pyralis) luciferase, Escherichia coli β-galactosidase, and fluorescent protein (GFP) have been used as reporter genes. Among these, a system using luminescence of the luciferase gene is particularly used at present because of its high sensitivity and easy activity measurement.

  There are unintended factors between reporter assay samples, such as transfection efficiency, cell death, cell variation, and other factors that cause differences in reporter expression levels. Therefore, in addition to the reporter gene linked to the test sequence, it is necessary to standardize between samples using a reporter molecule with different substrate specificity (reactivity) linked to the control promoter as an internal standard. In such cases, Renilla luciferase has been cloned and used to measure the expression of two or more genes simultaneously, in combination with the above genes, or with different substrate specificities. .

In the method, the cis-acting base linked to the first reporter gene is standardized between the samples with the reporter protein activity for the second internal standard, and the activity of the first reporter protein between the samples is compared. Analyzes the activity of array elements. However, in such a comparison, the kinetic comparison of multiple promoter activities is indirect. Furthermore, this comparison may include false positives and false negatives due to variations in experimental operations. In particular, in the analysis of promoter polymorphisms such as SNP, it is not possible to understand the effects of SNP by simply analyzing one gene expression information. Therefore, comparison is made with the same cell, under the same conditions, and with the gene expression of a third party. There is a need. Therefore, there is a need for a method that overcomes these problems.
WO2004 / 099421 MF Egan, M. Kojima, JH, Callicott, TE Goldberg, BS Kolachana, A. Bertolino, E. Zaitsev, B. Gold, D. Goldman, M. Dean, B. Lu, D. R Weinberger; The BDNF val66met polymorphism affects activity-dependent secretion of BDNF and human memory and hippocampal function.Cell Cell. 2003 Jan 24; 112 (2): 257-69.

  An object of the present invention is to provide a method for more directly comparing and evaluating a polymorphic promoter with a normal promoter and analyzing the contribution of mutations and polymorphisms to transcriptional activity. Furthermore, it is to provide a method for directly evaluating a difference in drug response due to mutation or polymorphism, and a method for screening a drug.

In the patent document 1, the present inventors have disclosed a method for simultaneously measuring three promoter activities using green and orange photoproteins derived from Iriomote butter and red photoprotein derived from a railworm. As a result of further earnest research, the present inventors compared the polymorphic promoter with the normal promoter more directly, analyzed the contribution of the mutation and polymorphism to the transcriptional activity, and also analyzed the mutation and polymorphism. The present inventors have found that it is possible to more directly evaluate the difference in drug response due to the drug, and have completed the present invention.
That is, the present invention has the following configuration.

  Item 1. Having three or more reporter genes under the control of separate promoters, the first reporter gene under the control of a constant expression promoter, the second reporter gene under the control of a normal promoter, and the remaining reporters A mammalian cell into which a gene construct in which a gene is under the control of a mutant promoter is transiently or stably expressed.

  Item 2. Having three or more reporter genes under the control of separate promoters, the first reporter gene is under the control of a constant expression promoter, the second reporter is under the control of a first gene polymorphic promoter, Mammalian cells that have been introduced to transiently or stably express a gene construct in which the remaining reporter is under the control of another gene polymorphism promoter.

  Item 3. Having three or more reporter genes under the control of separate promoters, the first reporter gene is under the control of a constant expression promoter, the second reporter gene is under the control of a responsive promoter, and the remaining reporters A mammalian cell into which a gene construct in which a gene is under the control of a non-responsive promoter is transiently or stably expressed.

  Item 4. A promoter comprising culturing mammalian cells according to any one of Items 1 to 3 under different culture conditions, measuring the transcriptional activity of a promoter linked to a reporter gene, and comparing the transcriptional activity of the promoter between samples. Analysis method of activity.

  Item 5. The mammalian cells according to any one of Items 1 to 3 are cultured in the presence or absence of a drug candidate compound, the transcriptional activity of a promoter linked to a reporter gene is measured, and the transcriptional activity of the promoter between samples is compared. A method for evaluating the influence of the candidate compound.

  Item 6. Item 3. The mammalian cell according to Items 1 and 2, wherein the reporter gene is selected from chloramphenicol acethyltransferase, growth hormone, alkaline phosphatase, β-galactosidase, luciferase, and fluorescent protein.

  Item 7. Item 7. The mammalian cell according to Item 6, wherein the reporter gene comprises one or more luciferases.

  Item 8. Item 8. The mammalian cell according to Item 7, wherein the luciferase comprises two or more luciferases.

  Item 9. The method according to claim 7 or 8, wherein the luciferase comprises a luciferase derived from a luminescent beetle.

  Item 10. The method of claim 9, wherein the one or more luciferases have different emission wavelength characteristics.

  With the method of the present invention and mammalian cells, using three reporter genes, two types of promoter activities, particularly a normal promoter and a mutant promoter, a responsive promoter and a non-responsive promoter, and a control promoter are used as internal standards. Thus, it has become possible to more directly compare the kinetics of drug responses and the like, and to efficiently analyze promoter polymorphisms. In this system, three types of photoproteins are used as three reporter genes, and light separation measurement is performed. By measuring three reporter protein activities in one reaction, more rapid and / or large amounts of polymorphisms or treatments are performed. It is possible to analyze the conditions.

  The present invention is described in detail below.

  The “promoter” in the present invention is a cis-acting base sequence element (a gene expression control sequence such as a promoter, enhancer, silencer, or the like, or a base sequence that is considered to be possible. Herpes simplex virus thymidine kinase (HSVtk). It may be a cis-acting base sequence linked to a promoter derived from Cytomegalovirus (CMV) or a minimal unit derived from Cytomegalovirus (CMV), which is cloned from mammalian or viral genomic DNA, RNA, library, or the like.

  The activity of the reporter protein linked to the constant expression promoter is desirably used to standardize transfection efficiency between samples, the number of cells, and variations in the growth state of cells. As the constant expression promoter, virus-derived HSVtk promoter, CMV promoter, SV40 promoter, mammalian cell-derived housekeeping gene promoter, and the like are used.

  Mutant promoters and gene polymorphic promoters are promoters in which one or more bases are substituted (or different), deleted or inserted, and are promoters obtained from differences in individuals or races. Further, in order to examine the contribution of the nucleotide sequence of a specific site of the promoter to the activity, it may be a promoter in which a base is artificially substituted, deleted, or inserted in a site-specific manner. Mutagenesis sites such as TATA box, CAAT box, GC box, transcription factor binding site, SRE (serum responsive element), TRE (TPA responsive element), etc. may affect promoter activity. A site. Furthermore, it is also possible to compare the dynamics of a polymorphic promoter that is frequently observed in a certain disease population and the normal promoter of a healthy person.

  By performing such an assay, it is possible to examine the variation in transcriptional activity caused by the polymorphic promoter or the function of the (normal) promoter.

  For example, by introducing mutations (including deletions) into the SRE and TRE sites of c-fos, responsiveness of c-fos due to PMA (Phorbol 12-myristate 13-acetate) of c-fos, serum, and growth factors Can be observed.

  In the past, gene coding region mutations (cSNPs) have been well studied, but promoter mutations have not been well investigated. Since the present invention enables detailed examination of promoter polymorphism and gene expression, it is expected to be useful for analysis of promoter polymorphism and elucidation of the function of the promoter in the future. In addition, the involvement of promoters (polymorphisms) can be elucidated in more detail in the relationship between diseases and genes and the relationship between individual effects of drug effects.

  For example, in the cytosolic phosphoenolpyruvate carboxykinase (PDK1) gene promoter of type 2 diabetic patients, a C → G SNP is observed at position −232, and the position −232 is not down-regulated by insulin when compared to the C promoter, and is in a steady state. There is a report that the increase of the expression was recognized. Such a change in responsiveness due to SNP can be found more directly and more efficiently by evaluating the polymorphic promoter and the normal promoter in the same cell.

  Responsive promoters include cAMP response element (CRE), TPA response element (TRE), heat shock element (TPA (PMA), cAMP, serum, etc., which have the ability to induce expression depending on the presence of various inducers. HSE), glucocorticoid response element (GRE), metal response element (MRE), steroid response element (ERE), foreign drug response element (XRE), serum response element (SRE), etc. Examples include promoters, promoters exhibiting tissue-specific expression inducibility, promoters exhibiting expression inducibility specific to development / differentiation states, and the like. Stimulate such a responsive promoter in a sample by coexisting a second reporter gene under the control of a promoter responsive to a stimulus and the remaining reporter gene under the control of a non-responsive promoter It is possible to evaluate with higher accuracy such as a test of whether or not such a drug is contained and drug screening. To explain a little more about the more accurate assessment, in a sample with only a non-responsive promoter, a stimulus was actually applied to the cell, demonstrating that the signal to that stimulus was actually in the cell but did not respond. This includes the possibility that a response was not recognized because a stimulus could not be successfully applied to the sample because there was no signal to do so. The responsive promoter alone may have been stimulated by other factors.

  These promoters are linked upstream of the reporter gene. The reporter gene is preferably changed to a mammalian codon usage for efficient expression in mammalian cells. Furthermore, it is preferable that a Kozak sequence is inserted in order to improve translation efficiency.

  Methods for introducing these gene constructs into mammalian cells include commercially available lipofection reagents, chemical methods such as calcium phosphate, physical methods such as microinjection and electroporation, and biological methods using viral vectors. It may be. The transcriptional activity of the promoter is relativeized by measuring the activity or amount of reporter protein synthesized in mammalian cells.

  Examples of mammals include humans, mice, rats, cows, horses, sheep, monkeys, pigs, mice, rats, hamsters, guinea pigs, rabbits, and dogs, preferably humans.

  The reporter gene is selected from chloramphenicol acethyltransferase, growth hormone, alkaline phosphatase, β-galactosidase, luciferase, and fluorescent protein. For example, the activity of chloramphenicol acethyltransferase is measured by a reaction using chloramphenicol and the luciferase is a reaction using luciferin as a substrate. Examples of the fluorescent protein include GFP (Green Fluorescent Protein) and modified types into which various amino acid substitutions are introduced. The fluorescent protein is irradiated with excitation light, and the relative amount is measured by measuring fluorescence. However, analysis using fluorescent proteins is said to have low quantitativeness. More preferably, luciferase is used. The luminescence measurement of luciferase is simple and highly quantitative.

  Examples of the luciferase gene include firefly-derived luciferase, red beetle-derived luciferase, Renilla luciferase, railway insect luciferase, dinoflagellate-derived luciferase, and Iriomote botul-derived luciferase. More preferably, a luminescent beetle-derived luciferase using the same substrate, D-luciferin as a substrate, is preferred. More preferably, the emission spectrum is selected from green, orange-emitting luciferase derived from Iriomote butter having a smaller half-value width, red, green luciferase derived from railway insects, and the emission spectrum is not affected by pH.

  For luciferase activity measurement, cells are disrupted by a cell lysing agent containing a surfactant or sonication or the like, mixed with a luminescent substrate solution containing luciferin, ATP, etc., and luminescence is measured. It is also possible to add luciferin to the cell culture medium and measure luminescence as it is. When a plurality of luminescent luciferases are used, the activity of a plurality of reporter proteins (luciferases) can be measured in one reaction by separating and measuring with a luminometer with a light separation function.

  Examples of analysis include, for example, investigating the effect of transcriptional activity of the promoter polymorphism on the transcriptional activity of the normal and mutant promoters by analyzing the fluctuations in the activity of the normal and mutant promoters more directly. Compounds that only affect either can be screened more directly.

  Examples of other analyzes include, for example, screening for compounds that act more specifically and stimulating response elements in unknown samples by comparing the activity variations of response element promoters and non-responsive element promoters. It is possible to check whether a compound is present.

  The analysis result can be used for drug discovery.

Hereinafter, the effects of the present invention will be further clarified by illustrating examples of the present invention.
Example 1 Cloning of c-fos promoter Using 20 ng of human genomic DNA as a template, oligonucleotides 1 and 2 (PRORIGO) (SEQ ID NOs: 1 and 2) were used with KOD-plus (Toyobo) in the presence of 5% DMSO. PCR amplified. The amplified fragment, red light emitting luciferase vector pSLR-test (Toyobo) was treated with restriction enzymes Bgl II and EcoR I, and ligated using Ligation high (Toyobo) (pSLR-cfos1).
Example 2 Cloning of mutant (deletion) c-fos promoter
PCR amplification was performed with KOD-plus (Toyobo) using oligonucleotide 3 (PRORIGO) (SEQ ID NO: 3) and 2 using pSLR-cfos as a template. The amplified fragment, orange-emitting luciferase vector pSLO-test (Toyobo) was treated with restriction enzymes BglII and EcoRI and ligated using Ligation high (Toyobo) (pSLO-cfos2, pSLO-cfos3).
Example 3 Transfection and culture of CHO-K1 cells
CHO-K1 cells (1 × 10 ⁵ cells) were seeded in 12-well plates and cultured in Ham's F12 (Nissui Pharmaceutical) + 10% FCS. The next day, 0.1 μg of pSLG-HSVtk in each well (vector with luminescent luciferase linked to a constant expression promoter, Toyobo), 0.5 μg of pSLR-cfos1 and 0.4 μg of pSLO-cfos2 diluted with 100 μl Opti-MEM (GIBCO) The resulting mixture was mixed with 3 μl of GeneJuice (Novagen) and transfected in serum-free medium Ham's F12. The next day, untreated, 1 μM PMA (TPA, Sigma) and 10% FCS were added, and the cells were further cultured for 3 hours.
Example 4 Measurement of luciferase activity After rinsing the cells with PBS (-), the cells were lysed with the Picker Gene cell lysing agent Lcβ (Toyo Ink), added with a luminescent substrate (Toyo Ink), and the relative luminescence of green, orange, and red luminescent luciferases The amount was measured using a luminometer with a light separation function, AB-2250 type luminescence sensor MCA (Ato). Figure 2 is a graph showing the relative ratio of red luciferase (SLR) and orange luciferase (SLO), with the untreated sample as 1, normalized between samples based on the relative luminescence of green luciferase. In the same sample where responsiveness to the addition of serum or PMA is observed with the promoter, this is a graph suggesting that the responsiveness is lost with the mutant (deletion type) promoter.
Example 5 Cloning of annexin6 promoter PCR amplification with KOD-plus (Toyobo) in the presence of 5% DMSO using 20 ng of human genomic DNA as a template and oligonucleotides 4 and 5 (PRORIGO) (SEQ ID NOs: 4 and 5) did. The amplified fragment, red light emitting luciferase vector pSLR-test (Toyobo) was treated with restriction enzymes Bgl II and EcoR I, and ligated using Ligation high (Toyobo) (pSLR-annexin6).
Example 6 Transfection and culture of CHO-K1 cells
CHO-K1 cells (1 × 10 ⁵ cells) were seeded in 12-well plates and cultured in Ham's F12 (Nissui Pharmaceutical) + 10% FCS. The next day, 0.1 μg of pSLG-HSVtk in each well (vector with luminescent luciferase linked to a constant expression promoter, Toyobo), 0.5 μg of pSLR-annexin6, 0.4 μg of pSLO-cfos diluted with 100 μl Opti-MEM (GIBCO) The resulting mixture was mixed with 3 μl of GeneJuice (Novagen) and transfected in serum-free medium Ham's F12. The next day, untreated, 10% FCS was added, and further cultured for 3 hours.
Example 7 Measurement of luciferase activity After rinsing cells with PBS (-), the cells were lysed with Pickagene cell lysing agent Lcβ (Toyo Ink), luminescent substrate (Toyo Ink) was added, and the relative luminescence of green, orange and red luminescent luciferases The amount was measured using a luminometer with a light separation function, AB-2250 type luminescence sensor MCA (Ato). Fig. 3 is a graph showing the relative ratio of red luciferase (SLR) and orange luciferase (SLO) with the untreated sample as 1, normalized between samples based on the relative luminescence of green luciferase, and serum stimulation. The response-type c-fos promoter (SLO) suggests that the non-response type annexin-6 promoter that does not contain a serum stimulation response element has lost its responsiveness within the same sample where responsiveness to serum addition is observed It is a graph.

The method of directly comparing a plurality of transcriptional activities using the same cell sample using a plurality of reporter genes of the present invention is a system for directly comparing a normal promoter and a mutant promoter and analyzing a gene polymorphism. Furthermore, it can be used as a drug screening system based on direct comparison to drug response and a test system for the presence or absence of stimulating factors in unknown samples, and contributes greatly to industry such as drug discovery.

It is a comparison figure on the sequence of a normal type c-fos promoter and a deletion type c-fos promoter. This graph shows the relative ratio of red luciferase (SLR) and orange luciferase (SLO) with the untreated sample taken as 1, based on the relative luminescence of green luciferase, and the normal c-fos promoter In the same sample where responsiveness to the addition of serum or PMA is observed, the polymorphic (deletion type) promoter c-fos2 is a graph suggesting that responsiveness is lost. This is a graph showing the relative ratio of red luciferase (SLR) and orange luciferase (SLO) relative to the sample based on the relative luminescence of green luciferase, with the untreated sample being 1, and the serum stimulation response type c- The fos promoter (SLO) is a graph suggesting that responsiveness is lost in the non-responding type annexin-6 promoter containing no serum stimulation response element in the same sample in which responsiveness to the addition of serum is observed.

Claims (10)

  Having three or more reporter genes under the control of separate promoters, the first reporter gene under the control of a constant expression promoter, the second reporter gene under the control of a normal promoter, and the remaining reporters A mammalian cell into which a gene construct in which a gene is under the control of a mutant promoter is transiently or stably expressed.   Having three or more reporter genes under the control of separate promoters, the first reporter gene is under the control of a constant expression promoter, the second reporter is under the control of a first gene polymorphic promoter, Mammalian cells that have been introduced to transiently or stably express a gene construct in which the remaining reporter is under the control of another gene polymorphism promoter.   Having three or more reporter genes under the control of separate promoters, the first reporter gene is under the control of a constant expression promoter, the second reporter gene is under the control of a responsive promoter, and the remaining reporters A mammalian cell into which a gene construct in which a gene is under the control of a non-responsive promoter is transiently or stably expressed.   Culturing the mammalian cells according to any one of claims 1 to 3 under different culture conditions, measuring the transcriptional activity of a promoter linked to a reporter gene, and comparing the transcriptional activity of the promoter between samples. Method for analyzing promoter activity.   The mammalian cells according to any one of claims 1 to 3 are cultured in the presence or absence of a drug candidate compound, the transcriptional activity of a promoter linked to a reporter gene is measured, and the transcriptional activity of the promoter between samples is compared. A method for evaluating the influence of the candidate compound.   The mammalian cell according to any one of claims 1 to 3, wherein the reporter gene is selected from chloramphenicol acethyltransferase, growth hormone, alkaline phosphatase, β-galactosidase, luciferase, and fluorescent protein.   The mammalian cell of claim 6, wherein the reporter gene comprises one or more luciferases.   The mammalian cell of claim 7, wherein the luciferase comprises two or more luciferases.   The method according to claim 7 or 8, wherein the luciferase comprises a luciferase derived from a luminescent beetle.   The method according to claim 9, wherein three or more luciferases have different emission wavelength characteristics.

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