WO2013054846A1 - 哺乳動物細胞を用いた変異原性試験法 - Google Patents
哺乳動物細胞を用いた変異原性試験法 Download PDFInfo
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- WO2013054846A1 WO2013054846A1 PCT/JP2012/076325 JP2012076325W WO2013054846A1 WO 2013054846 A1 WO2013054846 A1 WO 2013054846A1 JP 2012076325 W JP2012076325 W JP 2012076325W WO 2013054846 A1 WO2013054846 A1 WO 2013054846A1
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- 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
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
- G01N33/5017—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity for testing neoplastic activity
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- C12Q2600/00—Oligonucleotides characterized by their use
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- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4703—Regulators; Modulating activity
Definitions
- the present invention relates to a mutagenicity test method for confirming the mutagenicity and thus carcinogenicity of a test substance to mammalian cells, and a recombinant vector and a transformant used in this method.
- Chemical substances that come into contact with humans, such as food, cosmetics, and pharmaceuticals, and substances contained in products that are discarded in the environment are required to be non-carcinogenic. Since many mutagenic substances have carcinogenicity, it is essential to evaluate whether or not the substance to be used has mutagenicity.
- the Ames test using Salmonella employed in the Chemical Substances Control Law and the Pharmaceutical Affairs Law is the most widely used. Since this test is a test using microorganisms, chemical substances determined to be positive for mutagenicity in this test do not necessarily give the same results to humans.
- tests using cultured mammalian cells and tests using individual animals such as mouse lymphoma TK test, chromosomal aberration test, and micronucleus test, have been established as mutagenicity test methods. It is cumbersome and takes at least two days before results are obtained.
- the present inventors possessed a recombinant vector comprising (1) a p53 binding sequence, a minimal promoter that can function in mammalian cells, and a reporter gene linked so that it can be expressed by this minimal promoter.
- a mutagenicity test method including a step of determining whether or not a test substance affects the expression level of a reporter gene in a transformant has been developed (Patent Document 1).
- the main object of the present invention is to provide a test method capable of confirming the mutagenicity of mammals including humans accurately, simply and in a short time.
- the present inventor has conducted research by contacting a transformant having a recombinant vector containing a p53 binding sequence, a minimal promoter and a reporter gene with a test substance for a shorter time than before. It was found that a mutagenicity test can be carried out by a mutagenicity test method including a process. In particular, it has been found that the contact time with a test substance can be significantly shortened by using a combination of a specific recombinant vector and a host cell. The present invention has been completed based on the above findings.
- the present invention provides the following sections: Item 1. (1) In the presence of a test substance, a transformant carrying a recombinant vector comprising a p53-binding sequence, a minimal promoter that can function in mammalian cells, and a reporter gene linked so that it can be expressed by this minimal promoter. Culturing for 4 to 10 hours at (2) By comparing the expression level of the reporter gene in the transformant cultured in the above step (1) with the expression level of the reporter gene in the transformant that does not contact the test substance, the test substance is a reporter in the transformant.
- a mutagenicity test method comprising a step of determining whether or not an expression level of a gene is affected.
- Item 2. The method according to Item 1, wherein the reporter gene is luc2 and the transformant is obtained by introducing the recombinant vector into TK6 cells.
- Item 3. The method according to Item 1 or 2, wherein the minimal promoter is an SV40 promoter.
- Item 4. The p53 binding sequence, the minimal promoter, and the reporter gene are arranged in the recombinant vector in the order of the p53 binding sequence, the minimal promoter, and the reporter gene from the upstream side. Method.
- Item 5 Any one of Items 1-4, wherein the recombinant vector is a pGL4-Luc2 plasmid in which the p53-binding sequence and the minimal promoter are inserted, and the transformant is a product obtained by introducing the recombinant vector into TK6 cells. The method according to claim 1.
- p53 binding sequence is 5'-WRRCWWGYYY-3 ': SEQ ID NO: 1 Items 1 to 5 having the nucleotide sequence shown in (In SEQ ID NO: 1, R represents a nucleotide containing a purine base, W represents a nucleotide containing adenine or thymine, and Y represents a nucleotide containing a pyrimidine base) The method as described in any one of.
- p53 binding sequence is 5'-WRRCWWGYYY WRRCWWGYYY-3 ': SEQ ID NO: 2 Item 1 to 6 having the base sequence shown in (In the SEQ ID NO: 2, R represents a nucleotide containing a purine base, W represents a nucleotide containing adenine or thymine, and Y represents a nucleotide containing a pyrimidine base) The method as described in any one of.
- Item 8 The method according to Item 7, wherein the p53-binding sequence has three base sequences represented by SEQ ID NO: 2.
- Item 9 The method according to Item 1, wherein the expression level of the reporter gene is a relative value with respect to a standard gene.
- a transformant which is a TK6 cell harboring a recombinant vector comprising a p53 binding sequence, a minimal promoter that can function in mammalian cells, and a reporter gene linked so as to be expressed by the minimal promoter.
- a mutagenicity test kit comprising the transformant according to Item 11 or Item 10.
- the mutagenicity of mammals including humans can be confirmed more accurately and simply than before.
- the mutagenicity test method of the present invention does not directly evaluate phenotypic changes due to DNA damage, but evaluates biological reactions due to DNA damage, it can detect DNA damage with high sensitivity. .
- the method of the present invention does not use microorganisms as in the conventional mutagenicity test, but uses a reaction system against DNA damage in mammalian cells including humans. It accurately reflects gender.
- the entire test can be performed within about one day, and the time can be greatly shortened.
- DMSO is a group to which only a solvent is added, ADM-0.003, ADM-0.01, ADM-0.03, ADM-0.1, ADM-0.2, ADM-0.7 are adriamycin 0.003 ⁇ g / mL, 0.01 ⁇ g / mL, 0.03 ⁇ g / mL, 0.1 ⁇ g / mL, respectively. , 0.2 ⁇ g / mL and 0.7 ⁇ g / mL. 6 is a graph showing luciferase activity against adriamycin in various human-derived cells for the pGL4-p53R2 ⁇ 3-SV40-Luc2 transformant of Example 6.
- FIG. 2 is a graph showing luciferase activity against adriamycin in various human-derived cells for the PGV-p53R2x3-Luc transformant of Reference Example 1.
- 5 is a graph showing luciferase activity against adriamycin in various human-derived cells for the PGV-p53R2x3-Luc transformant of Reference Example 2. This shows the base sequence of pGL4.11 plasmid (Sequence of pGL4.11 plasmid). Shows the base sequence of pGL4.11 plasmid.
- Example 9 it is a graph which shows a time-dependent change of luciferase activity when a pGL4-p53R2x3-Luc2P stable transformant is contacted with adriamycin.
- the mutagenicity test method of Example 10 it is a graph which shows the luciferase activity when a pGL4-p53R2x3-Luc2P stable transformant is contacted with each concentration of adriamycin. It is a graph which shows the luciferase activity with or without correction
- the vertical axis represents “Luciferase activity (% of Solvent control)”, that is, the luciferase of the control solvent control sample. Measured value of luciferase activity expressed as a relative value to activity.
- the present invention is described in detail below: (I) Recombinant vector
- the transformant used in the method of the present invention is a recombinant vector comprising a p53 binding sequence, a minimal promoter that can function in mammalian cells, and a reporter gene linked so that it can be expressed by this minimal promoter. Holding.
- the p53-binding sequence p53 protein is activated by phosphorylation of a specific site when DNA is damaged by chemical substances, ultraviolet rays, or gamma rays, and functions to regulate transcription by binding to the transcriptional regulatory region of a specific gene group. It is a transcription factor.
- the amino acid sequence of p53 protein is known.
- the amino acid sequence of human (Homo sapiens) p53 protein is described in GenBank accession No. NP_001119584.
- the amino acid sequence of the mouse (Mus musculus) p53 protein is described in GenBank accession No. NP_035770.
- Examples of the p53-binding sequence include DNA sequences that exist in mammalian cells upstream of a target gene group whose transcriptional activity is regulated by p53 upon DNA damage or in a transcriptional regulatory region in an intron.
- a p53-binding sequence of a gene group in which activated p53 phosphorylated by DNA damage recognizes and binds to its transcriptional regulatory region to activate transcription and increase the transcription amount.
- genes whose transcription is activated by p53 include p53R2 involved in DNA repair, p21 / Waf-1 involved in cell cycle regulation, p53AIP1, Bax, p53DINP1 involved in regulation of apoptosis, and the like.
- a p53-binding DNA sequence of a gene group whose transcription is promoted by p53 in a natural mammalian cell is usually a consensus sequence 5′-WRRCWWGYYY -3 ′ (SEQ ID NO: 1: R represents a nucleotide having a purine base, W represents a nucleotide containing adenine or thymine, Y represents a nucleotide containing a pyrimidine base, and the sequence 5′-WRRCWWGYY WRRCWWGYYY-3 ′ (SEQ ID NO: 2: R represents a nucleotide having a purine base) W represents a nucleotide containing adenine or thymine, and Y represents a nucleotide containing a pyrimidine base).
- the recombinant vector used in the present invention preferably contains one or more consensus sequences of SEQ ID NO: 1 as p53 binding sequences.
- the recombinant vector used in the present invention preferably contains one or more consensus sequences of SEQ ID NO: 2 as p53 binding sequences.
- the recombinant vector may contain one or more base sequences in which up to several, particularly 1 to 2 nucleotides are substituted in the base sequence of SEQ ID NO: 2 as a p53 binding sequence.
- the recombinant vector used in the present invention is a base sequence in which up to several nucleotides are substituted in the base sequence of SEQ ID NO: 2 or the base sequence of SEQ ID NO: 2 (preferably the base sequence or sequence of SEQ ID NO: 3) as a p53 binding sequence More preferably, the base sequence of No. 3 comprises about 3 base sequences in which up to several nucleotides are substituted. When two or more consensus sequences of SEQ ID NO: 2 are contained, they may be directly linked, and about 1 to 20 nucleotides may be sandwiched between them.
- intron 1 of p53R2 contains 5'- TGACATGCCCAGGCATGTCT-3 '(SEQ ID NO: 3 Nature, 404, 42-49 (2000)), and intron 1 of Bax is 5'.
- p53AIP1 intron 1 is 5'- TCTCTTGCCCGGGCTTGTCG-3' (SEQ ID NO: 5: Cell, 102,849-862 (2000)) 5'- GAACTTGGGGGAACATGTTT-3 '(SEQ ID NO: 6: Mol.
- the p53 binding sequence is preferably SEQ ID NO: 3 included in the above consensus sequence (SEQ ID NO: 1), but the base sequence of SEQ ID NO: 4, 5, 6 or 7 (particularly the base sequence of SEQ ID NO: 5) You may use the arrangement
- a plurality of base sequences of SEQ ID NOs: 3, 4, 5, 6 or 7 may be included in combination.
- a base sequence in which the sequence of SEQ ID NO: 3, the sequence of SEQ ID NO: 4 and the sequence of SEQ ID NO: 5 are linked can be mentioned.
- a DNA sequence containing a consensus sequence can be chemically synthesized, or can be prepared by amplifying and cloning by PCR using primers designed based on the above consensus sequence using a mammalian chromosomal DNA as a template.
- a binding sequence of another transcription factor activated by DNA damage can be used instead of the p53 binding sequence.
- transcription factor-binding sequences of other target genes whose transcriptional activity is regulated by DNA damage in mammalian cells can also be used.
- Examples of such a target gene include a gene encoding a kinase protein such as ATM or ATR.
- ATM is a kinase that phosphorylates p53 in response to DNA damage. Therefore, a transcription factor binding sequence existing in the upstream region of the ATM gene can be used in place of the p53 binding sequence.
- ATR is a kinase that phosphorylates p53 in response to DNA damage mainly by ultraviolet rays. Therefore, a transcription factor binding sequence existing in the upstream region of the ATM gene can be used in place of the p53 binding sequence.
- Minimum promoter The minimum promoter that can be expressed in mammalian cells is also called a core promoter, and is usually a region found in a relatively narrow portion near the transcription start site of a gene.
- the minimal promoter is a main functional element involved in transcriptional regulation, and does not contain other functional elements involved in transcriptional regulation such as recognition sequences of other transcriptional regulatory factors, or such functionality. Even if it contains an element, it is a sequence that does not substantially change the transcriptional regulatory function of the p53 binding sequence and minimal promoter.
- a known mammalian minimum promoter can be used without limitation.
- a minimal promoter base sequence include a TATA box, which is a DNA region involved in determining the transcription start site by RNA polymerase II and maintaining the minimum transcription level, and a base sequence near the transcription start point, Specifically, for example, the base sequence of the 5 ′ upstream region of the mouse metallothionein I gene (Genbank Accession No. J00605), the base sequence of the 5 ′ upstream region of the chicken ovalbumin gene (Genbank accession No.
- HSV herpes simplex virus
- tk thymidine kinase gene 5 'upstream region base sequence
- SV40 simian virus
- These minimal promoter sequences are preferably used as the minimal promoter in the recombinant vector of the present invention.
- SV40 is particularly preferable.
- a DNA sequence in which about 1 to several nucleotides are added, deleted or substituted in these DNA sequences can be used as long as it functions as a minimal promoter in mammalian cells.
- the recombinant vector used in the present invention may contain a broader promoter region including the minimal promoter as long as the transcriptional control function by the p53 binding sequence and the minimal promoter is not essentially changed.
- DNA consisting of these sequences can be prepared by chemical synthesis based on the base sequence.
- the reporter gene refers to a gene capable of qualitatively or quantitatively confirming the expression of the gene.
- the reporter gene for example, known reporter genes can be widely used.
- a gene capable of measuring the expression level based on the enzyme activity and the like of the transcription product (reporter protein) is preferable in terms of easy measurement of the expression level.
- reporter gene for example, firefly (Photinus pyralis) Genes encoding enzyme proteins such as luciferase, Renilla reniformis luciferase, ⁇ -galactosidase, chloramphenicol acetyltransferase, alkaline phosphatase, ⁇ -glucuronidase, luminescence such as green fluorescent protein (GFP) Examples include a gene encoding a protein.
- enzyme proteins such as luciferase, Renilla reniformis luciferase, ⁇ -galactosidase, chloramphenicol acetyltransferase, alkaline phosphatase, ⁇ -glucuronidase, luminescence such as green fluorescent protein (GFP) Examples include a gene encoding a protein.
- GFP green fluorescent protein
- reporter genes may be those contained in commercially available reporter plasmids.
- a preferred reporter gene includes, for example, the luc2 gene.
- the luc2 gene is a gene encoding a modified luciferase derived from firefly luciferase.
- the luc2 gene is not particularly limited as long as it is a gene consisting of a base sequence encoding a protein having luciferase activity.
- the luc2 gene not only the nucleotide sequence (SEQ ID NO: 8) of the plasmid shown in FIG. 1 but also the portion indicated as luc2 gene1sequence (luc2 gene sequence), Those in which about several nucleotides are added, deleted or substituted are also included.
- the luc2 gene also includes a base sequence encoding an amino acid sequence in which a tag sequence, a signal sequence, etc. are added to the N-terminal or C-terminal of the amino acid sequence encoded by the base sequence represented by SEQ ID NO: 8.
- Specific examples include the luc2P gene, which is a modified product that is modified so that the amount of luciferase protein accumulated in the cell is reduced by adding a proteolytic sequence (hPEST) to the C-terminus.
- the luc2P gene is not limited to the nucleotide sequence (SEQ ID NO: 19) of the portion shown as luc2P gene sequence (luc2P gene sequence) in the plasmid shown in FIG.
- SEQ ID NO: 19 the nucleotide sequence of the portion shown as luc2P gene sequence (luc2P gene sequence) in the plasmid shown in FIG.
- a base sequence encoding a protein having about one nucleotide added, deleted or substituted and having luciferase activity is also included.
- the luc2 gene includes those obtained by modifying the base sequence represented by SEQ ID NO: 8 or 19 so as to further increase the expression level of luciferase.
- the expression level of luciferase (for example, the base sequence represented by SEQ ID NO: 8 or SEQ ID NO: 19 and a modified firefly luciferase thereof) is, for example, a reporter plasmid containing a luciferase gene first in a host cell (for example, described later).
- Basal level luciferase in the same manner as above except that a positive control substance (eg, adriamycin) is added and the expression level of the luciferase gene is measured, and then no positive control substance is added.
- the gene expression level can be measured and evaluated by taking the ratio of these expression levels (S / N ratio).
- a positive control substance is brought into contact with a host cell into which a reporter plasmid containing a luciferase gene has been transiently or stably introduced, and then washed with PBS ( ⁇ ) or the like, Add a cell lysing agent into the cell to lyse the cells, add a luciferase substrate (for example, luciferin if the luciferase is firefly luciferase) to the cell lysate, and use a luminometer To quantify (positive control substance added group). Next, the expression level of the basal level luciferase gene is quantified in the same manner as described above except that no positive control substance is added (control group).
- a luciferase substrate for example, luciferin if the luciferase is firefly luciferase
- the expression level of luciferase can be evaluated by calculating the ratio of the luminescence level (expression level) of the positive control substance added group to the control group.
- the S / N ratio when using a variant is higher than the S / N ratio when using luciferase, it can be evaluated that the expression level has increased due to the modification.
- the recombinant vector used in the present invention may contain a cell selectable marker gene that can function in mammalian cells so that the retention of the recombinant vector in the cell can be confirmed.
- a “cell selection marker gene” is a gene that encodes a phenotype that can serve as a mark when distinguishing between cells transformed with DNA containing the gene and non-transformed cells. “Functional in animal cells” means that the trait can be expressed in animal cells, and “genes capable of functioning in animal cells” mean that under the control of a promoter capable of initiating transcription in animal cells. It means a gene that is in a state that can be expressed in animal cells.
- cell selection marker genes effective in animal cells include genes that can confer resistance to drugs that suppress or inhibit the growth of animal cells.
- specific examples include a neomycin resistance-conferring gene (aminoglycoside phosphotransferase gene), a hygromycin resistance-conferring gene (hygromycin phosphotransferase gene), and a blasticidin S resistance-conferring gene (blastcidin S deaminase gene).
- a blasticidin S resistance-conferring gene is preferable in that selection of transformed cells can be performed in a shorter period of time.
- the recombinant vector used in the present invention comprises a p53 binding sequence, a minimal promoter that can function in mammalian cells, and a reporter gene linked so that it can be expressed by this minimal promoter. That is, a reporter gene is linked to the downstream of a transcriptional regulatory region containing a p53 binding sequence and a minimal promoter so as to allow expression. Either the transcription factor binding sequence or the minimal promoter may be present upstream.
- Examples of the recombinant vector include a vector in which the above-described luc2 gene sequence is linked as a reporter gene.
- -1 to several nucleotides may exist between the p53 binding sequence and the minimal promoter. Further, between the transcriptional regulatory region including the p53 binding sequence and the minimal promoter and the reporter gene, about 1 to several nucleotides may be present within a range in which the reporter gene can be expressed.
- a base sequence encoding a peptide expressed together with the reporter protein may be present upstream or downstream of the reporter gene.
- Such peptides include His-tag, myc-tag, FLAG-tag, HA-tag and other tag sequences; p53 binding sequences, a minimal promoter that can function in mammalian cells, and expression by this minimal promoter.
- Examples thereof include a signal sequence such as a recombinant vector (for example, hPEST sequence) containing a linked reporter gene, a nuclear translocation signal sequence (for example, NLS sequence), and a secretory signal sequence.
- the recombinant vector used in the present invention uses a commercially available expression vector for mammalian cells having a minimal promoter and a gene insertion site downstream thereof, and a p53 binding sequence and a reporter gene are contained in this vector. It may be inserted.
- Examples of such commercially available mammalian expression vectors include pUC18 and pUC19.
- the recombinant vector used in the present invention may be one in which a p53 binding sequence is inserted into a vector that is commercially available as a commercially available reporter plasmid.
- reporter plasmids examples include luciferase plasmids pGL4-Luc2, pGL4-Luc2P, pT81lu, pSluc2, and pGL3.
- the recombinant vector used in the present invention is preferably a pGL4-Luc2 plasmid in which the p53-binding sequence and the minimal promoter are inserted, or a pGL4-Luc2P plasmid in which the p53-binding sequence and the minimal promoter are inserted.
- the pGL4-Luc2 plasmid and the pGL4-Luc2P plasmid are commercially available from Promega.
- Examples of the pGL4-Luc2 plasmid include a plasmid consisting of the base sequence represented by SEQ ID NO: 9 (pGL4.10 plasmid).
- the base sequence represented by SEQ ID NO: 9 is shown in FIGS.
- the pGL4-Luc2 plasmid used as a raw material for the recombinant vector in the present invention includes a plasmid in which about 1 to several nucleotides are added, deleted or substituted in the base sequence represented by SEQ ID NO: 9.
- examples of the pGL4-Luc2P plasmid include a plasmid consisting of the base sequence represented by SEQ ID NO: 20 (pGL4.11 plasmid).
- the base sequence represented by SEQ ID NO: 20 is shown in FIG.
- the pGL4-Luc2P plasmid used as a raw material for the recombinant vector in the present invention includes those in which about 1 to several nucleotides are added, deleted or substituted in the base sequence represented by SEQ ID NO: 20.
- the pGL4-Luc2P plasmid is a plasmid containing a base sequence encoding an hPEST sequence downstream of the coding region of the reporter protein in the pGL4-Luc2 plasmid.
- the plasmid was modified so that the amount of luciferase protein accumulated in the cell decreased by adding a proteolytic sequence (hPEST) to the C terminus of the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 8. Encodes the variant (luc2P gene).
- hPEST proteolytic sequence
- the recombinant vector used in the present invention is preferably compact in size because it is easy to handle and the frequency of gene recombination within a vector molecule or between molecules or dropout from a chromosome in a stably transformed cell is reduced.
- the size is preferably about 2 to 10 kb.
- the recombinant vector used in the present invention is not only a full-length plasmid but also a part thereof as long as it contains a minimal promoter that can function in mammalian cells and a reporter gene linked so that it can be expressed by this minimal promoter. It may be a gene construct.
- the recombinant vector of the present invention can be suitably used as a recombinant vector for preparing a transformant to be subjected to a mutagenicity test described later.
- the transformant of the present invention is a cell carrying the above-described recombinant vector of the present invention.
- the transformant of the present invention may temporarily hold the above recombinant vector or may hold it stably.
- the host cell into which the recombinant vector is introduced is not particularly limited as long as it is a mammal.
- a mammalian cell in which wild-type p53 is expressed and functions normally can be used, but even if it is an abnormal cell in which p53 function is reduced, deleted or mutated, the wild-type p53 gene If p53 is expressed by separately introducing p53, it can be used.
- p53 gene expression is further introduced by introducing another wild-type p53 gene. What is necessary is just to use it, increasing the quantity.
- established cell lines that can be stably passaged are preferred. Such cell lines are not particularly limited and known ones can be used. As an established cell line that normally expresses p53 and can be stably passaged, the relationship between DNA damage and p53 activation in humans can be reproduced, and the mutagenicity of test substances against human cells can be accurately evaluated. Although human-derived cells are preferably used, human lymphoblast-derived TK6 cells are particularly preferable. In particular, the combination of the above-described recombinant vector using the pGL4-Luc2 plasmid or the pGL4-Luc2P plasmid and TK6 cells is preferable because the contact time with the test substance can be greatly shortened.
- TK6 cells are represented by ATCC number CRL-8015.
- ATCC number CRL-8015 ATCC number CRL-8015.
- the recombinant vector of the present invention may be transiently or stably introduced into a host cell.
- “Stable introduction” can also be said to be “inserted into the genome of a mammalian cell” or “integrated into the genome of a mammalian cell”. It is preferable to introduce stably because it is not necessary to consider the transformation rate.
- a stably expressing cell in which a vector is stably introduced into a host cell is preferable from the viewpoint of work efficiency and reproducibility of mutagenicity tests.
- Vector introduction methods are already well known. Specifically, for example, about 10 5 to 10 7 host cells are first seeded in a petri dish having a diameter of about 6 to 10 cm, and 5% CO 2 is used using a MEM medium containing about 5 to 10% of serum. It can be cultured for several hours to overnight at 37 ° C. under saturated humidity conditions. What is necessary is just to introduce
- the amount of DNA to be introduced is preferably 0.1 to 1.0 ng, more preferably 0.2 to 0.5 ng per 10 5 cells.
- the DNA introduction method general methods such as electroporation method, calcium phosphate method, lipofection method and the like can be adopted without limitation. From the viewpoint of easy operation and reproducibility, the electroporation method is preferable.
- the transformant When the vector is introduced by these methods, the transformant is usually in a state where the reporter gene can be transiently expressed. Therefore, the transformant may be subjected to a mutagenicity test immediately after DNA introduction.
- cells in which the expression vector is stably expressed can be prepared by means known to those skilled in the art.
- cells stably transfected with a recombinant vector can be selected by the following method. After introduction of the vector, for example, a host into which the recombinant vector has been appropriately introduced, using as an index the activity of a cell selection marker gene such as drug resistance or the activation of a reporter gene by a positive control substance or the presence of these genes can be detected What is necessary is just to select a cell.
- a cell in which the luciferase activity is recognized at least twice the basal level when a positive control substance is added is regarded as a cell in which the reporter gene is activated.
- a means using a virus vector capable of integrating a transgene into the genome of a mammalian cell such as a retrovirus vector or a lentivirus vector, can also be mentioned. Specific methods for implementing such means are known to those skilled in the art.
- the introduction amount of the recombinant vector is preferably 0.1 to 1.0 (ng / well) and more preferably 0.2 to 0.5 (ng / well). preferable.
- the transformant of the present invention can be suitably used as a cell for a mutagenicity test described later.
- a TK6 cell that temporarily retains a recombinant vector comprising a p53 binding sequence, a minimal promoter that can function in mammalian cells, and a luc2 gene linked so as to be expressed by the minimal promoter;
- B a TK6 cell that stably holds a recombinant vector comprising a p53 binding sequence, a minimal promoter that can function in mammalian cells, and a luc2 gene linked so as to be expressed by the minimal promoter;
- C a TK6 cell that temporarily retains a recombinant vector comprising a p53 binding sequence, a minimal promoter that can function in mammalian cells, and a luc2P gene linked so that it can be expressed by the minimal promoter;
- D A TK6 cell stably retaining a recombinant vector comprising a p53 binding sequence,
- the mutagenicity test method of the present invention comprises: (1) a step of culturing the transformant described above in the presence of a test substance; and (2) an expression level of a reporter gene in the transformant contacted with the test substance and a transformant that does not contact the test substance. And comparing the expression level of the reporter gene to determine whether the test substance affects the expression level of the reporter gene in the transformant.
- Test substance The type of test substance is not particularly limited. In addition to a low molecular compound, a high molecular compound, etc., for example, it may be a mixture of a plurality of types of substances such as foods, processed foods, wastes, and waste incinerators.
- a transformant to be cultured in the presence of a test substance is first seeded in a cell culture container and cultured for about 8 hours to overnight (in the case of using floating cells such as TK6 cells, 0.5% It is preferable to culture for about 1 hour.
- the culture temperature may be a temperature suitable for the cell, usually about 36 to 38 ° C., preferably about 37 ° C.
- the culture solution is not particularly limited, and a culture solution suitable for cells may be used.
- the number of cells is not particularly limited. For example, when a 96-well plate is used, about 1 ⁇ 10 4 to 1 ⁇ 10 5 cells per well are usually seeded.
- a test substance is added to the cell culture medium.
- the test substance is added as it is or dissolved in a solvent.
- a solvent for example, distilled water, a buffer solution such as PBS ( ⁇ ) that does not affect the cells, dimethyl sulfoxide (DMSO), ethanol, methanol and the like can be used.
- the test substance may be added so that the final concentration in the cell culture medium is usually about 0.1 to 1% by weight.
- a buffer solution such as PBS (-) that does not affect cells
- an organic solvent such as methanol.
- the concentrated product may be added so that the final concentration is usually about 0.1 to 1% by weight.
- the culture After adding the test substance to the cell culture medium, the culture is continued preferably in the same temperature range as described above, usually about 4 to 10 hours, preferably about 5 to 9 hours. In one embodiment of the present invention, the culture can be performed for about 6 to 10 hours, preferably about 7 to 9 hours.
- step (1) A step of culturing the transformant described above for about 4 to 10 hours in the presence of a test substance.
- the expression level of the reporter gene is compared between the transformant contacted with the test substance and the transformant not contacted.
- the specific aspect of a process (2) is illustrated.
- the expression level of the reporter gene in both cells can be measured, and both measured values can be compared, or the reporter gene expression level in both cells can be directly qualitatively compared visually.
- the expression level of the reporter gene to be compared between the transformant contacted with the test substance and the transformant not contacted is the expression level of another standard gene of the measured reporter gene expression level (internal It may be a relative value with respect to (standard).
- Such an expression level of the reporter gene is also referred to as an expression level of the reporter gene normalized or corrected with the standard gene.
- each reporter gene may be measured by a method according to the type of reporter gene. For example, when the reporter gene is a firefly luciferase gene, each cell is washed with PBS ( ⁇ ) or the like, then a cell lysing agent containing a surfactant is added to lyse the cell, and luciferin, which is a luciferase substrate, is added to the cell.
- the luciferase activity can be measured by adding to the lysate and quantifying the amount of luminescence due to the decomposition of luciferin using a luminometer.
- the reporter assay can be performed manually, but it should be performed quickly and easily by using a so-called high-throughput screening (tissue culture engineering, vol123, No.13,521-524, US Pat. No. 5,670,113) that is performed automatically using a machine. Can do.
- the standard gene is not particularly limited as long as it is a gene different from the reporter gene.
- specific examples of the standard gene include a gene endogenous to a cell such as a housekeeping gene (eg, GADPH gene, ⁇ -actin gene, etc.), or a known foreign reporter gene that is different from the reporter gene.
- a reporter gene is used as a standard gene, it is preferably a reporter gene linked so that it can be expressed by a promoter that functions constantly in mammalian cells, such as SV40 promoter, TK promoter, CMV promoter.
- the standard gene is preferably a gene whose expression level can be measured by the same method as the reporter gene of the present invention.
- the reporter gene of the present invention is a firefly-derived luciferase or a variant thereof
- a luciferase derived from other than firefly particularly a luciferase gene encoding a luciferase protein whose substrate is other than luciferin can be used.
- Examples of such a luciferase gene include a luciferase gene derived from a sea urchin bamboo (Sea pansy, scientific name Renilla reniformis).
- the Renilla luciferase gene includes a nucleotide sequence encoding a protein having luciferase activity, in which about 1 to several nucleotides are added, deleted or substituted in the wild-type nucleotide sequence. Renilla luciferase usually uses coelenterazine as a substrate.
- a transformant that expresses the standard gene of the present invention is prepared according to the method of preparing a transformant that holds a recombinant vector containing the reporter gene of the present invention.
- the transformant of the present invention is, for example, a transformant holding a recombinant vector containing the reporter gene of the present invention and a recombinant vector containing the standard gene.
- the transformant of the present invention temporarily holds the recombinant vector containing the reporter gene of the present invention
- the recombinant vector containing the standard gene also temporarily contains the reporter gene of the present invention.
- the recombinant vector is stably retained, it is preferable that the recombinant vector containing the standard gene is also stably retained.
- the reporter gene expression level per cell of the transformant contacted with the test substance is not contacted with the reporter gene per cell of the transformant.
- the test substance concentration is significantly and significantly increased compared to the expression level, it can be determined that the test substance is a mutagenic substance or a carcinogenic substance.
- the criteria for determining whether or not there is a significant increase can be appropriately set based on a significant difference test using a known carcinogen, for example, a reporter gene per cell of a transformant contacted with a test substance
- a known carcinogen for example, a reporter gene per cell of a transformant contacted with a test substance
- the expression level is 130% or more in TK6 cells compared to the expression level of the reporter gene per cell of the transformant that is not contacted, it can be determined that the test substance is a mutagenic substance or a carcinogenic substance.
- the reporter gene expression level per cell of the transformant contacted with the test substance is not the contact reporter per cell of the transformant. If the concentration of the test substance is significantly decreased compared to the gene expression level, it can be determined that the test substance is a mutagenic substance or a carcinogenic substance.
- Judgment criteria for whether or not there is a significant decrease can be appropriately set based on a significant difference test using a known carcinogen, for example, a reporter gene per cell of a transformant contacted with a test substance
- a known carcinogen for example, a reporter gene per cell of a transformant contacted with a test substance
- the test substance can be determined to be a mutagenic substance or a carcinogenic substance.
- an antimutagenic substance or an anticarcinogenic substance can also be selected by bringing a test substance into contact with a transformant together with a known mutagenic substance. That is, the reporter gene expression level of the control transformant, the expression level of the reporter gene of the transformant contacted only with the mutagenic substance, and the reporter gene of the transformant contacted with the mutagenic substance and the test substance Compared with the expression level, the amount of change in reporter gene expression due to contact with the mutagenic substance is decreased by further contact with the test substance, preferably with respect to the reporter gene expression level of the control transformant When it does not increase significantly, it can be determined that the test substance is an antimutagenic substance or an anticarcinogenic substance. Examples of such substances include antioxidant substances.
- the aspect of using the relative value with respect to the expression level of another standard gene as the expression level of the reporter gene is one of the particularly preferable aspects of the present invention from the viewpoint of improving the accuracy of determination.
- a mutagenicity test on the test substance is performed by the method of the present invention.
- the mutagenicity test method of the present invention comprises: A mutagenicity test kit comprising a TK6 cell harboring a recombinant vector comprising a p53 binding sequence, a minimal promoter capable of functioning in mammalian cells, and a luc2 gene linked so as to be expressed by the minimal promoter. .
- a mutagenicity test kit comprising a TK6 cell harboring a recombinant vector comprising a p53 binding sequence, a minimal promoter capable of functioning in mammalian cells, and a luc2 gene linked so as to be expressed by the minimal promoter.
- this kit is not particularly limited.
- appropriate reagents and various containers can be included.
- Specific examples include a medium for culturing cells, a reagent for measuring luciferase activity (a reagent for lysing cells, a substrate for firefly luciferase, etc.) and a container for appropriately storing this;
- tools such as petri dishes for cell culture.
- the kit of the present invention may include a procedure manual including a description relating to the procedure for carrying out the mutagenicity test of the present invention.
- the procedure manual preferably describes that the culture is continued for about 4 to 10 hours, preferably about 5 to 9 hours after contacting the cells with the test substance.
- Example 1 (Preparation of recombinant vector) A nucleotide sequence (5'-CTGACATGCCCAGGCATGTCTTGACATGCCCAGGCATGTCTTGACATGCCCAGGCATGTCTA-3 ': SEQ ID NO: 10) containing a p53-binding sequence of the p53R2 gene intron expressed by DNA damage, and a complementary base sequence (5' Oligonucleotides consisting of -GATCTAGACATGCCTGGGCATGTCAAGACATGCCTGGGCATGTCAAGACATGCCTGGGCATGTCAGGTAC-3 ': SEQ ID NO: 11) were respectively synthesized by a DNA synthesizer and annealed to obtain double-stranded DNA.
- Plasmid Mini Mini kit QIAGEN
- RVprimer3 primer Promega
- a DNA sequence (ABI PrismTABigDye Terminater v3.0; manufactured by Applied Biosystems) was confirmed using the designed reverse primer (5'-CTTAATGTTTTTGGCATCTTCCA-3 ': SEQ ID NO: 12).
- the reporter plasmid in which the p53 binding sequence was correctly inserted was named pGL4-p53R2x3-Luc2.
- Example 2 (Preparation of recombinant vector; repertoire of promoter; preparation of reporter plasmid containing SV40 minimal promoter) After digesting the plasmid PGV-p53R2x3-Luc prepared by the method described in Japanese Patent No. 4243716, Example 1 with restriction enzymes BglII and HindIII, electricity using low melting point agarose (NuSieve 3: 1 Agarose; manufactured by BMA) was used.
- the target 198 bp DNA was recovered from the gel of the band part, and was named as SV40 minimal promoter (5'-TGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTATGTTTTATTTATGCACTTGTTCCTT
- the reporter plasmid pGL4-p53R2x3-Luc2 prepared in Example 1 was digested with restriction enzymes BglII and HindIII, and then subjected to electrophoresis using low melting point agarose, and the target 4257 bp DNA was recovered from the gel of the band part. About 3 ng of this DNA was mixed with 10 ng of the above-mentioned SV40 minimal promoter DNA and bound with T4 ligase, and then this reaction solution was introduced into an E. coli M JM109 competent cell.
- each plasmid was purified from several colonies of Escherichia coli showing ampicillin resistance using PlasmidPMini kit (manufactured by QIAGEN), and the DNA sequence was confirmed using RVprimer3 primer and reverse primer.
- the reporter plasmid in which the SV40 minimal promoter sequence was correctly inserted was named pGL4-p53R2x3-SV40-Luc2.
- Example 3 (Preparation of recombinant vector; repertoire of promoter; preparation of reporter plasmid containing tk minimal promoter) After digesting plasmid PGV-p53R2x3-tk-Luc prepared by the method described in Patent No. 4243716, Example 3 with restriction enzymes BglII and HindIII, low melting point agarose (NuSieve 3: 1 Agarose; manufactured by BMA) is used.
- the target 181 bp DNA was collected from the gel of the band portion, and named the tk minimal promoter (5'-TAAGAAAATATATTTGCATGTCTTTAGTTCTATGATGACACAAACCCCGCCCAGCGTCTTGTCATTGGGGAATTCGAACACGACGCCGCACGCGTCGGCTCTCACGCGTCG;
- the reporter plasmid pGL4-p53R2x3-Luc2 prepared in Example 1 was digested with restriction enzymes BglII and HindIII, and then subjected to electrophoresis using low melting point agarose, and the target 4257 bp DNA was recovered from the gel of the band part. About 3 ng of this DNA was mixed with 10 ng of the above-mentioned tk minimal promoter DNA and bound with T4 ligase, and then this reaction solution was introduced into an E. coli JM109 competent cell.
- each plasmid was purified from several colonies of Escherichia coli showing ampicillin resistance using PlasmidPMini kit (manufactured by QIAGEN), and the DNA sequence was confirmed using RVprimer3 primer and reverse primer.
- the reporter plasmid in which the tk minimal promoter sequence was correctly inserted was named pGL4-p53R2x3-tk-Luc2.
- Example 4 (Preparation of recombinant vector; repertoire of promoter; preparation of reporter plasmid containing TATA minimal promoter)
- An oligonucleotide consisting of a base sequence (5′-GATCTCGACTATAAAGAGGGCAGGCTGTCCTCTAAGCGTCACCACGACTTCA-3 ′: SEQ ID NO: 15) containing a TATA minimal promoter, and a base sequence complementary to this base sequence (5′-AGCTTGAAGTCGTGGTGACGCTTAGAGGACAGCCTGCCCTCTTTATAGTCGA-3 ′: SEQ ID NO: 16)
- Oligonucleotides were synthesized with a DNA synthesizer and annealed to form double-stranded DNA, which was named the TATA minimal promoter.
- the reporter plasmid pGL4-p53R2x3-Luc2 prepared in Example 1 was digested with restriction enzymes BglII and HindIII, and then subjected to electrophoresis using low melting point agarose, and the target 4257 bp DNA was recovered from the gel of the band part. About 3 ng of this DNA was mixed with 10 ng of the above-mentioned TATA minimal promoter DNA and ligated with T4 ligase, and then this reaction solution was introduced into an E. coli M JM109 competent cell.
- each plasmid was purified from several colonies of Escherichia coli showing ampicillin resistance using PlasmidPMini kit (manufactured by QIAGEN), and the DNA sequence was confirmed using RVprimer3 primer and reverse primer.
- the reporter plasmid in which the TATA minimal promoter sequence was correctly inserted was named pGL4-p53R2x3-TATA-Luc2.
- Example 5 Reactivity of pGL4-p53R2 ⁇ 3-SV40-Luc2
- the plasmid pGL4-p53R2 ⁇ 3-SV40-Luc2 prepared in Example 2 was transiently introduced into TK6 cells by electroporation. Next, using a medium containing 10% fetal bovine serum (FBS) in RPMI1640 medium (manufactured by Nissui Pharmaceutical Co., Ltd.), a cell density of 70 to 96 in a 96-well plate for luciferase luminescence measurement and culture (Corning # 3610) It seed
- FBS fetal bovine serum
- RPMI1640 medium manufactured by Nissui Pharmaceutical Co., Ltd.
- Adriamycin which is known to intercalate with DNA and cleave DNA, was brought into contact with this transformant to confirm whether the amount of luciferase production increased. More specifically, the obtained transformant cells were divided into 7 groups, of which 6 groups had final adriamycin dissolved in DMSO of 0.003 ⁇ g / mL, 0.01 ⁇ g / mL, 0.03 ⁇ g / mL, 0.1 It added so that it might become microgram / mL, 0.2 microgram / mL, and 0.7 microgram / mL (final DMSO density
- DMSO having the same volume as that of the adriamycin solution was added to serve as a negative control.
- these cells were cultured at 37 ° C. for 4, 8 and 16 hours.
- 25 ⁇ l of a cell lysate (manufactured by Promega) was added / welled, and left at room temperature for 15 minutes.
- This plate was set in a luminometer LB96P (manufactured by Bertoled) with an enzyme substrate automatic injector, and 30 ⁇ L / well of substrate solution (manufactured by Promega) was automatically dispensed to measure luciferase activity.
- luciferase activity was measured in the same manner as above except that the plasmid PGV-p53R2x3-Luc prepared in Example 1 was used instead of pGL4-p53R2 ⁇ 3-SV40-Luc2 did.
- Example 6 Comparison between different human-derived cells
- the plasmid pGL4-p53R2 ⁇ 3-SV40-Luc2 prepared in Example 2 was transiently introduced into the TK6 cells.
- a medium containing 10% fetal bovine serum (FBS) in RPMI1640 medium (manufactured by Nissui Pharmaceutical Co., Ltd.)
- FBS fetal bovine serum
- RPMI1640 medium manufactured by Nissui Pharmaceutical Co., Ltd.
- a cell density of 70 to 96 in a 96-well plate for luciferase luminescence measurement and culture (Corning # 3610) is used. It seed
- the amount of pGL4-p53R2 ⁇ 3-SV40-Luc2 to be introduced was adjusted to be 0.25 ng / well.
- human breast cancer cell line MCF-7 human liver cancer cell line HepG2, human lung cancer cell line A549, human colon cancer cell line HCT116, human fetal kidney cell line HEK293, and human skin-derived fibroblasts in which wild-type p53 is normal
- cells were seeded in a 96-well plate for luciferase luminescence measurement and culture (Corning # 3610) and cultured overnight at 37 ° C using a medium containing 10% fetal bovine serum (FBS). did.
- FBS fetal bovine serum
- TK6 cells Using the obtained transformants of MCF-7 cells, HepG2 cells, A549 cells, HCT116 cells, HEK293 cells, and NB1RGB cells, luciferase activity was measured in the same manner as the above TK6 cells. The results are shown in FIG. In TK6 cells into which pGL4-p53R2 ⁇ 3-SV40-Luc2 was introduced, very high luciferase activity was observed after 8 hours of contact with adriamycin. In contrast, MCF-7 cells, HepG2 cells, A549 cells, HCT116 cells, HEK293 cells, and NB1RGB cells showed only a slight luciferase activity after 8 hours of contact with adriamycin.
- Example 7 (Preparation of recombinant vector; repertoire of reporter gene; preparation of reporter plasmid containing luc2P gene) Except for using pGL4.11 (manufactured by Promega) as a plasmid containing the firefly luciferase gene, the same procedure as in Examples 1 and 2 was performed, and the plasmid was named reporter plasmid pGL4-p53R2x3-SV40-Luc2P.
- Example 8 Construction of reporter gene stably expressing cells
- a medium containing fetal bovine serum (FBS) at a final concentration of 10% in RPMI1640 medium (manufactured by Nissui Pharmaceutical Co., Ltd.), seed the cells in a 96-well plate (Corning # 3595) at 1 cell / well. did.
- FBS fetal bovine serum
- the culture is continued for one month while appropriately changing the medium, and the obtained cell colonies are cultured in a 96-well plate (Corning # 3595) for culturing and for luciferase luminescence measurement and culturing for luciferase assay.
- the plate was divided into 96-well plates (Corning # 3610) and further cultured for 24 hours.
- luciferase activity was measured for the cells divided for luciferase assay according to the method of Example 5, and the cell in which luciferase activity was observed was named TK6 / p53R2x3-Luc2P.
- Example 9 (Reactivity of TK6 / p53R2x3-Luc2P)
- the stable expression cell TK6 / p53R2x3-Luc2P prepared in Example 8 is used for medium for luciferase luminescence measurement and culture using a medium containing 10% fetal bovine serum (FBS) in RPMI1640 medium (manufactured by Nissui Pharmaceutical).
- FBS fetal bovine serum
- RPMI1640 medium manufactured by Nissui Pharmaceutical
- Adriamycin dissolved in DMSO was added to a final concentration of 0.2 ⁇ g / mL (final DMSO concentration 0.3% (v / v)).
- the cells were cultured at 37 ° C. for 2, 4, 6 and 8 hours. Subsequently, after each culturing time, luciferase activity was measured according to the method of Example 5.
- Example 10 Reactivity of TK6 / p53R2x3-Luc2P
- the stably expressing cells TK6 / p53R2x3-Luc2P prepared in Example 8 were confluent at a cell density of 70-80% in a 96-well plate for luciferase luminescence measurement and culture (Corning # 3610). Sowing.
- Adriamycin was brought into contact with this transformant, and it was confirmed whether or not the amount of luciferase production increased. More specifically, the obtained transformant cells were divided into 7 groups, of which 6 groups had final adriamycin dissolved in DMSO of 0.003 ⁇ g / mL, 0.01 ⁇ g / mL, 0.03 ⁇ g / mL, 0.1 It added so that it might become microgram / mL, 0.2 microgram / mL, and 0.7 microgram / mL (final DMSO density
- Example 2 the same procedure was performed except that the plasmid pGL4-p53R2 ⁇ 3-SV40-Luc2 prepared in Example 2 was transiently introduced into TK6 cells and cultured at 37 ° C. for 8 hours after addition of adriamycin or DMSO. The luciferase activity was measured.
- Example 11 Construction of cells stably expressing the internal standard gene
- the cell TK6 / p53R2x3-Luc2P prepared in Example 8 was transiently treated with the pRL-SV40-Rluc plasmid (plasmid having Renilla luciferase (Rluc) ligated downstream of the SV40 promoter) according to the method of Example 8. Introduced. Next, according to the method of Example 8, cells stably expressing Luc2P and Rluc were obtained and named TK6 / p53R2x3-Luc2P / Rluc.
- Example 12 (Correction by internal standard) The cells TK6 / p53R2x3-Luc2P / Rluc prepared in Example 11 were seeded in a 96-well plate for luciferase luminescence measurement / culture according to the method of Example 9.
- 5-aza-2'-deoxycytidine was brought into contact with the above two types of cells. Specifically, each of the obtained cells was divided into 7 groups, and 6 groups of them were respectively prepared with 0.1 ⁇ g / mL, 0.2 ⁇ g / mL, and 0.7 ⁇ g / mL of 5-aza-dC dissolved in DMSO. It added so that it might become mL, 2.2 microgram / mL, 6.7 microgram / mL, and 20 microgram / mL (final DMSO density
- the expression level of Luc2P gene was determined as a relative value to the expression level of Rluc gene. Specifically, using a luminometer LB96P (Berthold) with an automatic enzyme substrate injector, the expression level of the Luc2P gene was measured by first dispensing 30 ⁇ L of a substrate solution for firefly luciferase measurement (manufactured by Promega) in a pothole. Thereafter, the expression level of the Rluc gene was determined by further dispensing 30 ⁇ L / well of a Renilla luciferase measurement substrate solution (Promega).
- 5-aza-dC is known to induce DNA demethylation by inhibiting the activity of DNA methyltransferase and increase the transcriptional activity of cells.
- mutagenicity is not great. Therefore, it can be seen that the accuracy of the mutagenicity test of the present invention can be improved by correcting the Luc2P activity with the internal standard.
- Example 1 Plasmid PGV-p53R2x3-Luc prepared by the method described in Japanese Patent No. 4243716, Example 1 was transiently introduced into TK6 cells. Next, using a medium containing 10% fetal bovine serum (FBS) in RPMI1640 medium (manufactured by Nissui Pharmaceutical Co., Ltd.), a cell density of 70 to 96 in a 96-well plate for luciferase luminescence measurement and culture (Corning # 3610) is used. Seeding was carried out to 80%.
- FBS fetal bovine serum
- RPMI1640 medium manufactured by Nissui Pharmaceutical Co., Ltd.
- cells were seeded in a 96-well plate for luciferase luminescence measurement and culture (Corning # 3610) and cultured overnight at 37 ° C. using a medium containing fetal bovine serum (FBS) at a final concentration of 10%.
- FBS fetal bovine serum
- Reference example 2 The culture time of the transformant was the same as in Reference Example 1 except that the culture time was 16 hours for TK6 cells, 24 hours for MCF-7, HepG2, A549, HCT116, and HEK293, and 18 hours for NB1RGB. Then, plasmid PGV-p53R2x3-Luc was introduced, cultured, and luciferase activity was measured. The results are shown in FIG.
- Patent Document 1 The technology related to Patent Document 1 developed by the present inventors has been awarded the Japan Toxicology Society Award. This award is given to young researchers who are members of the Society who have made excellent contributions such as the practical application of newly developed technology in the development of toxicity assessment technology, and this technology is awarded to only three people a year. Highly evaluated.
- the present invention further improves the technique according to Patent Document 1 and greatly shortens the test time, and thus is very useful in the industry.
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Abstract
Description
本出願は、2011年10月11日に出願された、日本国特許出願第2011-224292号明細書(その開示全体が参照により本明細書中に援用される)に基づく優先権を主張する。
項1.(1) p53結合配列と、哺乳動物細胞で機能可能な最小プロモーターと、この最小プロモーターにより発現できるように連結されたレポーター遺伝子とを含む組み換えベクターを保持する形質転換体を、被験物質の存在下で4~10時間培養する工程、及び
(2) 上記工程(1)で培養した形質転換体におけるレポーター遺伝子の発現量と被験物質を接触させない形質転換体におけるレポーター遺伝子の発現量とを比較することにより、被験物質が形質転換体におけるレポーター遺伝子の発現量に影響を及ぼすか否かを判定する工程
を含む変異原性試験方法。
5'-WRRCWWGYYY -3':配列番号1
に示す塩基配列(配列番号1中、Rはプリン塩基を含むヌクレオチドを示し、Wはアデニン又はチミンを含むヌクレオチドを示し、Yはピリミジン塩基を含むヌクレオチドを示す)を有するものである項1~5のいずれか一項に記載の方法。
5'-WRRCWWGYYY WRRCWWGYYY -3':配列番号2
に示す塩基配列(配列番号2中、Rはプリン塩基を含むヌクレオチドを示し、Wはアデニン又はチミンを含むヌクレオチドを示し、Yはピリミジン塩基を含むヌクレオチドを示す)を有するものである項1~6のいずれか一項に記載の方法。
(I)組み換えベクター
本発明の方法で用いる形質転換体は、p53結合配列と、哺乳動物細胞で機能可能な最小プロモーターと、この最小プロモーターにより発現できるように連結されたレポーター遺伝子とを含む組み換えベクターを保持している。
p53蛋白質は、化学物質、紫外線又はガンマ線などによるDNA損傷時に、特定部位がリン酸化されることにより活性化し、特定遺伝子群の転写調節領域に結合して、その転写を調節する機能を有する転写因子である。
哺乳動物細胞で発現可能な最小プロモーターは、コアプロモーターともいわれ、通常、遺伝子の転写開始部位付近の比較的狭い部分にみられる領域である。また、最小プロモーターは、転写調節に関わる主たる機能性エレメントであり、例えば他の転写調節因子の認識配列のような転写調節に関わる他の機能性エレメントを含まないか、または、このような機能性エレメントを含んでいてもそれがp53結合配列と最小プロモーターとによる転写調節機能を本質的に変化させることのない配列である。
本発明において、レポーター遺伝子とは、その遺伝子の発現を定性的又は定量的に確認できる遺伝子をいう。レポーター遺伝子としては、例えば公知のレポーター遺伝子を広く使用できる。特に、発現量の測定が容易である点で、その転写産物(レポーター蛋白質)の有する酵素活性等に基づいて発現量を測定できる遺伝子が好ましく、このようなレポーター遺伝子として、例えばホタル(Photinus pyralis)ルシフェラーゼ、ウミシイタケ(Renilla reniformis)ルシフェラーゼ、β-ガラクトシダーゼ、クロラムフェニコールアセチルトランスフェラーゼ、アルカリホスファターゼ、β-グルクロニダーゼのような酵素蛋白質をコードする遺伝子、緑色蛍光蛋白質(GFP :green fluorescent protein)のような発光蛋白質をコードする遺伝子等が挙げられる。
本発明で用いる組み換えベクターは、細胞における組み換えベクターの保持を確認できるように、哺乳動物細胞で機能可能な細胞選択マーカー遺伝子が含まれていてよい。「細胞選択マーカー遺伝子」とは、その遺伝子を含むDNAで形質転換された細胞と非形質転換細胞とを見分ける際に目印となり得る表現形質をコードする遺伝子である。「動物細胞で機能可能な」とは、動物細胞で前記形質を発現することができることを意味し、「動物細胞で機能可能な遺伝子」とは、動物細胞で転写開始能を有するプロモーターの制御下にあることにより動物細胞で発現可能な状態にある遺伝子をいう。
本発明で使用する組み換えベクターは、p53結合配列と、哺乳動物細胞で機能可能な最小プロモーターと、この最小プロモーターにより発現できるように連結されたレポーター遺伝子とを含む。すなわち、p53結合配列と最小プロモーターとを含む転写調節領域の下流に、レポーター遺伝子が発現可能に連結されている。転写因子結合配列と最小プロモーターとはいずれがより上流に存在していてもよい。組み換えベクターとしては、例えば、レポーター遺伝子として前述したluc2遺伝子配列が連結されたベクターを挙げることができる。
本発明の形質転換体は、上記説明した本発明の組み換えベクターを保持する細胞である。本発明の形質転換体は、上記組み換えベクターを一時的に保持していてもよく、安定的に保持していてもよい。
形質転換体を調製するに際し、組み換えベクターを導入する宿主細胞は哺乳動物であるかぎり、特に限定されない。宿主細胞としては、野生型p53が発現し、正常に機能している哺乳動物細胞を用いることができるが、p53機能が低下、欠損又は変異している異常細胞であっても、野生型p53遺伝子を別途導入することによりp53を発現させてやれば使用することができる。また、野生型p53が発現している細胞であっても、その発現量が少ない場合や組み換えベクター中のプロモーター活性が弱すぎる場合等は、さらに野生型p53遺伝子を別途導入してp53遺伝子の発現量を増大させて使用すればよい。
形質転換体の調製に当たっては、本発明の組み換えベクターを宿主細胞に一過的又は安定的に導入すればよい。「安定的に導入」とは、「哺乳動物細胞のゲノムに挿入」若しくは「哺乳動物細胞のゲノムに組み込む」と換言することもできる。形質転換率を考慮する必要がない点で、安定的に導入することが好ましい。また、ベクターを宿主細胞に安定的に導入した安定発現細胞は、作業効率の点及び変異原性試験の再現性の点からも好ましい。
(a)p53結合配列と、哺乳動物細胞で機能可能な最小プロモーターと、この最小プロモーターにより発現できるように連結されたluc2遺伝子とを含む組み換えベクターを一過的に保持するTK6細胞;
(b)p53結合配列と、哺乳動物細胞で機能可能な最小プロモーターと、この最小プロモーターにより発現できるように連結されたluc2遺伝子とを含む組み換えベクターを安定的に保持するTK6細胞;
(c)p53結合配列と、哺乳動物細胞で機能可能な最小プロモーターと、この最小プロモーターにより発現できるように連結されたluc2P遺伝子とを含む組み換えベクターを一過的に保持するTK6細胞;
(d)p53結合配列と、哺乳動物細胞で機能可能な最小プロモーターと、この最小プロモーターにより発現できるように連結されたluc2P遺伝子とを含む組み換えベクターを安定的に保持するTK6細胞。
本発明の変異原性試験方法は、
(1) 上記説明した形質転換体を、被験物質の存在下で培養する工程と、(2) 被験物質を接触させた形質転換体におけるレポーター遺伝子の発現量と被験物質を接触させない形質転換体におけるレポーター遺伝子の発現量とを比較することにより、被験物質が形質転換体におけるレポーター遺伝子の発現量に影響を及ぼすか否かを判定する工程と
を含む方法である。
被験物質の種類は特に限定されない。低分子化合物、高分子化合物等の他に、例えば、食品、食品加工物、廃棄物、ゴミ焼却物等の複数種類の物質の混合物であってもよい。
以下に、工程(1)の具体的態様を例示する。本発明において、被験物質の存在下で培養させる形質転換体は、先ず、形質転換体を細胞培養容器に播種し、8時間~一晩程度(TK6細胞等の浮遊細胞を用いる場合、0.5~1時間程度)培養しておくことが好ましい。培養温度は、その細胞に適した温度とすればよく、通常は36~38℃程度、好ましくは37℃程度とすればよい。培養液は特に限定されず、細胞に適した培養液を使用すればよい。細胞数は特に限定されないが、例えば96穴プレートを使用する場合は、通常1穴あたり1×104~1×105個程度の細胞を播種すればよい。
(1) 上記説明した形質転換体を、被験物質の存在下で4~10時間程度培養する工程である。
その後、被験物質を接触させた形質転換体と接触させていない形質転換体との間で、レポーター遺伝子の発現量を比較する。以下に、工程(2)の具体的態様を例示する。比較するに当たっては、両細胞のレポーター遺伝子の発現量をそれぞれ測定し、両測定値を比較することができ、又は、両細胞のレポーター遺伝子発現量を目視などにより直接定性的に比較することもできる。あるいは、被験物質を接触させた形質転換体と接触させていない形質転換体との間で比較するレポーター遺伝子の発現量は、測定したレポーター遺伝子の発現量の、別の標準遺伝子の発現量(内部標準)に対する相対値であってもよい。このようなレポーター遺伝子の発現量は、標準遺伝子で標準化若しくは補正したレポーター遺伝子の発現量ともいう。
本発明の変異原性試験方法は、
p53結合配列と、哺乳動物細胞で機能可能な最小プロモーターと、この最小プロモーターにより発現できるように連結されたluc2遺伝子とを含む組み換えベクターを保持するTK6細胞を含む、変異原性試験用キットである。本発明のキットにより、前記本発明の変異原性試験を簡便に実施することができる。
DNA損傷で発現するp53R2遺伝子イントロンのp53結合配列を3回繰り返し含む塩基配列(5’-CTGACATGCCCAGGCATGTCTTGACATGCCCAGGCATGTCTTGACATGCCCAGGCATGTCTA-3’:配列番号10)からなるオリゴヌクレオチド、及びこの塩基配列と相補的な塩基配列(5’-GATCTAGACATGCCTGGGCATGTCAAGACATGCCTGGGCATGTCAAGACATGCCTGGGCATGTCAGGTAC-3’ :配列番号11)からなるオリゴヌクレオチドをDNA合成機にてそれぞれ合成し、これらをアニーリングさせて2本鎖DNAとした。
特許第4243716号、実施例1に記載の方法にて作成したプラスミドPGV-p53R2x3-Lucを制限酵素BglII及びHindIIIで消化した後、低融点アガロース(NuSieve 3:1 Agarose;BMA製)を用いた電気泳動に供し、バンド部分のゲルから目的の198bpのDNAを回収し、SV40最小プロモーターと名づけた(5’-TGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATCGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAA-3’:配列番号13)。
特許第4243716号、実施例3に記載の方法にて作成したプラスミドPGV-p53R2x3-tk-Lucを制限酵素BglII及びHindIIIで消化した後、低融点アガロース(NuSieve 3:1 Agarose;BMA製)を用いた電気泳動に供し、バンド部分のゲルから目的の181bpのDNAを回収し、tk最小プロモーターと名づけた(5’-TAAGAAAATATATTTGCATGTCTTTAGTTCTATGATGACACAAACCCCGCCCAGCGTCTTGTCATTGGCGAATTCGAACACGCAGATGCAGTCGGGGCGGCGCGGTCCCAGGTCCACTTCGCATATTAAGGTGACGCGTGTGGCCTCGAACACCGAGCGACCCTGCAGCGACCCGCTTAAA-3’:配列番号14)。
TATA最小プロモーターを含む塩基配列(5’- GATCTCGACTATAAAGAGGGCAGGCTGTCCTCTAAGCGTCACCACGACTTCA-3’:配列番号15)からなるオリゴヌクレオチド、及びこの塩基配列と相補的な塩基配列(5’- AGCTTGAAGTCGTGGTGACGCTTAGAGGACAGCCTGCCCTCTTTATAGTCGA-3’ :配列番号16)からなるオリゴヌクレオチドをDNA合成機にてそれぞれ合成し、これらをアニーリングさせて2本鎖DNAとし、TATA最小プロモーターと名付けた。
ヒトリンパ芽球細胞株TK6細胞の野生型p53が正常であることを、シーケンス用プライマーである5’-ACACGCTTCCCTGGATTG-3’(配列番号17)及び5’-CTGTCAGTGGGGAACAAGAAGTGGAGA-3’(配列番号18)を用いたDNAシーケンス(ABI Prism BigDye Terminater v3.0;アプライドバイオシステムズ製)により確認した。
前記と同様にしてヒトリンパ芽球細胞株TK6の野生型p53が正常であることを確認した。当該TK6細胞に実施例2で作製されたプラスミドpGL4-p53R2×3-SV40-Luc2を一過的に導入した。次いで、RPMI1640培地(日水製薬製)に牛胎児血清(FBS)を終濃度10%含む培地を用いて、ルシフェラーゼ発光測定用兼培養用96穴プレート(コーニング製#3610)に細胞密度が70~80%コンフルエントとなるよう播種した。尚、導入するpGL4-p53R2×3-SV40-Luc2量は0.25ng / 穴ずつとなるよう調製した。
ホタルルシフェラーゼ遺伝子を含むプラスミドとしてpGL4.11(Promega製)を用いること以外、上記実施例1及び2に準じた操作を行い、レポータープラスミドpGL4-p53R2x3-SV40-Luc2Pと名づけた。
TK6細胞に実施例7で作製したプラスミドpGL4-p53R2x3-SV40-Luc2Pを一過的に導入した。次いで、RPMI1640培地(日水製薬製)に牛胎児血清(FBS)を終濃度10%含む培地を用いて、培養用96穴プレート(コーニング製#3595)に細胞が1個/穴となるよう播種した。その後、適宜培地を交換しながら1ヶ月間培養を継続し、得られた細胞コロニーを培養継続用として培養用96穴プレート(コーニング製#3595)に、及びルシフェラーゼアッセイ用としてルシフェラーゼ発光測定用兼培養用96穴プレート(コーニング製#3610)に分割し、さらに24hr培養した。その後、ルシフェラーゼアッセイ用に分割した細胞について、実施例5の方法に準じて、ルシフェラーゼ活性を測定し、ルシフェラーゼ活性が認められた細胞をTK6/p53R2x3-Luc2Pと名づけた。
実施例8で作製した安定発現細胞TK6/p53R2x3-Luc2Pを、RPMI1640培地(日水製薬製)に牛胎児血清(FBS)を終濃度10%含む培地を用いて、ルシフェラーゼ発光測定用兼培養用96穴プレート(コーニング製#3610)に細胞密度が70~80%コンフルエントとなるよう播種した。
実施例9の方法に準じて、実施例8で作製した安定発現細胞TK6/p53R2x3-Luc2Pを、ルシフェラーゼ発光測定用兼培養用96穴プレート(コーニング製#3610)に細胞密度が70~80%コンフルエントとなるよう播種した。
実施例8で作製した細胞TK6/p53R2x3-Luc2Pに、pRL-SV40-Rlucプラスミド(SV40プロモーターの下流にウミシイタケルシフェラーゼ(Rluc)が連結されたプラスミド)を、実施例8の方法に準じて一過的に導入した。次いで、実施例8の方法に準じて、Luc2P及びRlucを安定して発現する細胞を取得し、TK6/p53R2x3-Luc2P/Rlucと名付けた。
実施例11で作製した細胞TK6/p53R2x3-Luc2P/Rlucを、実施例9の方法に準じてルシフェラーゼ発光測定用兼培養用96穴プレートに播種した。
TK6細胞に、特許第4243716号、実施例1に記載の方法で作成したプラスミドPGV-p53R2x3-Lucを一過的に導入した。次いで、RPMI1640培地(日水製薬製)に牛胎児血清(FBS)を終濃度10%含む培地を用いて、ルシフェラーゼ発光測定用兼培養用96穴プレート(コーニング製#3610)に細胞密度が70~80%となるよう播種した。
形質転換体の培養時間を、TK6細胞については16時間、MCF-7、HepG2、A549、HCT116、及びHEK293については24時間、NB1RGBについては18時間培養とする以外、上記参考例1と同様にして、プラスミドPGV-p53R2x3-Lucの導入、培養、及びルシフェラーゼ活性の測定を行った。結果を図6に示す。
配列番号9 プラスミド
配列番号12 プライマー
配列番号17 プライマー
配列番号18 プライマー
配列番号19 luc2P遺伝子
配列番号20 プラスミド
Claims (9)
- (1) p53結合配列と、哺乳動物細胞で機能可能な最小プロモーターと、この最小プロモーターにより発現できるように連結されたレポーター遺伝子とを含む組み換えベクターを保持する哺乳動物細胞の形質転換体を、被験物質の存在下で4~10時間培養する工程、及び
(2) 上記工程(1)で培養した形質転換体におけるレポーター遺伝子の発現量と被験物質を接触させない形質転換体におけるレポーター遺伝子の発現量とを比較することにより、被験物質が形質転換体におけるレポーター遺伝子の発現量に影響を及ぼすか否かを判定する工程
を含む変異原性試験方法。 - 前記レポーター遺伝子がluc2であり、かつ前記形質転換体がTK6細胞に当該組換えベクターを導入したものである、請求項1に記載の方法。
- 前記最小プロモーターがSV40プロモーターである、請求項1に記載の方法。
- p53結合配列、最小プロモーター、及びレポーター遺伝子が、前記組み換えベクターに、上流側から、p53結合配列、最小プロモーター、及びレポーター遺伝子の順に配置されている、請求項1に記載の方法。
- p53結合配列が、
5'-WRRCWWGYYY -3':配列番号1
で示される塩基配列(配列番号1中、Rはプリン塩基を含むヌクレオチドを示し、Wはアデニン又はチミンを含むヌクレオチドを示し、Yはピリミジン塩基を含むヌクレオチドを示す)を有するものである請求項1に記載の方法。 - p53結合配列が、
5'-WRRCWWGYYY WRRCWWGYYY -3':配列番号2
で示される塩基配列(配列番号2中、Rはプリン塩基を含むヌクレオチドを示し、Wはアデニン又はチミンを含むヌクレオチドを示し、Yはピリミジン塩基を含むヌクレオチドを示す)を有するものである請求項1に記載の方法。 - 前記レポーター遺伝子の発現量が、標準遺伝子に対する相対値である、請求項1に記載の方法。
- p53結合配列と、哺乳動物細胞で機能可能な最小プロモーターと、この最小プロモーターにより発現できるように連結されたレポーター遺伝子とを含む組み換えベクターを保持するTK6細胞である、形質転換体。
- 請求項8に記載の形質転換体を含む、変異原性試験用キット。
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JP2003024080A (ja) * | 2001-07-19 | 2003-01-28 | Univ Tokyo | p53依存性アポトーシス誘導タンパク質、およびアポトーシス調節剤のスクリーニング方法 |
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2012
- 2012-10-11 EP EP12839616.5A patent/EP2767592B1/en active Active
- 2012-10-11 US US14/350,741 patent/US20140308675A1/en not_active Abandoned
- 2012-10-11 JP JP2013538568A patent/JP6066914B2/ja active Active
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JP2005000024A (ja) * | 2003-06-10 | 2005-01-06 | Nissin Food Prod Co Ltd | 哺乳動物細胞を用いた変異原性試験法 |
JP4243716B2 (ja) | 2003-06-10 | 2009-03-25 | 日清食品ホールディングス株式会社 | 哺乳動物細胞を用いた変異原性試験法 |
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Cited By (3)
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JP2015223107A (ja) * | 2014-05-27 | 2015-12-14 | オリンパス株式会社 | プロモーターアッセイ |
WO2016017039A1 (ja) * | 2014-07-31 | 2016-02-04 | 株式会社トランスジェニック | 炎症レポーターシステム |
US10765094B2 (en) | 2014-07-31 | 2020-09-08 | Transgenic Inc. | Inflammation reporter system |
Also Published As
Publication number | Publication date |
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EP2767592A1 (en) | 2014-08-20 |
JPWO2013054846A1 (ja) | 2015-03-30 |
EP2767592A4 (en) | 2015-06-17 |
JP6066914B2 (ja) | 2017-01-25 |
EP2767592B1 (en) | 2017-06-28 |
US20140308675A1 (en) | 2014-10-16 |
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