JP2016202044A - Mutagenesis study method using transformed cells - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide methods for assessing the mutagenesis of a substance, and to provide recombinant vectors used for performing the methods.SOLUTION: The present invention relates to a recombinant vector comprising a mutagenic detection sequence consisting of a multiple of 3 bases without stop codon, wherein an initiation codon, a mutagenesis detection sequence, and a reporter gene sequence without an initiation codon are arranged in this order. A recombinant vector in which the reporter gene is inserted expressibly and a mutagenesis study method using the recombinant vector are provided.SELECTED DRAWING: None

Description

  The present invention relates to a method for evaluating the mutagenicity of a substance (mutagenicity test method), a recombinant vector used in the method, and a transformant.

  With the development of science and technology in recent years, various new chemical substances have been developed. Not all of these chemicals have a negative impact on the environment, the human body, etc., but there are substances that cause mutations that can cause cancer and other inherited diseases. Therefore, it is important to confirm the mutagenicity of a chemical substance, and in particular, it is necessary to quickly and accurately test the mutagenicity of a newly developed chemical substance.

One of the currently popular mutagenicity tests is the Ames test (Non-patent Document 1). The Ames test is mutagenic using the indication that a histidine-requiring mutant of Salmonella typhimurium changes to a histidine-unrequired strain by a backmutation induced by contact with the mutagen. This is the most reliable and widely used test method. This test can detect various mutations including frameshift mutations caused by base pair substitution and insertion / deletion of base pairs. In addition, according to this method, the number of colonies generated varies depending on the strength of mutagenicity. Therefore, this method can be said to be a simple method in that mutagenicity can be evaluated simply by counting the number of colonies. However, there are concerns that it is not always possible to extrapolate the results of the Ames test to animals, etc., because there are significant differences in metabolic systems between bacteria such as Salmonella and higher eukaryotic cells derived from animals.

In general, microorganisms such as bacteria have few drug-metabolizing enzymes (CYP enzymes, etc.). Therefore, when detecting the mutagenicity of a test substance against metabolites, a 9,000 x g supernatant of liver homogenate (S9 ) And a test substance are pre-incubated and then used for a test using microorganisms. Under these circumstances, the micronucleus test method (Non-patent document 2) or the comet assay method (Non-patent document 3), which is a test method using cultured mammalian cells instead of microorganisms, is often used. Yes. In addition, a mutagenicity test using a transgenic rat introduced with a gene containing a lambda phage reporter gene has also been reported (Patent Document 1).
These tests are considered to be easier to evaluate the influence of the test substance on humans than the Ames test in that mammalian cells are used. However, these methods require the skill of the tester in terms of detecting abnormal cell morphology and the like, and in the need to produce transgenic animals. Accordingly, there is a need for a new test method that is easier to operate and easy to evaluate.

  As a simple method using mammalian cells, a mutagenicity test method using a transcriptional regulatory mechanism by p53 has been reported (Patent Documents 2 and 3). This method is excellent as a simple method for evaluating the effect of mutagen on mammalian cells, but it is for evaluating mutagenicity through the transcriptional regulatory function of p53. The development of a simple evaluation method for detecting specific damage is desired.

JP2003-52276 JP2005-24 WO2013 / 054846

Ames et al., Mutation Research., 31: 347-364, 1975 Hayashi et al., J Agric Food Chame. 54: 6881-3887, 2006 Olive et al., Radiat Res., 122: 86-94, 1990

In view of the above circumstances, the present inventors have attempted to develop a mutagenicity test method that is simpler than conventional methods.
That is, an object of the present invention is to provide a mutagenicity test method using eukaryotic cells, a recombinant vector used in the method, and a transformed cell containing the vector.

  The inventors expressed a modified GFP gene in which a target sequence (hereinafter referred to as a mutagenicity detection sequence) for detecting DNA damage was inserted immediately below the start codon (ATG) of the fluorescent protein (GFP) gene. A recombinant vector that can be inserted is prepared, and the mutagenicity of the test substance against animal cells transformed with this vector is evaluated, and it is found that the mutagenicity can be evaluated easily and with high sensitivity. The present invention has been completed.

That is, this invention is the following (1)-(9).
(1) A recombinant vector containing a mutagenicity detection sequence comprising no base codon and consisting of a multiple of 3 bases,
A recombinant vector in which an initiation codon, a mutagenicity detection sequence, and a reporter gene sequence not including the initiation codon are arranged in this order, and the reporter gene is inserted so that it can be expressed.
(2) The recombinant vector according to (1) above, wherein the mutagenicity detection sequence is composed of 50 bases or more.
(3) In the mutagenicity detection sequence, a part of the sequence in the antisense strand is composed only of thymine and cytosine, according to any one of (1) and (2) above Recombinant vector.
(4) The recombinant vector according to any one of (1) to (3) above, wherein the reporter gene encodes a fluorescent protein.
(5) The recombinant vector according to any one of (1) to (4) above, wherein a partial sequence of the mutagenicity detection sequence is the sequence represented by SEQ ID NO: 1.
(6) A cell comprising the recombinant vector according to any one of (1) to (5) above.
(7) The cell according to (6) above, wherein the cell is an animal cell (8) A mutagenicity test method comprising the following steps (a) and (b):
(A) contacting the cell according to (6) or (7) with a test substance,
(B) Whether the test substance affects the expression of the reporter gene by comparing the expression level of the reporter gene in the cell contacted with the test substance and the expression level of the reporter gene in the cell not contacted with the test substance (9) A kit for carrying out the mutagenicity test method according to (8) above, comprising at least the recombinant vector according to any one of (1) to (5) above.

  By using the mutagenicity test method using the recombinant vector according to the present invention, it is possible to quickly evaluate mutagens that may affect eukaryotes without complicated operations. it can. Moreover, it is not necessary to add substances other than the test substance to the culture solution.

It is the figure which showed the base sequence of the insertion position in the vector in which the mutagenicity detection sequence (sequence number 1) is inserted. It is the figure which showed the procedure of the exposure experiment. It is a microscope picture of the cell produced for the mutagenicity test. A is a 293 cell into which no gene has been introduced, B is a 293 cell into which only the GFP gene has been introduced (GFP293 cell), and C is a 293 cell into which a mutagenic detection sequence and GFP gene have been introduced (T-GFP293 cell). . A, B, and C, respectively, the left is an image observed in a bright field, and the right is an image observed by fluorescence. It is the figure which showed the GFP intensity | strength area | region used by the analysis by a cell analyzer. It is the graph which showed the decreasing rate of the fluorescence from T-GFP293 cell after exposure to a test substance. In each graph, the vertical axis represents the decrease rate (%) of fluorescence from T-GFP293 cells, and the horizontal axis represents the concentration of the test substance exposed. It is the graph which compared the result obtained by the mutagenicity test method of this invention, and the result obtained by another test method about the detection limit concentration of a mutagen.

A first embodiment of the present invention is a recombinant vector comprising a mutagenicity detection sequence comprising no base codon and comprising a multiple of 3 bases,
A recombinant vector in which a start codon, a mutagenicity detection sequence, and a reporter gene sequence not including the start codon are arranged in this order from the 5 ′ side, and the reporter gene is inserted so that it can be expressed.
In the first embodiment of the present invention, a “mutagenicity detection sequence” is linked adjacent to the start codon, and immediately after that, a reporter gene not containing the start codon (ie, the reporter gene alone can be expressed). A recombinant vector is provided in which a DNA construct linked with a gene that does not contain a proper start codon is inserted so that it can be expressed in a state in which the reporter gene can function. The “mutagenicity detection sequence” is a sequence for detecting a mutation caused by a test substance, for example, substitution, insertion, deletion, or base modification such as dimer formation. The mutagenicity detection sequence in a state where no mutation has occurred is a multiple of 3 bases and does not contain a termination codon (TAA, TAG that matches the translation frame frame of the protein in the mutagenicity detection sequence) And no TGA), the reporter gene following this sequence can be expressed normally (ie, green fluorescence is obtained when using the GFP gene). However, when a mutation is caused in the mutagenicity detection sequence by the test substance, displacement of the reading frame occurs due to substitution, insertion, deletion, etc., and as a result, a termination codon occurs in the mutagenicity detection sequence, Alternatively, the reporter gene cannot be normally expressed, for example, a polypeptide having a sequence different from the amino acid sequence of the reporter gene is expressed, and the activity of the protein encoded by the reporter gene varies (that is, When using the GFP gene, green fluorescence cannot be obtained). In addition, when base modification such as dimer formation is induced in the mutagenicity detection sequence, the transcription process stops at the modified part, and the reporter gene is not expressed (that is, when using the GFP gene, Green fluorescence cannot be obtained).
The initiation codon and the mutagenicity detection sequence, and the mutagenicity detection sequence and the reporter gene may be directly linked, or may be indirectly linked by inserting a base sequence such as a restriction enzyme site. When indirectly linking, it is necessary to insert a base sequence having the number of bases that enables expression of the reporter gene.

  In other words, the mutagenicity detection sequence has a function to detect various mutations caused by a test substance as generation of a stop codon, frame shift, or base modification, and to detect it as a change in reporter gene expression. I'm in charge. Therefore, the detection sensitivity increases as the length of the mutagenic detection sequence (the number of bases) increases, but the protein translated from the mutagenic detection sequence is translated from the reporter gene. Because it is expressed by fusion with a protein, mutagenicity is achieved so that a protein that does not adversely affect cells is expressed without impairing the original activity of the protein expressed from the reporter gene. It is desirable to determine the configuration and length of the detection sequence. As a preliminary experiment, a person skilled in the art will detect the mutagenicity by preliminarily expressing the produced recombinant vector in a desired cell and knowing in advance the influence of the protein expressed from the mutagenicity detection sequence. The availability of the sequence can be easily determined. However, if the mutagenicity detection sequence is too short, it is expected that it will be difficult to detect the mutation induced by the test substance. For example, a sequence composed of 50 bases or more is desirable. As shown in Examples described later, when the sequence of SEQ ID NO: 1 is used as a mutagenicity detection sequence, the evaluation of mutagenicity can be carried out simply and with high sensitivity. Therefore, although not limited thereto, a sequence that partially includes the sequence of SEQ ID NO: 1 can be exemplified as a mutagenicity detection sequence.

  Further, the antisense strand of the mutagenicity detection sequence may be composed of only thymine and cytosine in order to detect base modification, particularly thymidine dimer formation. Base modifications, thymidine dimers, and the like occur in the antisense strand, affecting transcription and making the protein untranslatable. As a result, the expression of the reporter gene is reduced, and the mutagenicity of the test substance can be evaluated.

  The “reporter gene” is a gene that can be inserted into the recombinant vector according to the embodiment of the present invention, and is expressed in the introduced host cell under the condition that no mutagen is present. Any substance that can be detected qualitatively or quantitatively can be used. The sequence of the reporter gene inserted into the recombinant vector of the embodiment of the present invention does not include an initiation codon for starting translation of the protein encoded by the reporter gene. Since the reporter gene is used to examine the effect of the mutagen using the change in the amount of the expression product as an index, if the translation initiation codon of the reporter gene exists downstream from the mutagen detection sequence, the mutagen When any mutation is introduced into the detection sequence, expression of the reporter gene may be induced.

Examples of the reporter gene include, but are not limited to, a gene encoding a fluorescent protein, a gene encoding an enzyme that generates luminescence by reacting with a substrate, and an antibiotic resistance gene. As the reporter gene, any protein can be used as long as the protein encoded by the reporter gene exhibits some activity. For example, when a gene encoding a fluorescent protein is used as a reporter gene, it is not necessary to use a DNA encoding the full length of the fluorescent protein as a reporter gene as long as it has a function of emitting fluorescence. It may be used as a gene.
When a gene encoding a fluorescent protein is used as a reporter gene, its expression can be quantitatively detected by measuring the fluorescence from the translation product of the reporter gene (for example, it can be measured by a cell analyzer or the like). The gene encoding the fluorescent protein that can be used in the embodiment of the present invention is not particularly limited, and any gene that can be selected by those skilled in the art can be used. As a reporter gene encoding a fluorescent protein that can be used in the present embodiment, for example, a fluorescent protein derived from a jellyfish such as a green fluorescent protein (GFP) and a derivative thereof (for example, BFP, CFP, YFP, RFP, etc.) In addition, a gene encoding a fluorescent protein derived from coral or sea anemone can be mentioned.
In addition, when a gene encoding an enzyme that produces luminescence by reacting with a substrate is used as a reporter gene, the enzyme expressed from the reporter gene is reacted with the substrate, and the resulting luminescence is measured, for example, with a luminometer By doing so, the expression level from the reporter gene can be detected. Examples of such reporter genes include genes encoding firefly luciferase, Renilla luciferase and the like.
In addition, in some cases, antibiotic resistance genes (neomycin resistance-conferring gene, hygromycin resistance-conferring gene, blasticidin S resistance-conferring gene, etc.) can be used as reporter genes.

In the recombinant vector of this embodiment, in addition to the above-described components, a promoter for protein expression is present.
The promoter that can be used here is not particularly limited, and an appropriate promoter may be used corresponding to the host cell into which the recombinant vector according to the present invention is introduced. For example, when the host is an animal cell, CMV promoter, SRα promoter, CAG promoter, SV40 promoter and the like can be used, and when the host is an insect cell, polyhedrin promoter, P10 promoter and the like can be used.

The second embodiment of the present invention is a cell (transformed cell) transformed with the recombinant vector of the first embodiment. As a cell transformed with the recombinant vector according to the present invention, a eukaryotic cell is preferable, and a higher eukaryotic cell such as an animal cell is particularly preferable. Animal cells that can be used here are not particularly limited, and any animal cells can be used as long as they are derived from animals. Examples of cells that can be used are, for example, human-derived HEK293 cells, mouse-derived NIH3T3 cells, hamster-derived CHO cells, BHK cells, African green monkey-derived COS-1 cells, COS-7 cells, Vero cells, etc. Can be mentioned.
As a method for introducing the recombinant vector into the host cell, any method that can be carried out by those skilled in the art may be selected. For example, electroporation method (Cytotechnology, 3, 133 1990), lipofection method (Proc. Natl. Acad. Sci. USA, 84, 7413 1987), etc. can be used.
The transformed cells of this embodiment include both cells that express the reporter gene transiently and cells that stably express, but cells that stably express are more desirable.
The transformed cell according to this embodiment is preferably cultured in an environment where no mutagen is present, and it is confirmed that the reporter gene is normally expressed in the cell.

The third embodiment of the present invention
The following steps (a) and (b);
(A) contacting the cell according to the second embodiment (the cell containing the recombinant vector according to the first embodiment) with a test substance;
(B) Whether the test substance affects the expression of the reporter gene by comparing the expression level of the reporter gene in the cell contacted with the test substance and the expression level of the reporter gene in the cell not contacted with the test substance Determining whether or not,
A mutagenicity test method including

The test substance is not particularly limited, and may be one kind or a mixture of plural kinds of substances.
A cell into which a reporter gene for detecting a mutagen is introduced by the recombinant vector of the first embodiment (hereinafter referred to as a transformed cell) is subcultured before contacting with a test substance, and is not limited. Those cultured for about 8 hours to overnight may be used. The culture temperature may be a temperature suitable for the transformed cells to be used, and the number of cells to be seeded is not particularly limited, but for example, 1.0 × 10 ⁴ to 1.0 × 10 ⁴ per well of a 24-well plate ^It may be about ⁵ .
Next, a test substance is added to the medium of transformed cells. The test substance may be added as it is or dissolved in an appropriate solvent depending on the physical properties of the substance. The solvent to be used is preferably one that does not affect the transformed cells, and is not limited. For example, in addition to a buffer solution such as water and PBS, an organic solvent such as DMSO, ethanol, and methanol depends on the concentration to be added to the medium. Can be used. The amount of the test substance to be added also varies depending on the optimum concentration of the test substance to be used. For example, the test substance can be added so that the final concentration is about 0.1 to 1.0% by weight.
After adding a test substance to the culture medium of a transformed cell, it is cultured for an appropriate time, for example, about 12 to 72 hours, preferably about 24 to 48 hours.

Thereafter, the expression level of the reporter gene in the transformed cell contacted with the test substance is compared with the expression level of the reporter gene in the transformed cell not contacted with the test substance.
The expression level of the reporter gene can be measured using a known method or the like according to the characteristics. For example, when the reporter gene is a gene encoding a fluorescent protein, the intensity of fluorescence emitted from the fluorescent protein in the transformed cell can be measured using a cell analyzer or the like, and the expression level can be quantified. If the reporter gene is a luciferase that produces a luminescent substance as a result of the enzymatic reaction, add the enzyme substrate luciferin to the cell lysate of the transformed cell, and measure the amount of luminescence by luciferin degradation using a luminometer. And the expression level can be quantified.

  The ratio of the expression level of the reporter gene in the transformed cell contacted with the test substance to the expression level of the reporter gene in the transformed cell not contacted with the test substance depends on the concentration of the test substance to be added. The test substance can be determined to be a mutagenic substance when the test substance fluctuates to the extent that it is determined that there is a significant difference.

The fourth embodiment of the present invention is a kit for carrying out the mutagenicity test method according to the present invention.
The kit of this embodiment includes at least the recombinant vector according to the first embodiment. Moreover, the cell transformed with the said recombinant vector may be contained. In addition, reagents necessary for introducing the recombinant vector into cells, instructions for using the kit, and the like may also be included.

  Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

1. Experimental method 1-1. Reagent In this example, a special grade reagent manufactured by Wako Pure Chemical Industries, Ltd. was used unless otherwise specified. In addition, enzymes manufactured by Takara Shuzo Co., Ltd. were used unless otherwise specified.

1-2. Construction of mutagenicity detection sequences and recombinant vectors For construction of a recombinant vector to detect mutagens, pAcGFP1-N1 (Clontech), an expression vector for mammals with a CMV promoter and a green fluorescent protein gene (AcGFP1) )It was used.
First, in order to remove the multicloning site in this vector, PCR was performed using the following primer set and pAcGFP1-N1 as a template.
AcGFP1_del_F1; 5'-ATCCCCCGGGAGATCTGTGAGCAAGGGCGCCGA-3 '(SEQ ID NO: 2)
AcGFP1_del_R2; 5'-CACGTCAGATCTCGATAGCGCGGATCCCATGGTGGCGATC CTCCTGAATTCGAGTCCGGTAGCGCTAGC-3 '(SEQ ID NO: 3)
The obtained PCR product was digested with BamHI and BglII and then self-ligated to construct plasmid pMC-G.
In addition, the restriction enzyme XbaI recognition sequence immediately under the AcGFP1 gene of this plasmid is methylated by the influence of dam methylase and cannot be digested with XbaI. Therefore, this pMC-G was introduced into HST04 E. coli (TaKaRa), a competent cell that does not cause methylation for the purpose of demethylation so that it can be digested with XbaI, and the demethylated pMC-G was purified. After digestion with BamHI and XbaI and agarose electrophoresis, the gel containing the AcGFP1 gene was excised and the AcGFP1 gene was purified.

On the other hand, in order to remove XbaI even under the influence of dam methylase, the following procedure was performed to change the TC sequence following the XbaI recognition sequence of pMC-G to CC for the purpose of removal.
First, PCR was performed using pMC-G as a template and the following primer set.
pMC-TBG-dAcGFP_F1; 5'-GAGCTCTAGACCATAATCAGCCATACCACATTTGTAGAG-3 '(SEQ ID NO: 4)
pMC-TBG-dAcGFP_R1; 5'-TGAACAGCTCGGCGCCCTTG-3 '(SEQ ID NO: 5)
In the obtained PCR product, the XbaI recognition sequence is replaced with TCTAGACC, and the AcGFP1 gene is removed. This PCR product was digested with BamHI and XbaI and ligated with the previous AcGFP1 gene to construct plasmid pMC-G2.

Next, a sequence (Thymine Dimer Target Site; TDTS) capable of detecting DNA damage that forms a dimer on a pyrimidine base was designed (FIG. 1). This TDTS sequence was designed so that the antisense strand was a 59-base sequence composed only of thymine and cytosine. TDTS_U (SEQ ID NO: 6) was synthesized as the sense strand and TDTS_L (SEQ ID NO: 7) was synthesized as the antisense strand to prepare a DNA fragment having the TDTS sequence.
TDTS_U ； 5'-GATCCCTCGAGGAAGAAGAGGAGGGAAAAGGGAAAAAGAAAAGAAAAGAAAAAAGGAAAGAGAAAGGTACCA-3 '
TDTS_L; 5'-GATCTGGTACCTTTCTCTTTCCTTTTTTCTTTTCTTTTCTTTTTCCCTTTTCCCTCCTCTTCTTCCTCGAGG-3 '
First, 0.1 μg / μl TDTS_U and 0.1 μg / μl TDTS_L were added 10 μl at a time to a 1.5 ml microtube, heated in a 96 ° C. heat block for 30 seconds, and allowed to stand at room temperature for 1 hour. The resulting double-stranded DNA has a sticky end sequence generated when cleaved with BamHI and BglII. Using this terminal sequence, the obtained double-stranded DNA was inserted into pMC-G2 to prepare a recombinant vector (pMC-TG) for detecting a mutagen (FIG. 1).

1-3. Cultured cells for mutagenicity test The cells used in this Example were adherent cells, Flp-In ^TM 293 T, which is a stable strain in which the Flp-In system is incorporated into cells derived from human kidney cells (HEK 293 cells). -REx cell lines (Life Technologies; hereinafter referred to as “293 cells”) were used. For the cell culture, the complete medium described in the attached manual of 293 cells was used. For cell passage, 60 mm dishes, 24-well plates or 48-well plates were used.
Remove the complete medium from the dish and plate of 70-80% confluent cells, add 300 μl to 4 ml of 1 × PBS (pH = 7.4), gently agitate, and remove the supernatant twice. went. After that, trypsin / EDTA (T4049-500ML, GIBCO) is added in 300 μl to 4 ml, incubated in a CO ₂ incubator (RCO3500TABB, Revco Technologies) for 5 minutes, and the cells were examined using an inverted microscope (TS100, Nikon). After confirming the detachment, 300 μl to 500 μl of complete medium was added, followed by centrifugation at 4 ° C. and 1,000 rpm for 5 minutes. Then, the supernatant was removed, 5 ml of complete medium was added to the cells, and the cells were resuspended. The cell suspension was added to each well in a new 60 mm dish, 24-well plate, or 48-well plate, and replanted. .

Cell transformation was performed using the lipofection method as follows.
50 μl Dulbecco's modified Eagle's medium (DMEM, GIBCO) and 1 μl of Lipofectamine (registered trademark) 2000 Transfection Reagent (Life Technologies) were mixed in a 1.5 ml microtube (lipofection solution). In addition, 50 μl of DMEM and 1 μl of 1 μg / μl of plasmid were mixed in different 1.5 ml microtubes (plasmid solution). These lipofection solution and plasmid solution were incubated at room temperature for 5 minutes, mixed in total, and incubated at room temperature for an additional 20 minutes. The whole amount of this solution was dropped onto 293 cells cultured in a 12-well plate (NUNC) using a medium not containing antibiotics. After 4 hours, the medium was removed and replaced with a new culture medium. Thereafter, the cells were cultured in a CO ₂ incubator for 3 to 4 days. Transformed cells were screened over a long period of time using G418 (Geneticin (registered trademark), Life Technologies), an amyloglycoside antibiotic, as a selective drug, thereby removing wild strains and transiently expressed strains. After most of the transformed cells in one well of the 12-well plate became occupied, the transformants were collected using the penicillin cup method, and the cells were transferred to a 96-well plate (NUNC) for cloning. . Fluorescence observation of cells cloned with an inverted microscope (TE300, Nikon) was performed, the cell lines emitting fluorescence were subcultured, and cells transfected with pAcGFP1-N1 and pMC-TG were GFP293 cells and T-GFP293 cells, respectively. And stored in -80 ° C. or liquid nitrogen according to a conventional method until use.

1-4. Test Substances As test substances, DNA is directly targeted and mitomycin C (MMC), 5-fluorouracil (5-FU), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), which are widely used as positive target reagents for mutagenicity tests, In addition to N-methyl-N-nitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU), 1-nitropyrene (1-NP), which is recognized as a suspected carcinogen, was used. Information on these test substances is shown in Table 1.
Each test substance was dissolved in an appropriate solvent, sterilized by filtration through MILLEX (registered trademark) GV Filter Unit 0.22 μm (Millipore), and stored at −20 ° C. until use. These test substances were used for exposure experiments in addition to a medium not containing antibiotics at the time of use. However, when dimethyl sulfoxide (DMSO) was used as the solvent, the test substance was added so that the DMSO concentration in the culture solution became a final concentration of 0.5%.

1-5. Exposure experiment An exposure experiment was conducted using T-GFP293 cells in which the mutagenicity detection sequence (SEQ ID NO: 1) and the GFP gene were inserted on the chromosome, and GFP293 cells in which only the GFP gene was inserted on the chromosome. It was.
Cells that have become 80% confluent in a 60 mm dish are detached with trypsin / EDTA, seeded with 7.5 × 10 ⁴ cells per well of a 24-well plate, and 1 in a CO ₂ incubator. Day incubation was performed. After the incubation, all the medium was removed, 400 μl of test substance whose concentration was changed stepwise was added, and again exposed for 3 days in a CO ₂ incubator (FIG. 2). The experiment was performed using 6 samples for one concentration.

1-6. Analysis by Cell Analyzer Cells exposed to the test substance for 3 days were observed for the state of cells using a fluorescence microscope. Next, the entire medium in each well was removed, and after washing with PBS, all of the washing solution was removed. Subsequently, 100 μl of trypsin / EDTA was added and incubated for 10 minutes in a CO ₂ incubator. After confirming that the cells were released from the plate, 400 μl of PBS solution containing formaldehyde at an arbitrary ratio was added and resuspended, and transferred to a 1.5 ml microtube. This cell suspension is shielded from light for 1 hour on ice, and then the cell suspension is used with a 41 μm nylon mesh (NY41-HC, NYTAL) and a disposable test tube (93-5001-6, Sansho). Cells were isolated and the GFP intensity of each cell was measured with a cell analyzer (EC800, Sony). The mutagenicity was evaluated by examining the ratio of the number of cells in which the cell size and internal structure were constant and the GFP fluorescence disappeared.

1-7. Statistical analysis For the data obtained, the mean value ± standard deviation (SD) of each group was calculated, and the uniformity of each group was tested by the Bartlett test (Snedecor and Cochran, 1989a) (significant Level: 1% on both sides). If the variance is uniform, use Dunnett's test (Dunnett, 1955, 1964) to test the mean difference between the control group and each exposed group (significance level: 5 and 1% on both sides). The steel test (Steel, 1959) was used to test the difference in the average rank between the control group and each exposure group (significance level: 5 and 1% on both sides).

2. Result 2-1. Production of Mutagenicity Test Cells First, it was confirmed whether green fluorescence was generated from GFP293 cells and T-GFP293 cells. For GFP293 cells, all cells observed in one field of the fluorescence microscope emitted uniform and strong green fluorescence (FIG. 3B). This suggests that FP293 cells could express genetically uniform GFP. Similarly, when green fluorescence was observed for T-GFP293 cells into which a mutagenic detection sequence was introduced, it was found that green fluorescence was uniformly emitted (FIG. 3C). Therefore, it was confirmed that the GFP in which a peptide having a length of 24 amino acids translated from the mutagenicity detection sequence (SEQ ID NO: 1) was added immediately under the start codon was a protein emitting green fluorescence.

2-2. Exposure experiment In order to examine the ratio of the number of cells in which fluorescence from GFP disappeared after each test substance was exposed to cells, a normal GFP fluorescence intensity range was first defined.
Using 293 cells, GFP293 cells, and T-GFP293 cells, gating on the area where the area of the area where 50% of FS and SS values of 200 or more are present is minimized To obtain measurement results. The result is shown in FIG. Since 293 cells do not have GFP fluorescence, the region where the fluorescence intensity is weak is defined as the non-fluorescence region and the strong region is defined around the point where the base of the fluorescence intensity of the normally distributed histogram is in contact with the baseline. A normal fluorescent region was set (FIG. 4). According to this definition, the results of an exposure experiment in which each test substance was added were evaluated.

In order to investigate the usefulness of T-GFP293 cells, T-GFP293 cells and GFP293 cells were used to examine differences in sensitivity to MMC. The experiment was performed by changing the concentration of MMC from 25 to 250 nM in an arithmetic progression of 25 nM. As a result, it was found that the ratio of the number of cells in the non-fluorescent region increased in T-GFP293 cells with a significant difference at a concentration of 125 nM or more with respect to the control (Table 2). On the other hand, GFP293 cells increased with a significant difference from the control only when 250 nM MMC was added (Table 2). Therefore, it was confirmed that T-GFP293 cells can detect mutagenicity with higher sensitivity than GFP293 cells. In other test substance exposure experiments, experiments were performed using only T-GFP293 cells.

Next, in order to obtain the detection limit concentration of mutagenicity for MMC of T-GFP293 cells, the regression linear equation obtained from the value of the region where the number of cells in the non-fluorescent region did not increase in a concentration-dependent manner and the concentration-dependent Assuming that the concentration at the intersection with the regression line equation obtained with the value of the region that increases gradually is the detection limit concentration, it was found to be 117 nM (FIG. 5). Similarly, the ratio of the number of cells in the non-fluorescent region decreased with 5-FU in a concentration-dependent manner, and the detection limit concentration was calculated to be 13.6 μM. Furthermore, EMS, MNU, ENU, and 1-NP also showed concentration-dependent changes, and their detection limit concentrations were 2.1 mM, 2.1 mM, 2 mM, and 20 μM, respectively (FIG. 5).
MMS showed a concentration-dependent increase when exposed at concentrations of 50, 100, 125, 200, 500 and 1,000 μM (FIG. 6).

Regarding the detection limit concentration of mutagenic substances, the results obtained by the mutagenicity test method according to the present invention were compared with the results obtained by other methods, and it was confirmed that they had almost the same detection sensitivity. (Table 3, FIG. 6). FIG. 6 is a graph of the results of the test method according to the present invention and the results of other methods shown in the literature. Other methods shown here are Ames test, Micronucleus test, Sister chromatid exchange test, Umu test, Chromosomal aberration test, and irregular DNA synthesis test. (Unscheduled DNA synthesis test).
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  The present invention provides a method for easily detecting mutagenicity of eukaryotic cells such as animal cells, and is a technique that can be used in any industrial field where mutagenicity is evaluated.

Claims (9)

A recombinant vector containing a mutagenicity detection sequence that does not contain a termination codon and is a sequence consisting of multiples of 3 bases,
A recombinant vector in which an initiation codon, a mutagenicity detection sequence, and a reporter gene sequence not including the initiation codon are arranged in this order, and the reporter gene is inserted so that it can be expressed.   The recombinant vector according to claim 1, wherein the mutagenicity detection sequence is composed of 50 bases or more.   The recombinant vector according to claim 1 or 2, wherein in the mutagenicity detection sequence, a part of the sequence in the antisense strand is composed only of thymine and cytosine.   The recombinant vector according to any one of claims 1 to 3, wherein the reporter gene encodes a fluorescent protein.   The recombinant vector according to any one of claims 1 to 4, wherein a partial sequence of the mutagenicity detection sequence is a sequence represented by SEQ ID NO: 1.   A cell transformed with the recombinant vector according to any one of claims 1 to 5.   The cell according to claim 6, wherein the cell is an animal cell. A mutagenicity test method comprising the following steps (a) and (b):
(A) contacting the cell according to claim 6 or 7 with a test substance;
(B) Whether the test substance affects the expression of the reporter gene by comparing the expression level of the reporter gene in the cell contacted with the test substance and the expression level of the reporter gene in the cell not contacted with the test substance Step of determining whether or not   A kit for carrying out the mutagenicity test method according to claim 8, comprising at least the recombinant vector according to any one of claims 1 to 5.