JPH0630623B2 - Assays for rapid screening for mutagenicity and teratogenicity - Google Patents

Description

TECHNICAL FIELD The present invention relates to genetic engineering, and particularly to a target gene linked to a reporter gene (both genes can be expressed in animal cells in the assay of mutagens). The use of treated animal cells containing

(Prior Art) Many new drugs and drugs released into the environment or workplace require toxicity testing. Compounds generally must be assayed for damage to the embryo (teratogenic (teratogenic) activity) and damage to the differentiated animal (carcinogenic or mutagenic activity).

Traditionally, compounds have been tested in short-term tests (ST
Mutagenesis activity has been assayed using T), or animal studies. Most animal studies are conducted using rodent protocols developed by the National Cancer Institute in the early 1970s. This protocol was developed by Sontag et al.
USDep.HealthEduc, Welfare Publ. (NIH) Carcinog.Tec
h. Rep. Serv. 1,76 (1976). However, the correlation between the results obtained by the above two systems is poor and it is difficult to objectively evaluate. For example, as Tennant et al . Reported in Science, 236 , 933-941 (1987), four widely used STT methods and rodent carcinogenicity test results are only 60% useful. Do not match. The four test methods compared are as follows.
Salmonella (Salmonella) mutagenesis, SAL method (Hauosu (Haworth) et al, Environ.Mutagen. 5suppl.l) 3 (1983 )
And Mortelmans et al., Toxicol.App
l.Pharmacol. 75.137, (1984));
Method by chromosomal abnormality in Chinese hamster ovary cells (ABS method); Method by sister chromatid exchange in Chinese hamster ovary cells (SCE method) (The above two methods are Galloway et al . , Environ.mutagen.
1 (1985) and Galloway et al . Environ.mutagen. (1987) .
And the method using mouse lymphoma cells (MOLY method) (Myhr et al., Evaluation of
Short-term Tests for Carcinogens: Report of the Int
ernational Program on Chemical Safety's Collabor
ative Study on in vitro Assays, Progress in Mutatio
n Research Series , Vol. 5, pp. 55-568, edited by J. Ashby et al. (Elsevier, Amsterdam, 1985)).

A recent modification of the SAL method or the Ames in vitro assay was described by Oda et al. In Mutatio
n Research, 147, 219-229 (1985). Oda et al. Introduced the fused umuC '-' lacZ gene into Salmonella typhimurium .
The E. coli umu operon is responsible for mutagenesis by chemical and radioactivity and can induce mutations by reagents that damage DNA. Induction of umu by test compound, l
It is determined by the development of β-galactosidase activity resulting from the expression of acZ. SOS to detect reagents that damage DNA
A modified method similar to the chromotest method is known as Quiradet.
Lardet) et al . , Proc.Natl.Acad.Sci . (USA ) 79,5971-5975 (1
982). This method uses sfiA , one of the SOS genes, fused to lacZ on the E. coli chromosome.

It is completely different from the assay method that is fixed by the assay method in vitro,
The rodent assay has disadvantages. The most important problem is that it takes a long time to prove that a compound is carcinogenic. This is because this decision is based on the growth of the tumor after exposure to the test compound. In general, animal studies require a period of at least 12-18 months before a compound can be determined not to be carcinogenic. Another problem is genotoxic compounds (DN
It is not possible to distinguish between compounds that induce mutations in A to grow tumors and compounds that modify certain other non-DNA factors to grow tumors. Yet another problem with the rodent assay is that it is a stable method, not a quantitative method, under test conditions:
Only the minimum dose that induces tumor growth in the species tested can be determined.

So far, for non-genotoxic carcinogenic activity,
There are few assays that test compounds in vitro. One in vitro assay is disclosed in US Pat. No. 4,569,916 to Penman and Fey. This method is based on morphological changes induced in cell lines when exposed to test compounds.

Compounds have traditionally been assayed for teratogenic activity or inhibition of cell differentiation by exposing embryos to a test compound and then testing samples from various areas where abnormal or inhibition of proliferation has occurred. For details of the system used, see Flint, Teratology of the Limbs (edited by Marker et al.), Page 325 (Walter de Gruyter).
& Co., Berlin, (1980)) and Flint et al., J. Cell Sci. ,
61,247 (1983); Concepts in Toxicology, Volume 3 In Vitro
Embryotoxicity and Teratogenicity Tests (edited by Homburger et al.), (Karger, Basel, 1985);
Toxic.appl.Pharmac. 76.383 and J.Appl. Toxicol. 4,10
9 (1984). As reported, assays for cytotoxicity and cell differentiation have been performed in vitro with in vivo confirmatory tests. When conducting in vitro studies, at a minimum, cells from the midbrain and forelimb sections should be exposed to the compounds to test for adverse effects. Another in vitro assay for teratogenicity involves the use of vaccinia virus growth in primate cell cultures (Keller, Molecular Toxi.
Biology, 1, 261-276 (1987)), but it is said to predict human teratogenicity in the same manner as in vivo rodent assays.

None of the methods currently used to screen for oncogenic or teratogenic activity in vitro correlate very well with in vivo test results, or to be able to correlate test results. However, testing is time consuming, difficult to quantify, and has limited sensitivity.

(Object of the invention) Therefore, the object of the present invention is simple and quantifiable for mutagens, carcinogens and teratogens,
It is to provide a more sensitive assay method.

Another object of the invention is to provide an accurate measure of mutagenic or teratogenic activity that can be accurately correlated with in vitro observations.

SUMMARY OF THE INVENTION The present invention provides methods and assays using engineered animal cells, animal embryos or differentiated animals, each cell having a target gene and a linked reporter gene.
The assay method of the present invention for screening a compound that causes a mutation or change in the expression of a nucleic acid sequence in a eukaryotic cell includes providing a target gene and a reporter gene,
The product of expression of the target gene regulates expression of the reporter gene. Both target and reporter genes are engineered by splicing an animal promoter / enhancer and transcription termination signal into each coding sequence so that both genes can be expressed in animal cells. The target gene and the reporter gene are introduced into cultured animal cells or embryo cells by the DNA transfection method or the DNA microinjection method, respectively. In the absence of mutations, the product of the target gene inhibits reporter gene expression by interacting with regulatory sequences in the reporter gene. When the target gene is mutated to produce an inactive repressor or other regulatory protein, the reporter gene is no longer repressed and the reporter protein is expressed.

This reporter gene encodes a biologically active protein or antigen. The function and presence of those proteins or antigens can be easily monitored and quantified using standard biochemical methods. This target gene consists of a gene encoding a regulatory protein that can be linked to control the expression of the reporter gene. These genes may be repressors or other regulatory molecules. Mutational inactivation of the repressor gene results in transcription and translation of a defective repressor protein that cannot suppress reporter gene expression. Altering the operator region of the reporter gene has the same effect in a manner that prevents binding of the repressor protein. Expression of the reporter gene can then be monitored by assaying the known function of the gene product.

In the above case, the preferred reporter gene is E. coli containing β-galactosidase and containing an operator.
It is the lacZ gene. The target gene is the E. coli lacI gene, which encodes the lac repressor. In this test,
The treated animal cells are exposed to the mutagen. Before the mutation is induced, the expression of the β-galactosidase gene is suppressed by the binding of the lac repressor protein with the lac operator. The mutation alters the lacI gene to produce a defective lac repressor or alters the operator so that the repressor can no longer bind to the operator. The β-galactosidase gene is then expressed. Cells positive for β-galactosidase are β-galactosidase substrates 5
Addition of -bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) stains blue.

Detection of reporter gene expression is sensitive and quantitative, which facilitates analysis of the effects of test compounds. The assay system of the present invention for rapidly screening a compound that causes a mutation or change in the expression of a nucleic acid sequence in a eukaryotic cell includes a target gene and a reporter gene, and the product of the expression of the target gene is the product of the reporter gene. Regulates expression. By choosing the reporter gene, target gene, promoter / enhancer and operator sequences, and assay conditions, the assay system is capable of many embodiments. Three examples: transgenic animal cell lines for in vitro analysis of mutagens; transgenic animal cell lines for in vivo analysis of mutagens; and for in vivo analysis of teratogens. The transgenic animal embryo system of

(Structure of the Invention) The method of the present invention uses a set of two genes (that is, a target gene and a reporter gene) integrated into an animal or animal cell to induce mutagenesis activity, carcinogenic activity, or teratogenic activity A method for screening a compound having Exposure of the above animals or animal cells to compounds having any of the above activities results in mutations that alter the expression of the reporter gene. Both target and reporter gene constructs include the animal promoter / enhancer, protein coding sequences, and SV40 polyadenylation sequences.

This method has several advantages over conventional methods of screening compounds for mutagenic or teratogenic activity. The most important advantages are the ease of detection and the reduction in the number of false positives. Mutations in the gene encoding the reporter protein have been used for assaying mutation activity for a long time, but this mutation phenomenon prevents the protein from being expressed. Detecting single cells or a small number of cells that do not express a protein while being surrounded by cells that express the protein is a difficult and tedious task, and tends to cause a high error rate. In contrast, in the method of the invention, when a mutation occurs, a reporter molecule that would otherwise not be expressed is finally expressed.

As used in this application, unless otherwise stated, "animal cell"
The term is used to include cells in cell culture, embryos, and cells of differentiated animals. As used herein, the term "mutagenizer" is used to include toxins, carcinogens, teratogens, and other reagents that alter DNA or RNA sequences or expression, unless otherwise indicated.

Reporter genes are selected based on the following criteria:
(I) The reporter gene product must not be deleterious or lethal to the transformed cells, (ii) the gene product must provide a simple and sensitive detection system for its quantification. (Iii) The untransformed cells must have a low constitutive background level of product or activity of the gene being assayed. Reporter genes encoding enzymes, antigens, or other biologically active proteins that can be easily monitored by biological means are preferred. These include
β-galactosidase [Norton, PA and Coffin J.
M.), Mol.Cell.Biol. , 5,281-290 (1985)], peroxidase and luciferase [de Wet Jay.
De Wet, JR et al . , Mol.Cell.Biol. , 7,725
-737 (1987)] is included. In a preferred embodiment, the copy number of the reporter gene in a nuclear animal cell is only 1.
Is one. However, more than one copy may be utilized to increase the amount of reporter gene product. Although not usually required or desired, the same animal cell can contain more than one reporter gene.

The target gene is selected from the group of sequences encoding regulatory molecules that bind to sequences controlling the expression of the reporter gene. These sequences can be repressors or other regulatory molecules, including antisense RNA. In the most preferred embodiment, the lacI repressor gene is used as a target for mutagenesis. When the repressor gene is inactivated by a mutational phenomenon, transcription and translation of a defective repressor protein that can no longer control the expression of the reporter gene, the lacZ gene, which encodes β-galactosidase, occurs. Moreover, when the operator region of the reporter gene is changed so as to prevent binding of the repressor protein, the same effect as described above is produced. Derepression of the reporter gene can then be monitored by assaying gene products of known function.

The bacterial lac operator-reporter system is preferred. The reason is that this system is one of the most basic and thoroughly studied examples of protein-nucleic acid interactions that regulate gene transcription. For this, Coulombre and Miller
(Coulondre and Miller), Mol.Biol. , 117,577 (1977),
And Miller, Ann. Rev. Genet. , 17, 215 (1983). This bacterial regulatory system is transfected into mammalian cells and induces isopropyl β-D.
-Expression has been detected by adding thiogalactoside (IPTG). This is reported in Hu and Davidson, Cell 48,555 (1987), and Brown et al, Cell 49,603 (1987). An important difference between the conventional method, which used the lac operator-repressor system, and the method of the present invention is:
Mutation is used, not induction, to derepress the reporter gene and express a protein that functions alone as an indicator. Another difference is that in a preferred embodiment, a single copy of the functional target gene per cell is
Is to be introduced into the genome.

For repressor proteins that control expression of the reporter gene, the operator sequence must be constructed at a position between the transcription start site and the start codon ATG in the reporter gene. Original lac operator sequence (5'-GGAATTGTGAGCGGATAACAATCC-3 ') or mutant lac operator [eg, sequence that binds repressor 8 times tightly (5'-ATTGTGAGCGCTCACAAT-3')]
Can be used to construct the vector.

The lacI gene has a GTG start codon instead of ATG found in animal cells. This GTG codon can be converted to ATG by in vitro site-directed mutagenesis using the method of Hugh and Davidson, Cell , 48,555 (1987). This modified lacI gene is then inserted into an appropriate expression vector containing a eukaryotic promoter and SV40 polyadenylation site.

Several promoters are good candidates for the construction of expression vectors. Two promoters are commonly used in the methods of the invention. The first type of promoter consists of a ubiquitous promoter (eg, histone gene, ribosomal protein gene, and β-actin gene). The second type of promoter consists of tissue-specific promoters (including SV40 early promoter, Rous sarcoma virus (RSV) long terminal overlap (LTR), and cytomegalovirus (CMV) early gene promoter) . All of these promoters are known to express genes in animal cells.

To construct cells for use as an in vitro mutagenesis model, strong viral promoters (eg RSV-
LTR) is used to drive both the lacI and lacZ genes in animal cells. As a versatile in vivo mutagenesis model, most of the major organs of transgenic mice must express mutagenic target genes and reporter genes. To construct a transgenic animal system, it is preferable to use ubiquitous promoters and enhancers. As a result, the effect of the test compound on the tissue and metabolic properties of various organs containing the target gene can be detected regardless of the specificity of the test substance. The preferred promoter to use as the ubiquitous promoter to drive the lacI gene is the histone 3.2 gene promoter. It is a mouse single copy gene and has a very high transcriptional activity. It is expressed at high levels and accounts for 30-40% of histone H3 mRNA isolated from various mouse tissues.

For mammalian cells and animal tissues where the histone 3.2 gene promoter does not provide optimal expression, other viral or cellular promoters can be selected for proper expression of the lacI gene. Examples of good candidates for forming DNA constructs are human ribosomal protein S14 gene, mouse ribosomal protein S16 gene, rat cytoplasmic β-actin gene, and human β-actin gene. These genes have been cloned, their nucleotide sequences have been determined, and are available for use.

In a preferred embodiment, the reporter gene used with the lacI regulatory gene is a gene encoding the β-galactosidase derived from the bacterial lacZ gene. Expression of β-galactosidase is easily detected using histochemical techniques. In another embodiment, the gene encoding the SV40 large tumor (T) antigen is used as a reporter gene instead of the β-galactosidase gene and immunoperoxidase method detects cells expressing the SV40 T antigen. A construct with the complete sequence encoding the SV40 T antigen is prepared for insertion into a suitable expression vector using methods similar to those used to prepare the lacZ reporter system. The complete transcriptional unit of the SV40 T antigen is then physically linked to the functional transcriptional unit of the lacI gene and transferred into appropriate cells and tested for gene expression.

The principles and standard methods of molecular cloning are described in T. Maniatis et al. (Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY) and is well known to those skilled in the art. This method includes
Techniques included include: DNA and RAN preparation, cloning vector preparation, ligation, transformation of competent cells, in situ filter hybridization selection and screening [David et al.
et al), Advanced Bacterial Genetics (Cold Spring Ha
rbor Laboratory, Cold Spring, NY). Furthermore, separation of DNA by gel electrophoresis, mapping of restriction enzyme cleavage sites,
And techniques for modifying DNA fragments by enzymatic modification are used. Most restriction enzymes, vectors and reagents are commercially available and are available. Conventional vectors and E. coli strains are used. For example, pBR322, pUC sequence, λ-WES, M13mp, DH5, LE392, JM109,
And HB101 etc.

The chain termination method is used to determine the nucleotide sequence and
The splicing site of the construct is identified. This method is described by Sanger et al, Proc.Natl.Acad.Sci.US.
A, 74 , 5463 (1977). Many reagent kits and detailed protocols are commercially available. Since most of the nucleotide sequences of the above-mentioned vector, promoter, and gene to be used are known, oligonucleotides of known sequence are used as primers in sequencing experiments. These nucleotides are typically 15 to 20 nucleotides in length and are described in Messing et al . , Nucleic Acids Res. 9,3
It is very convenient to determine the sequence of a specific region of interest using the method of 09 (1981). Single-stranded DNA and double-stranded D
Any of the NAs can be sequenced this way.

Oligonucleotides used as linkers when determining the sequence of DNA are synthesized using an automatic DNA synthesizer. The service can be obtained from commercial sources such as Genetic Designs, Inc. (Houston, TX). Oligonucleotides with more than 30 nucleotides are then subjected to polyacrylamide gel electrophoresis to confirm high purity.

DNA is transfected into cells by one of the standard published techniques to form stable transformants. Standard methods include calcium phosphate precipitation, DEAE-dextran, electroporation, and protoplast fusion. These methods are detailed below.

Calcium Phosphate Precipitation Method: Graham and Van d'Eve before transferring DNA to cells
er Eb), Virology, 52, 456 (1973). 40 to 50 μg DNA for 0.5 × 10 ⁶ cells plated on 100 mm dish using salmon sperm DNA or calf thymus DNA as a carrier
Is used. Equal volume of 2 x CaCl ₂ (250 mM CaCl ₂ and 1
0.5m 2x Hepes solution with 0mM Hepes, pH7.0)
(280 mM NaCl, 50 mM Hepes, and 1.5 mM Na ₂ HPO ₄ , pH 7.0)
And mix the DNA. After 30 to 40 minutes, a solution in which a white granular precipitate appeared was added dropwise to the above cells and uniformly distributed.
Leave at 37 ° C for 4-16 hours. Remove the medium,
Shock these cells for 3 minutes with glycerol in 15% PBS. 10% after removing glycerol
DMEM containing fetal bovine serum is supplied to the cells and left in the incubator.

Protein samples are prepared for Western blot analysis by lysing cells and separating proteins by SDS PAGE. These proteins are found in Ausubeck et al.
eck et al) Current Protocols in Molecular Biology (J
transferred to nitrocellulose by the electroblotting method described in Ohn Wiley and Sons, 1987). This filter is used for instant skim milk powder (100m
After blocking with 1 g) in PBS, the primary antibody is added and incubated at room temperature for 1 hour. The filters are washed extensively with phosphate buffered saline (PBS) and incubated with horseradish peroxidase-antibody conjugate for 1 hour at room temperature. The filter is washed thoroughly again with PBS and then the band of antigen is fixed by adding diaminobenzidine.

As the enzyme assay method, traditional biochemical methods such as protein purification method are adopted. DNA and RNA are analyzed by Southern and Northern blotting. Typically, the analytical sample is size-fractionated by gel electrophoresis. Then the DNA or RNA of the sample in the gel
Is a nitrocellulose or nylon membrane,
Transferred by blotting method. A blot, a replica of the sample pattern in the gel, is hybridized with Southern and Northern probes. The particular band of interest can then be visualized by a detection system such as autoradiography.

DNA can also be transferred using the DEAE-dextran method described in: Kimura et al. Kimura et al.
al), Virology, 49,394 (1972), and Son Pirac et al.
(Sompayrac et al), Proc.Natl.Acad.Sci.USA, 78,7575 (1
981). DNA is also available in Potter, Proc.Natl.Aca.
d.Sci.USA , 81,7161 (1984), the electroporation method, and Sandri-Goddin et al, Mole.
It can also be transferred by the protoplast fusion method of c. Cell. Biol. , 1,743 (1981).

In a preferred embodiment of the assay, treated animal cells are exposed to the test compound. Expression of the reporter gene is suppressed by linking the lac repressor to the reporter gene operator prior to mutagenesis. When the mutation phenomenon alters the lacI gene to produce a defective lac repressor or the operator changes, the repressor no longer binds to the operator. The reporter gene is then stimulated to synthesize reporter protein.

DNA constructs that are introduced into the animal's genome are processed using substantially the same method. These transgenic animals are used for in vivo mutagenesis studies. A preferred embodiment is a model of a transgenic mouse or rat having the lac repressor gene as a mutagenesis target linked to a stimulatory operator-containing reporter gene, the lac repressor gene being inactivated by mutagenesis. In some cases, the expression of the reporter gene is monitored. Both the target gene and the reporter gene are of bacterial origin, but can be expressed in animal cells when inserted into an animal gene expression vector. According to this transgenic model, the in vivo mutagenesis phenomenon can be easily and easily quantified. Thus, this model allows for more accurate assessment of toxic substance (eg, mutagen, carcinogen, and teratogen) risk than traditional rodent assays. This system is also more sensitive and rapid than standard rodent assays. This is because mutations or alterations in the target gene can be detected at the DNA and cell levels by histochemical methods before the tumor develops.

The present invention is further described by the following examples,
The present invention is not limited to this.

Example 1 In Vitro Animal Cell Mutagenesis Assay (a) Construction of System An example of a useful DNA construct is shown in FIG. This DNA construct is physically linked to each other, and each has a promoter / enhancer, a coding sequence and a transcription termination signal.
It includes individual functional transcription units. One unit is
The other unit is used to drive expression of the lac repressor gene, and the other unit is used to drive expression of the β-galactosidase gene. As shown in the figure, β
-The complete coding sequence for galactosidase contains the RSV-LIR promoter and SV4 used to form the lacZ structure.
0 The polyadenylation site is adjacent and forms a functional transcription unit of the reporter gene. Similarly, the lacI transcription unit contains RSV-LIR, lac repressor coding sequence and SV40.
Built at the polyadenylation site. This reporter transcription unit is then physically linked to the lacI transcription unit by cloning techniques.

The DNA construct containing both the lac repressor transcription unit and the β-galactosidase unit is then used to transfect embryonic cells or epithelial cells, as shown in FIG. Preferred cells are ATCC CRL-1
573 (human 293) and ATCC CCL-T.1 (LLC-MKz),
The American Typ, Rockville, Maryland, USA
It can be obtained from the e Culture Collection. This construct is co-transfected into cells with a neo gene as a selectable marker using known methods such as the calcium phosphate precipitation method described above. Cell colonies resistant to the antibiotic G418 were detected by growth in selective medium, cloned, and the presence of a functional lac repressor gene and β-galactosidase gene, as schematically shown in FIG. Be analyzed.

(b) Analysis and characterization of the system Transfected cells containing the non-functional lac repressor gene and the functional β-galactosidase gene showed 5-bromo-4, a substrate for β-galactosidase.
-Chloro-3-indolyl-β-D-galactopyranoside (X-gal) stains blue when added to cultures without the inducer IPIG. Desirable colonies containing both the functional, lac repressor gene and the β-galactosidase gene do not stain when X-gal alone is added, but should stain blue when IPIG and X-gal are added. . Successful expression of the lacI gene is indicated by Sams et al., J. Biol.chem., 260, 1185.
Page, (1985) Western blot analysis of the lac repressor protein; and Phosphorus and Ridges (Lin
and Riggs), J. Mol.biol. 72, 671 (1972), by a filter binding assay method for the repressor-operator DNA complex.

After successful expression of the lac repressor gene and the β-galactosidase gene in cultured cells, each colony
The copy number of the introduced gene can be analyzed.
Single copy of lacI gene per cell (diploid genome)
Is analyzed by Southern blot analysis of genomic DNA. Cells with a single copy are highly preferred over cells with more than one copy of the functional lacI gene. Because
The latter is because more than one mutation is required to inactivate all copies of the functional repressor gene, resulting in repressor status of the reporter gene. Since the mutation phenomenon is detected by the expression of the reporter gene, the copy number of the reporter gene is not so definitive, but in some cases, it is easier to detect if it exceeds 1 copy. The probability of obtaining a single copy sequence is the low concentration of target DN
It is increased by using A and carrier DNA sufficient to transfect the recipient cells.

(c) Screening of compounds for mutagenic activity Nitrosomethylurea (NMU) can be employed as a model for mutagens. Because Dubridge
Et al., Mol. Cell. Biol. 7, 379, (1987), which shows that this compound easily induces a mutation in the lacI gene in vitro. Cultured cells are treated with NMU for a period of time, and the cells are screened for reporter gene expression. The frequency of mutations is measured by counting the number of β-galactosidase positive cells in total cells. Similarly, the background frequency of mutations can be obtained by counting the number of β-galactosidase positive cells present in cultures that have not been exposed to mutagens.

Example 2: Mutagenesis assay with transgenic animals The first step in creating transgenic animals for use in in vitro assays is lacI in mouse fibroblast cultures.
Is to prepare a β-galactosidase reporter gene induced by the mutation and to detect its activity.
The cultures were ligated using the method described in Example 1.
Obtained by successfully transfecting the lacI-lacZ gene construct and then exposing it to the mutagen to be tested. Controls are cells that have untransfected cells and ligated genes that have not been exposed to mutagens. In an in vitro system, then
C57BL / the founder with the ligated lacI-lacZ gene construct
Production of 6J transgenic mice and then production of transgenic progeny carrying the gene construct is performed as described in FIG. Sexually mature transgenic offspring that are positive for gene transfer serve as controls and for mutagenesis studies.

(a) Preparation of DNA As shown in FIG. 3, a DNA construct was prepared in the same manner as described in Example 1 and used to prepare a transgenic mouse. The above construct has two functional transcription units physically linked to each other, each having a promoter / enhancer, a coding sequence and an SV40 polyadenylation site. One unit is used to drive the expression of the lac repressor gene and the other unit is used to drive the expression of the β-galactosidase gene. The mouse histone 3.2 promoter is utilized in the construct to drive ubiquitous expression of both the lac repressor gene and the β-galactosidase gene. Xho I, as shown in the final vector
The site and the Sal I site are used to clone the coding sequences for the lac repressor and β-galactosidase, respectively.

Target genes such as the lac repressor transcription unit and β
-A DNA fragment containing both the reporter gene such as the galactosidase transcription unit or the SV40T antigen unit is excised from the vector by digestion with restriction enzymes and then subjected to gel electrophoresis and affinity for microinjection as follows. It is isolated by column purification. The fragments are separated by agarose gel and the desired band is recovered by electroelution. Phenol /
Purify by extraction with chloroform, concentration by ethanol precipitation, then binding to an Elutip-d column, followed by elution. The DNA was then recovered by ethanol precipitation and sterilized. It is dissolved in 10 mM Tris buffer (pH 8) containing 1 mM EDTA and quantified by Hoechst's dye fluorescence assay or spectrophotometric method at 260 nm.

(b) Introduction of DNA into embryos The principles and experimental procedure for producing transgenic mice were as follows: Bridid Hogan, Frank Constantini and Elizabeth Lacy.
Elizabeth Lacy) (Coldspring Harbor Laboratory, Cold Spring Harbor, NY, USA) in "Manipulation of Mouse Embryos". The mouse zygote is 5 IU of serum gonadotropin in pregnant female mice.
For 48 hours after administration of human ciliated gonadotropin
It was collected from 6-week-old female C57B / 6J mice (Jackson Laboratory, Bar Harbor, ME, USA) that had been superovulated by administration of 51 U. Sensitized females were placed with C57B / 6J males and examined the next morning for the presence of vaginal plugs. Pregnant females to be used as receptors were selected for estrus and placed in CD-1 female mice with sterilized vasectomized CD-1 male mice (Charles River Laboratories, Massachusetts, USA). , Wilmington). The conjugate was collected in BMOC-2 medium modified to contain 5.1 g / NaCl and 5 mg / m bovine serum albumin. Cumulus cells are removed by treatment with 300 IU (Sigma) in hyaluronidase (Sigma type IV, PBS containing 1% PVP 40T) diluted to 60 IU / m in medium. The debris is removed by washing twice in the medium, and about 2 pl of DNA solution is injected into the pronucleus of males so that about 50% is alive.The injected conjugate is anesthetized with tribromoethanol. Until it moves to the female oviduct of the
Incubated at 37 ° C. in air with 5% CO ₂ .

As reported by Palmiter, Cell, 29, 701 (1982), integration of the gene construct
Generally, a head-to-tail tandem array
Occurs as a single copy or multiple copies of ay). A method for injecting a gene into a mouse embryo is described in Brins (Brins
ter) et al., Proc. Natl. Acad. Sci. USA , 82, 4438 (1985).
It is described in. The more important factors that increase the frequency of integration are: injected DNA
Concentration is a limiting factor for integration efficiency, 1-2 ng /
DNA concentrations or about 100-1,000 copies of DNA fragments appear optimal; more efficient when linear DNA molecules are cleaved with endonucleases to give nonblunt ends; and Injection into the nucleus rather than into protoplasts is necessary for high efficiency. Some other factors do not appear to affect the integration rate. Female and male pronucleus injections can accommodate the injected DNA, giving equal integration efficiency. When DNA is injected into the nucleus of a 2-cell embryo, it becomes a transgenic animal.
Injection into the pronucleus is not essential for triggering integration.

It has been suggested that the mouse strain used will affect the efficiency of integration. For example, eggs of the C57 x SJL hybrid have been shown to be more efficient than the inbred C57 line. Efficiency comparable to the above hybrids has been obtained with inbred CD-1 mice (Charles River). Inbred C57BL /
6J mice (Jackson Laboratories) can be used to eliminate segregation of latent background-modifying genes. Female inbred C57BL / 6J black mice were administered with follicle-stimulating hormone and luteinizing hormone to induce hormone-induced superovulation. These mice are then mated with inbred male mice.

Fertilized single cell embryos are washed from the oviduct and treated with hyaluronidase to remove cumulus oophorus cells from the embryo. Next, the target DNA is injected by microinjection into the pronucleus of a single-cell embryo. About 100 copies of lacI-1
The acZ DNA construct is injected into each embryo, then each embryo is transferred to the oviduct of a pseudopregnant foster mother to complete the gestation period. These embryos can differentiate and develop into whole animals.

(c) Selection and assay of transgenic mice for in vivo mutagenesis model Transgenic mice with a single copy of the bound functional lacI-lacZ gene construct are screened and selected by the following method. The tail section and / or the lobe of the liver,
Used as the source for separating genomic DNA. The copy number and structure of the introduced gene were analyzed by Southern blotting with DNA from the lac repressor gene or reporter gene used as a probe, and during the development of the transgenic mouse, within the introduced gene Check to see if rearrangements or modifications have occurred. Full lacI
Only mice with a single copy of the lacZ gene construct are further assayed.

To assay gene expression, appropriate tissues are collected from transgenic mice at necropsy. RNA and protein are extracted from these tissues and used for Northern blot assay, Western blot assay and repressor function assay to detect the expression of the lac repressor gene in the tissues of transgenic mice. The reporter gene should be repressed by the active lacI gene, so there is no functional lac repressor or the inducer IP
Expression of the reporter gene in tissue is not expected unless TG is added. Therefore, the use of the same promoter and enhancer in the structure of both the repressor and reporter genes is an important way to avoid "uncoupling" of the two genes. Functional lacI and lacZ in tissues of transgenic mice
The presence of the gene can also be assayed by the method described in Example 1 (b) using cells isolated from tail tissue.

Only a gene transfer system having a functional target gene at a ratio of 1 copy per cell is selected and propagated to form a homozygous gene transfer system as shown in FIG. The 1/4 homozygous gene transfer system that breeds F ₁ together and is expected in F ₂ is identified by the intensity of the Southern analysis signal. Homozygosity is confirmed by offspring reproductive studies and Southern analysis. Homozygous offspring carrying two copies of the introduced gene are propagated and crossed with a non-transgenic partner to produce homozygous test animals for mutagenesis assays.

(d) Assay of transgenic animals exposed to test compound For localization and distribution of positive cells in major organs of both control and mutagen-treated mice,
Quantitative data can be obtained. Tissues from the following organs were selected for morphometric analysis. The organs are the brain (tissues collected from the cerebellum, midbrain and forebrain), liver, spleen, kidneys, left ventricle of the heart, lungs, gonads, uterus,
The pancreas, fundus, duodenum, ileum, colon, and sternal bone marrow. Following fixation, tissue blocks are excised and histochemical reactions to β-galactosidase are performed on 8 micron cryostat sections or 30 micron vibratome sections to ensure random sampling. Serial sections separated by 40 microns are collected from each block. Counting of positive cells is performed by digitized morphometry using a biological image analyzer system and the Bio-quant II computer program (R & M Biometrics, Nashville, TN, USA). The hardware of this system is an image projection camera, Apple II, mounted on a standard Zeiss binocular microscope.
It consists of a microcomputer and a digitizing tablet. With this system, a sample is projected onto a computer screen and the operator selects a region for analysis and draws a sketch. An outline of the blood vessel and the space from which exists as the lumen of the hollow organ is outlined on the screen, and then the system is instructed to remove the area from the area to be analyzed. The strongly blue stained cells are counted using a digitizer and the number of positive cells per unit area of the tissue sample is automatically calculated. In this way, the number of positive cells per unit area of specific organs from untreated or treated animals can be counted and the data obtained are compared statistically. An additional major advantage of this system is the ability to identify and quantify specific cell lines that are particularly sensitive to a given mutagen.

Histochemical analysis is performed on tissues from treated and untreated male and female transgenic mice of the same strain. The morphometric method provides quantitative data on the number of β-galactosidase-positive cells per organ and animal sampled to inform the test compound, and the efficacy of the test method and the natural background of transgenic animals. It can be used to examine the frequency of ground mutations. The expression of the lacZ gene in cells is expressed by β-
It is assayed by the galactosidase staining method.

Immerse the tissue in cold 4% paraformaldehyde solution supplemented with 50 mM Na ₃ PO ₄ . The pH of this mixture is adjusted to 7.4. The soaked tissue is fixed in this mixture at 4 ° C overnight. The activity of the enzyme is not impaired by this fixation. The tissue is then rinsed in phosphate buffered saline (PBS) and 30 μm sections are cut with a vibratome or 8 μm sections with a cryostat. Next, the obtained section was cut with 1 mg / m of X-ga.
l, 5 mM potassium hexacyanoferrate (III), 5 mM potassium hexacyanoferrate (II), 2 mM MgCl ₂ , 0.02%
PBS containing NP-40 and 0.01% chlorate (pH 7.
Incubated in 3) at 37 ° C. in a 5% CO ₂ atmosphere. Bring this solution to an alkaline pH, and
Addition of ⁺⁺ is important to avoid background staining due to galactosidase activity naturally present in lysosomes. Cells in the test tissue that are positive for β-galactosidase stain blue. This blue color becomes visible within 1 hour and increases in intensity over the next 12 hours. This phenomenon can be visually observed in both freely moving vibratome sections or cryostat sections fixed to gelatin-coated glass slides. Once the desired color intensity was obtained, the vibratome sections were mounted on glass slides coated with 0.1% KCrSO ₄ and 1% gelatin and heated at 37 ° C. for 0.5 hour to dry. The sections were then dehydrated and washed in xylene for 2-3 minutes. Counterstain with methyl green, then place a coverslip over each part. Paraffin sections of all tissue inclusions can also be stained. This is because the post-embedding of this section does not impair the blue-positive staining. Therefore, paraffin-embedded sections can be stored and stored indefinitely as a permanent record of each test method.

When SV40 large T antigen or other antigen is used as a reporter gene, cytochemical immunoperoxidase method,
[For example, Hanahan, Nature , 315, 115 (198
5); Ornitz et al., Science , 238, 188 (198).
7); or Behringer, Proc.Natl.Acad.
Sci . USA , 85, 2648 (1988)]
Is used to detect expression of the reporter protein.

Example 3: Embryos of transgenic animals for teratogenic assay Embryos of transgenic animals having a target gene and a binding reporter gene are used for screening for teratogenic substances. This transgenic embryo model allows simple and rapid quantification of mutagenesis in a system similar to that of living human embryos, and is more accurate and accurate than teratogenic compounds compared to currently used methods. You should be able to assess the risk.

Transgenic animals, such as mice and pigs, carrying the lacI gene linked to the lacZ gene, generated by the method detailed in Example 2, are used as test animals to generate transgenic offspring. Embryos isolated at various stages from female transgenic pregnant animals with successful gene expression were exposed to test reagents and analyzed for reporter gene expression.

For example, a 10 mm transgenic pig embryo that develops during the 20-21 day gestation and that has primordia with almost all adult structures is exposed to potentially teratogenic substances.
The mutation caused by the teratogen inactivates the target gene and activates the reporter gene in certain cells / tissues of the embryo. These cells / tissues may be expressed in the histochemical assay described above, or Allen ND, et al., If the reporter protein is β-galactosidase or another suitable alternative to the reporter protein. “Transgenes as probes for activ
e chromosomal domains in mouse development ”, Natu
Re , 333, p. 852-855 (1988).

Modifications and alterations of detection methods and systems, target genes linked to reporter genes, and transgenic animals and transgenic animal cells for testing mutagens and teratogenic agents are described in It will be apparent to those skilled in the art from the description. Such modifications and variations are within the scope of the present claims.

SUMMARY OF THE INVENTION A rapid, sensitive and quantitative method and assay for rapidly screening compounds for mutagenic or teratogenic activity is provided. in this way,
Gene-transferred animal An animal in which a gene has been transferred to cells, adults or embryos is used. Each cell from cell culture, differentiated tissue from an animal, or animal embryo contains a target gene and a reporter gene. Both have an animal promoter / enhancer, a coding sequence and a transcription termination signal. Therefore, they can be expressed in animal cells. The product of the target gene can regulate the expression of the reporter gene by interacting with regulatory sequences in the reporter gene.

In a preferred embodiment, the target gene lacI (lac repressor) binds to the reporter gene lacZ (encoding β-galactosidase). It is detected by cytochemical or histochemical methods. Animal cells
The reporter gene is expressed when exposed to a compound that causes a mutation that alters the target gene or alters the operator of the reporter gene.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing the structure of the expression vector pRSV-I-pRSV-Z. This expression vector has the complete coding sequence for the lac repressor flanked by the RSV-LTR promoter and the SV40 polyadenylation site, which forms the functional transcription unit of the target gene; and the RSV-LTR promoter and SV40 polyadenylation. It has the complete coding sequence for the β-galactosidase enzyme flanked by ligation sites, which form the functional transcription unit of the reporter gene. FIG. 2 is a schematic diagram showing a method of constructing and testing transfected human embryonic cells or monkey epithelial cells containing pRSV-I-RSV-Z. FIG. 3 is a schematic diagram showing the structure of an expression vector inserted into mouse fibroblasts or embryos. This expression vector has two functional transcription sites physically linked to each other, each having a promoter, histone 3.2 and SV40 polyadenylation sites. One promoter was used to insert the lacI gene into the XhoI site of the final vector, and the other promoter was used to express the β-galactosidase gene.
It is used by inserting it into the Sal I site of the final vector. FIG. 4 is a schematic diagram showing a method of producing a transgenic mouse having a functional target gene and a reporter gene and performing an in vivo mutagenesis assay. FIG. 5 is a schematic diagram showing a method for breeding and producing transgenic mice used in an in vitro mutagenesis assay.

Claims (28)

[Claims] 1. A method for screening a compound that causes a mutation or a change in the expression of a nucleic acid sequence of a eukaryotic cell, which comprises providing a target gene and a reporter gene, wherein the product of the expression of the target gene is A screening method capable of regulating the expression of the reporter gene. 2. The reporter gene has a eukaryotic promoter, a regulatory sequence, a sequence encoding a reporter gene product, and a transcription termination signal, and the product of expression of the target gene is a regulatory sequence of the reporter gene. 2. The method of claim 1, which binds to and regulates expression of a sequence encoding the reporter gene product. 3. The target gene is a eukaryotic promoter,
The method according to claim 2, which has a sequence encoding a regulatory molecule and a transcription termination signal. 4. The method of claim 2, wherein the reporter gene product is selected from the group consisting of biochemically detectable protein and nucleic acid sequences. 5. The method of claim 2, wherein the regulatory sequence is an operator. 6. The regulatory product of the target gene is selected from the group consisting of a repressor and a nucleic acid sequence capable of binding to the reporter gene or gene product.
The method described in. 7. The method according to claim 6, wherein the product of the reporter gene is β-galactosidase and the regulatory molecule is the lac repressor protein. 8. The method according to claim 1, further comprising inserting the target gene and the reporter gene into a eukaryotic cell. 9. The method according to claim 8, wherein the eukaryotic cell is an animal embryo in an early stage of differentiation. 10. The method of claim 8, wherein the eukaryotic cell is in a fully differentiated animal tissue. 11. The method according to claim 8, further comprising controlling the copy number of the target gene and the reporter gene inserted into an animal cell. 12. The method according to claim 11, wherein the copy number of the functional target gene is limited to one. 13. exposing the animal cell to a compound;
9. The method of claim 8, further comprising: and comparing the expression of the reporter gene product in the exposed cell with the expression of the reporter gene product in a control cell that has not been exposed to the compound. 14. Exposing the embryo to a compound; allowing the embryo to undergo cell division for a period of time; and expressing the reporter gene product in the embryo with respect to expression of the reporter gene product in the embryo not exposed to the compound. 10. The method of claim 9, further comprising comparing with expression. 15. exposing said differentiated animal to a compound; metabolizing said compound and causing cell division in said animal; and expressing expression of the reporter gene product in tissue from said exposed animal. 11. The method of claim 10, further comprising comparing with expression of a reporter gene product in syngeneic tissue of a control animal that has not been exposed to the compound. 16. The expression of the reporter gene is measured by encoding the β-galactosidase with the reporter gene, providing a substrate for β-galactosidase, and staining with a ferricyanate-ferrocyanate solution. Further embraces,
The method of claim 8. 17. The method according to claim 8, further comprising measuring the expression of the reporter gene using an immunoglobulin bound with an enzyme and detecting the expression of the antigen by an immunochemical method. 18. A system for rapidly screening for a compound that causes a mutation or a change in the expression of a nucleic acid sequence in a eukaryotic cell, which comprises a target gene and a reporter gene, and the product of expression of the target gene is A screening system capable of regulating the expression of a reporter gene. 19. The reporter gene has a eukaryotic promoter, a regulatory sequence, a sequence encoding a reporter gene product, and a transcription termination signal, and the product of expression of the target gene regulates the reporter gene. 19. The system of claim 18, which binds to a sequence and regulates expression of the sequence encoding the reporter gene product. 20. The system according to claim 19, wherein the target gene has a eukaryotic promoter, a sequence encoding a regulatory molecule, and a transcription termination signal. 21. The system of claim 19, wherein the reporter gene product is selected from the group consisting of a biochemically detectable protein and a nucleic acid sequence complementary to the reporter gene sequence. 22. The system of claim 21, wherein the reporter gene product is selected from the group consisting of β-galactosidase, luciferase, peroxidase, and immunochemically detectable antigens. 23. The system of claim 20, wherein the regulatory gene product is selected from the group consisting of a repressor and a nucleic acid sequence capable of binding to the reporter gene or gene product. 24. The system of claim 23, wherein the regulatory molecule is the lac repressor protein and the regulatory sequence is the lacZ operator. 25. Histone 3.2 gene, human ribosomal protein S14 gene, mouse ribosomal protein S16 gene, rat β-actin gene, cytomegalovirus early gene promoter, SV40 early promoter, Rous sarcoma virus long terminal repeat, and Moloney leukemia virus. 19. The method further comprising at least one promoter selected from the group consisting of long terminal repeats.
The system described in. 26. The system of claim 18, further comprising a eukaryotic cell. 27. The system according to claim 26, wherein the eukaryotic cell is an embryo of an animal in an early stage of differentiation. 28. The system of claim 26, wherein the animal cell is in a fully differentiated animal tissue.