JPH02145200A - Inspection for rapidly screening mutagenicity and teratogenicity - Google Patents

Abstract

PURPOSE: To enable to highly sensitively and rapidly screen mutagenesis and teratogenesis by binding a product obtained by the expression of a target gene to a regulatory sequence of a reporter gene.

CONSTITUTION: A target gene such as Escherichia coil Lac1 gene encoding lac repressor and a reporter gene such as Escherichia coli lac Z gene encoding β-galactosidase and containing an operon are transduced into an animal cell such as a mouse embryo cell by a microinjection method. The transduced cell is cultured in a culture medium containing a mutagen 1P1G and 5-bromo-4- chloro-3-indolyl-β-D-galactopyranoside (X-gal), a substrate of the B-galactosidase, to obtain the galactosidase-positive cells where the culture medium is colored into a blue color with the expressed product. The positive cells are again treated with the X-gal, and the obtained lac 1-positive cells are treated with nitrosomethyl urea. The number of the β-galactosidase-positive cells in the whole cells is measured to assay the mutagenesis and the teratogenesis.

COPYRIGHT: (C)1990,JPO

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to genetic engineering, particularly in the assay of mutagens. Encompasses the use of treated animal cells containing a target gene linked to a reporter gene (both genes capable of expression in the animal cell).

BACKGROUND OF THE INVENTION Many new pharmaceutical products and agents released into the environment or workplace require toxicity testing. Compounds must generally be assayed for damage to embryos (teratogenic activity) and to differentiated animals (carcinogenic or mutagenic activity).

Traditionally, compounds have been tested in bacterial cell systems for short periods of time (
Mutagenic activity has been assayed using STT) or animal experiments. Most animal experiments began in the early 1970s at the National Cancer Institute.
This is done using a protocol for affidavits developed by the National Certification Institute. This protocol was developed by Zinterf et al.

It is. However, the correlation between the results obtained with the above two types of systems is poor and difficult to evaluate objectively. For example, Tennant et al.
ence, 236.933-941 (1987), there is only about 60% agreement between the four widely used STT methods and the results of sworn animal carcinogenicity tests. The four test methods compared are as follows: Salmonella mutagenesis method.

SAL method (Haworth et al., En
viron, Mutagen.

55uppl, 1)3 (1983) and Mortelmans' et al., Toxico
l, Appl, Pharmacol, 75
．． 137. (1984>, etc.); A method using chromosomal abnormalities in Chinese hamster ovary cells (ABS method): A method using sister chromatid exchange in Chinese hamster ovary cells (SCB method) (2)
How?

(1987)); and the mouse lymphoma cell method (MOLY method) (Meyer (My.
h r ) pp. 55-568, J.A. Ashby (
J. Ashby et al. (Blsevier, Amsterdam; 1985)).

A recent modification of the SAL method, or Ames in vitro assay, was described by Oda et al., Mutation Research, 14.
7. 219-229 (1985). Oda et al.
phimurium). The E. coli umu operon is amenable to chemical and radioactive mutagenesis, with mutations caused by DNA-damaging reagents.

It can be induced. Induction of umu by test compound, la
It is determined by the generation of β-galactosidase activity resulting from the expression of cZ. A modified method similar to the SOS chromotest method for detecting reagents that damage DNA is described by Quillardet et al., Proc.
tl, Acad.

Sci, (IIsA) 79.5971-5975
(1982). This method uses SO3
One of the genes, 5fiA, is located on the E. coli chromosome.
It is used by fusing it with cZ.

It is completely different from the in vitro assay method that fixes the
There are disadvantages to the sworn animal testing method.

The most important problem is the long time it takes to prove that a compound is carcinogenic. This is because this determination is based on tumor growth after exposure to the test compound. in general.

Animal studies require a period of at least 12-18 months before it can be determined that a compound is not carcinogenic. Another problem is the inability to distinguish between genotoxic compounds (compounds that induce mutations in DNA that cause tumors to grow) and compounds that change certain other non-DNA factors that cause tumors to grow. -It means not being able to come. A further problem with the animal assay is that it is a qualitative, not quantitative, method, and under the assay conditions it is possible to determine the lowest dose that induces tumor growth in the species being tested. It's nothing more than that.

To date, regarding non-genotoxic carcinogenic activity,
What are the assay methods for testing compounds in vitro?

There is no way. One of the in vitro assay methods.

Penman and Fey
No. 4,569,916.

This method is.

This method is based on the morphological changes induced in cell lines when exposed to a test compound.

Compounds have traditionally been assayed for teratogenic activity or inhibition of cell differentiation by exposing embryos to the test compound and then testing samples from various areas where abnormalities or inhibition of proliferation have occurred. Details of the system used can be found in Flint.

Teratology of the Limbs
(7-Edited by Merker et al.), p. 325 (
Waiter de Gruyter & Co, Berlin, (1980)) and Flint et al., J.
Ce1l Sci,.

Te5ts (edited by Hamburger et al.), (Karger.

and J. Appl, Toxicol, 4.109
(1984). As reported, assays for cytotoxicity and cell differentiation have been performed in vitro along with in vivo confirmatory tests. When performing in vitro studies, at a minimum, cells from the midbrain and forelimb regions must be exposed to the compound and tested for adverse effects. Other in vitro assays for teratogenicity include methods that utilize growth of cotton cinavirus in primate cell cultures (Keller,
Molecular Toxicology, 1°261-276 (1987)) has been reported to be as predictive of human teratogenicity as in vivo sworn animal assays.

With methods currently used to screen for carcinogenic or teratogenic activity in vitro.

None of the test results correlate very well with in vivo test results, or even if the test results can be correlated, they are time-consuming to test.

Difficult to quantify and has limited sensitivity.

(Object of the Invention) It is therefore an object of the present invention to provide a simple, quantifiable, and
The objective is to provide a more sensitive assay method.

Another object of the invention is to accurately correlate with in vitro observations. The objective is to provide an accurate measure of mutagenic or teratogenic activity.

(Summary of the Invention) The present invention provides a target gene and a reporter linked to each cell.
It has a gene. Engineered animal cells.

Methods and assays using animal embryos or differentiated animals are provided. The assay method of the present invention for screening for 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, and the product of expression of the target gene is the reporter gene. - Regulate gene expression. By splicing an animal promoter/enhancer and a transcription termination signal into each coding sequence, both target and reporter genes are manipulated so that both genes can be expressed in animal cells. A target gene and a reporter gene are introduced into cultured animal cells or embryonic cells by DNA transfection or DNA microinjection, respectively. In the absence of a mutation, the product of the target gene inhibits expression of the reporter gene by interaction with regulatory sequences in the reporter gene. When a mutation occurs in the target gene, producing 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 genes encoding regulatory proteins that can be linked to control the expression of the reporter gene. These genes may be repressors or other regulatory molecules. When a repressor gene is inactivated by mutation, transcription and translation of a defective repressor protein that cannot suppress the expression of the reporter gene occurs. Altering the operator region of the reporter gene produces 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.

A preferred reporter gene in the above case is the E. coli lacZ gene which encodes β-galactosidase and contains an operator. What is the target gene?

This is the E. coli lacI gene encoding the lac repressor. In this assay, treated animal cells are exposed to a mutagen. Before mutations were induced.

The expression of the β-galactosidase gene is suppressed by binding of the Iac repressor protein to the lac operator. The mutation changes the Iacl gene, resulting in a defective 1ac! Is J Breather produced?
Or the operator changes and the repressor can no longer bind to it.

The β-galactosidase gene is then expressed.

Cells positive for β-galactosidase are 5-bromo-4-chloro-3, a substrate for β-galactosidase.
-indolyl-β-D-galactopyranoside (X-ga
When 1) is added, it is dyed blue.

Detection of reporter gene expression is desensitized and quantitative, facilitating analysis of the effects of test compounds.

The assay system of the present invention for rapidly screening compounds that cause mutations or changes in the expression of nucleic acid sequences in eukaryotic cells includes a target gene and a reporter gene,
Products of expression of the target gene regulate expression of the reporter gene. Many embodiments of the assay system are possible depending on the choice of reporter gene, target gene, promoter/enhancer and operator sequences, and assay conditions. Three embodiments, namely:
transgenic animal cell lines for in vivo analysis of mutagens; transgenic animal cell lines for in vivo analysis of mutagens; and transgenic animal lines for in vivo analysis of teratogens. List it.

(Margins below) (Structure of the Invention) The method of the present invention uses a set of two genes (i.e., a target gene and a reporter gene) integrated into an animal or animal cell to generate a protein that has mutagenic activity, carcinogenic activity, and carcinogenic activity. , or a method of screening for compounds with teratogenic activity. Exposure of the animal or animal cell described above to a compound having any of the activities described above results in mutations that alter the expression of the reporter gene. Both the target gene and reporter gene constructs include an animal promoter/enhancer, a tanhakuo encoding sequence, and an SV40 polyadenylation sequence.

This method has several advantages over traditional methods of screening for compounds with mutagenic or teratogenic activity. The most important advantages are ease of detection and reduced number of false positives. Mutation of the gene encoding a reporter protein has long been used in assays for mutation activity, but this mutation phenomenon results in the protein not being expressed. Detecting a single cell or a small number of cells that do not express a certain protein while surrounded by cells that do express it is a difficult and tedious task, prone to high error rates.

In contrast, 9 in the method of the present invention, if a mutational event occurs, a reporter molecule that would otherwise not be expressed will eventually be expressed.

As used in this application, unless otherwise specified, "animal cells"
The term refers to cells in cell culture, embryos.

and differentiated animal cells. As used herein, the term "mutagen" refers to toxin 1 carcinogens, teratogens, unless otherwise specified.

and other agents that alter the sequence or expression of DNA or RNA.

Reporter genes are selected based on first-order criteria +
(i) the product of the U porter gene must not be harmful or lethal to the transformed cells; (ii)
) the gene product must provide a simple and sensitive detection system for its constant detection; (i)
ii) Untransformed cells should have low constitutive background levels of the 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 methods are preferred.

These include β-galactosidase [two-tone P.
A. and Coffin G. M. (Norton,
P, A, and Coff1n J, M,), M
o1. Ce1l, Biol.

5, 281-290 (1985) Peroxidase and Luciferase
al), Mol, Ce1l, Biol,
.

7, 725-737 (1987)]. In a preferred embodiment, the number of copies of the reporter gene in each animal cell is only one. however.

To increase the amount of reporter gene product.

More than one copy may be utilized. Although usually not required or desirable, it is possible for the same animal cell to contain more than one reporter gene.

The target gene is selected from the group of sequences encoding regulatory molecules that bind to sequences that control expression of the reporter gene. These sequences include antisense RNA.

It may be a repressor or other regulatory molecule. In a most preferred embodiment, the 1acI repressor gene is used as a target for mutagenesis. When the repressor gene is inactivated by mutational events, transcription and translation of the defective repressor protein occurs, which can no longer suppress the expression of the reporter gene, the lacZ gene encoding β-galactosidase. Additionally, when the operator region of the reporter gene is altered to prevent binding of the repressor protein, the same effect as described above occurs. Derepression of the reporter gene can then be monitored by assaying gene products of known function.

Bacterial lac operator-repressor systems are preferred. This is because this system is one of the most fundamental and thoroughly studied examples of protein-nucleic acid content interactions that regulate gene transcription. On this, see Coulondre and Miller,
Mol, Biol, 117.577 (1977
), and Miller, Ann, Rev, Genet
, 17.215 (1983).

This bacterial regulatory system has been transfected into mammalian cells and expression detected by addition of the inducer isopropyl β-lowthiogalactoside (IPTG). This is shown in Hu and Davidson, Ce1l,
48.555 (19g?), and Brown et al.
own et al), Ce11.49.603
(1987). Iac Operator Conventional method using IJ presser system and 1
An important difference with the method of the present invention is that mutation, rather than induction, is used to derepress the reporter gene and express a protein that has the function of acting alone as an indicator. Another difference is that in preferred embodiments, a single copy of the functional target gene per cell is introduced into its genome.

For repressor proteins that control the expression of a reporter gene, the operator sequence must be constructed at a location between the transcription start site and the start codon ATG in the reporter gene. The original Iac operator sequence (5'-GGAATTGTGAGCGGATAACA
ATCC-3'), or a mutant 1ac operator [e.g., a sequence that binds the repressor 8 times more tightly (5
'-ATTGTGAGCGCTCACAAT-3')
] can be used for vector construction.

The 1acl gene has a GTG start codon instead of the ATG found in animal cells. This GTG codon.

Hieu and Davidson, Ce1l, 48.55
5 (1987) by in vitro site-directed mutagenesis. This modified 1acf gene is then inserted into a suitable expression vector containing a eukaryotic promoter and an SV40 polyadenylation site.

A number of promoters are excellent candidates for construction of expression vectors. Two types of promoters are generally used in the methods of the invention.

The first type of promoter consists of 1M promoters (eg, histone genes, liposome protein genes, and β-actin genes).

The second type of promoter is one tissue-specific promoter (SV40 early promoter, Rous sarcoma virus (R
3V) and the early gene promoter of cytomegalovirus (CMV). All of these promoters are known to drive gene expression in animal cells.

To construct cells for use as in vitro mutagenesis models, use a strong viral promoter (e.g., R
3V-LTR) to activate both the Iacl and IacZ genes in animal cells.

As an in vivo mutagenesis model with many uses, most of the major organs of the transgenic mouse must express the mutagenic target and reporter genes. Preferably, the ubiquitous promoter and enhancer are used to construct transgenic animal systems. As a result, regardless of the specificity of the test substance.

The effects of test compounds on tissue and metabolic properties of various organs, including target molecules, can be detected. 1a
A preferred promoter for use as the ubiquitous promoter to drive the cl gene is the histone 3.2 gene promoter. This is a single copy gene in mice and has very high transcriptional activity. and histone H3, which is expressed at high levels and isolated from various mouse tissues.
It accounts for 30-40% of mRNA.

For mammalian cells and animal tissues where the histone 3.2 gene promoter does not provide optimal expression, other viral or cellular promoters may be selected and 1a
The cl gene can be appropriately expressed. Examples of good candidates for forming DNA constructs are the human ribosomal protein S14 gene, the mouse ribosomal protein S16 gene, the rat cytoplasmic β-actin gene, and the human β-actin gene. These genes have already been cloned.

Its nucleotide sequence has been determined and it can be used by one person.

In a preferred embodiment, the reporter gene used with the IacI regulatory gene is a gene encoding β-galactosidase from the bacterium lacZ. Expression of β-galactosidase is easily detected using nine histochemical techniques. In other embodiments, SV4
The gene encoding the 0 large tumor (T) antigen was used as a reporter gene instead of the β-galactosidase gene, and SV40 was detected by the immunoperoxidase method.
Cells expressing T antigen are detected. SV for insertion into a suitable expression vector using methods similar to those employed to prepare the lacZ reporter system.
A construct with the complete sequence encoding the 40 T antigen is prepared.

The complete transcription unit of the SV40 T antigen is then
physically linked to the functional transcription unit of the 1acl gene.

It is then transferred into appropriate cells and tested for gene expression.

Principles and standard methods of molecular cloning.

ToManiatis et al.
al ) (Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY)
ar C1onin3 and is widely known to those skilled in the art. This method includes the following techniques: DNA and RAN preparation, cloning vector preparation, ligation, transformation of competent cells,
Selection and screening by in 5 intu filter hybridization method [Depid et al.
ivid et al), Advancecl Ba
cterialGenetics (CoM Spri
ng Harbor Laboratory, -1
- Cold Spring, New York). Furthermore, techniques for separating DNA by gel electrophoresis, mapping restriction enzyme cleavage sites, and modifying DNA fragments by enzymatic modification are used.

Most restriction enzymes, vectors, and reagents are commercially available and can be done by one person.

Normal vectors and E, col
i) A bacterial strain is used. For example, pBR322, pU
c series, λWES, M13mp, Dt15. LB
392. JM109. and HBOI.

The chain termination method is used to determine nucleotide sequences, D
The splicing sites of the NA construct are confirmed. This method was described by Sanger et al.

has been reported. Many reagent kits and detailed protocols are commercially available. Since most of the nucleotide sequences of the vector promoters mentioned above and the genes used are known, they can be used as primers in sequencing experiments.

Oligonucleotides of known sequence are used.

These nucleotides are typically 15-20 nucleotides long and are described by Messing et al.
at), Nucleic Ac1ds Res, 9.
309 (1981) is very convenient for determining the sequence of a specific region of interest. -Book 1JI
Both DNA and double-stranded DNA can be sequenced using this method.

Oligonucleotides used as linkers in determining the sequence of DNA are synthesized using an automatic DNA synthesizer. This service is GenetiCDesi
gns, Inc., (Heuston, Texas). Oligonucleotides consisting of more than 30 nucleotides are then subjected to polyacrylamide gel electrophoresis to ensure high purity.

DNA is extracted by one of the standard published methods.
Transfected into cells and stable transformants are formed. Standard techniques include calcium phosphate precipitation, DB8-dextran, electroporation, and protoplast fusion. These methods are detailed below.

Calcium Phosphate Precipitation Method: Before transferring the DNA into the cells, the DNA is
and Van der Eb), Virolog
y, 52.456 (1973), with calcium phosphate. salmon sperm dna
Or use calf thymus DNA as a carrier, 10
40-50 μg of DNA is used for 0.5×10 cells plated in a 0 mm dish. equal volume of 2 x cac]2 (250 mM CaCl2 and 1
0,5 with 0mM Hepes, pH 7,0)
d 2X Hepes solution (280mM NaCl, 5
0mM 1lepes, and 1.5mM Na2HP
O4, pH 7,0) and the DNA. 30-4
The solution in which a white granular precipitate appeared after 0 minutes was added dropwise to the above cells, distributed uniformly, and incubated for 3 to 16 hours.
Leave at 7℃. Remove medium and add 15% of glycerol
Shock these cells for 3 minutes with PBS solution. After removing the glycerol.

The cells were fed with DMBM containing 10% fetal bovine serum.

Leave it in a warmer.

For protein samples, lyse cells and extract proteins from 5O3-P.
Prepared for Western blot analysis by separation using the AGIE method. These proteins are transferred to nitrocellulose using the electroblotting method described in Section A. After blocking the filter with instant skim milk powder (100 mj2 Ig in PBS), the next antibody was added.

Incubate for 1 hour at room temperature. The filter was thoroughly washed with phosphate buffered saline (PBS).

Incubate with horseradish peroxidase-antibody conjugate for 1 hour at room temperature. The antigen band is fixed by thoroughly washing the filter again with PBS and then adding diaminobenzidine.

Traditional biochemical methods such as enzyme assays and protein purification methods are employed. DNA and RNA are analyzed by signature and Northern blotting methods. Typically 9 analytical samples are size fractionated by gel electrophoresis. Next.

The sample DNA or RNA in the gel is transferred to a nitrocellulose or nylon membrane by a plotting method. Plots that are replicas of the sample pattern in the gel are hybridized with Southern and Northern assay probes. The specific bands of interest are then detected using a detection system such as autoradiography.

It can be visualized.

DNA again. It can also be transferred using the DBAR-dextran method described in Kimte et al.
imura et al), Virology,
49.394 (1972).

and Sompayrac et al.
al).

Proc, Natl, Acad, Sci, US
A, 78.7575 (1981).

DNA was further described by Potter, Pr.
oc, Natl.

Acad, Sci, USA, 81.7161 (
1984) and the electroporation method of Sandri-Goddin et al.
), Molec, Ce11. Biol...
It can also be transferred by the protoplast fusion method of 1.743 (1981).

In a preferred embodiment of the assay, treated animal cells are exposed to a test compound. By binding the lac repressor to the operator of the reporter gene prior to mutagenesis, expression of the reporter gene is suppressed. Due to mutational phenomenon, the 1acl gene changes and becomes defective 1a.
c If the IJ repressor is produced or the operator changes, the repressor no longer binds to the operator.

The reporter gene is then stimulated to synthesize reporter protein.

DNA constructs introduced into the animal's genome are processed using substantially the same methods. These transgenic animals are used for in vivo mutagenesis studies. A preferred embodiment uses lac as a mutagenic target linked to a stimulable operator-containing reporter gene.
A transgenic mouse or rat model carrying an IJ presser gene, in which the expression of the reporter gene is monitored when the 1ac IJ presser gene is inactivated by mutagenesis. Although both the target gene and the reporter gene are of bacterial origin,
When inserted into an animal gene expression vector, it can be expressed in animal cells. According to this transgenic model,
In vivo mutagenic phenomena can be easily and easily suppressed. Therefore, this model is suitable for toxic substances (e.g. mutagens).

Carcinogens and teratogens) can be evaluated more accurately than conventional testing methods using sworn animals. This shitism again. It is more sensitive and rapid than standard animal assays. This is because mutations or changes in target genes can be detected at the DNA and cellular level by one histochemical method before tumors develop.

(The following is a blank space) The present invention will be further explained with reference to the following examples.
The present invention is not limited to this.

Key points: In vitro mutation assay method for animal cells (a) System construction An example of a useful NA construct is shown in Figure 1.
The constructs are physically linked to each other and each has a promoter/
It contains two functional transcription units with an enhancer, a coding sequence and a transcription termination signal. One 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.

The complete coding sequence for β-galactosidase includes lac
The RSV-LIR promoter used to form the Z structure and the SV40 polyadenylation site are flanked to form a functional transcription unit of the reporter gene.

Similarly, 1ac1 transcription neat 1-7 is RSV-L
Constructed with IR, lac repressor coding sequence and SV40 polyadenylation site. Next, this reporter
The transcription unit was transformed into 1ac1 by cloning method.
physically coupled to the transfer unit.

A DNA construct containing both a lac repressor transcription unit and a β-galactosidase unit.

It is then used to transfect embryonic or epithelial cells, as shown in FIG. Preferred cells are ATCCCRL-1573 (human 293) and A
TCCCCL-T, 1 (LLC-MKz), the American, Rockville, Maryland, USA.
an Type Culture Collection
It can be obtained from. This construct is co-transfected into the II follicle with the neo gene as a two-selectable marker using known methods such as the calcium phosphate precipitation method described above. antibiotic G4
Cell colonies resistant to 18.

As shown schematically in FIG. 2, cells are detected by growth in selective media and analyzed for the presence of cloned and functional Iac repressor and β-galactosidase genes.

) Analysis and Characterization of the System Transfected cells containing a non-functional 1ac repressor gene and a functional β-galactosidase gene are transfected with 5-bromo-4, a substrate for β-galactosidase.
-Chloro-3-indolyl-β-D-galactopyranoside (X-gal) stains blue when added to cultures in the absence of the inducer IPIG. Desirable colonies containing both functional lac repressor and β-galactosidase genes will not stain when X-gal alone is added, but when IPIG and X-gal are added,
When gal is added, it should be dyed blue. 1a
Successful expression of the cl gene means that Sams (S
ans) et al., J.

Biol, chem, +260 volumes, 1185 pages,
(1985); and Lin and Riggs, J. Mo1.
．． biol, vol. 72, p. 671, (1972)
This can also be demonstrated by a filter binding assay for repressor-operator DNA complexes by .

After the lac repressor gene and β-galactosidase gene have been successfully expressed in cultured cells.

Each colony can be analyzed for copy number of the introduced gene. A single copy of the acl gene per cell (diploid genome) is analyzed by Southern blot analysis of the genome DN^. Cells with a single copy of the monofunctional 1acl gene are highly preferred over cells with 2 or more copies of the 1acl gene. because.

The latter requires more than one mutation to inactivate all copies of the 9-functional repressor gene, resulting in a repressed state of the reporter gene. Since mutation events are detected by the expression of the reporter gene, the copy number of the reporter gene is not very critical, but in some cases more than one copy makes detection easier. The probability that a single copy sequence will be obtained is determined by the low concentration of target D.
Enhancement is achieved by using NA and sufficient carrier DNA to transfect the recipient cells.

(C) Screening of compounds for mutagenic activity Nitrosomethylurea (NMU) can be taken as a model mutagen. Because Dubridge
ridge) et al., Mo1. Ce11. Biol,
, Vol. 7, p. 379 (1987), it has been shown that this compound readily induces mutations in the 1acI gene in vitro. Cultured cells are treated with NMU for a predetermined period of time, and the cells are
Screen for reporter gene expression.

The frequency of mutations is determined by counting the number of β-galactosidase positive cells among the 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 the mutagen.

! JIJLi = Mutagenesis assay method using transgenic animals The first step to create transgenic animals used for in vitro assays is to induce 1ac in a mouse fibroblast culture.
The purpose of the present invention is to prepare a β-galactosidase reporter gene induced by the mutation of 1 and to detect its activity. The above cultures are obtained by successfully transfecting the linked lacI-lacZ gene construct using the method described in Example 1 and then exposing them to the mutagens to be tested. Controls are untransfected cells and cells with linked genes that have not been exposed to mutagens. In an in vitro system, 9
Next, production of a progenitor C57BL/6J transgenic mouse carrying the linked 1acl-lacZ gene construct, and then production of transgenic progeny carrying the gene construct, as described in FIG. 4, is performed. Sexually mature transgenic progeny that are positive for gene transfer serve as controls and subjects for mutagenesis studies.

(a) Preparation of DNA As shown in Figure 3, a DNA construct was prepared in the same manner as described in Example 1 and used to generate transgenic mice. The above construct has two functional transcription units physically linked to each other, each having a promoter/enhancer, coding sequence and SV40 polyadenylation site.

One unit is used to drive expression of the lac repressor gene; the other unit is used to drive expression of the lac repressor gene.

It is used to express the β-galactosidase gene. The mouse histone 3.2 promoter is la
It is utilized in the construct to effect ubiquitous expression of both the c-repressor gene and the β-galactosidase gene. As shown in the final vector,
The XhoI and 5alI sites are used to clone the lac repressor and β-galactosidase coding sequences, respectively.

The DNA fragment carrying both the target gene, such as the lac repressor transcription unit, and the reporter gene, such as the β-galactosidase transcription unit, or the SV40 T antigen unit, is excised from the vector by restriction enzyme digestion and isolated from the primary As such, it is isolated by gel electrophoresis and affinity column purification for microinjection. The fragments are separated by agarose gel and the desired bands are collected by electroelution. D.N.
A was extracted with phenol/chloroform, concentrated by ethanol precipitation, and then bound to an Eluttp-d column.

It is then purified by elution. The DNA was then recovered by ethanol precipitation and sterilized. 1mM EDTA
(containing 10 mM) in Lys buffer (pH 8) and quantified by Hoechst dye fluorescence assay or spectrophotometry at 260 nm.

(b) Transfer of DNA into embryos The principles and experimental procedures for creating mice were described by Brigid Hogan, Frank Constantini and Elizabeth Lacey.

Frank Con5tantini and Eli
zabeth Lacy) (Coldspring)
"Manipulation of Mouse Embryos" by Harbor Laboratory, Cold Spring Harbor, NY, USA). Mouse zygotes were administered 5 IU of pregnant female mouse serum gonadotropin and, 48 hours later, 51 U of human ciliated gonadotropin.
The mice were harvested from 6-week-old C57B/6J female mice (Jackson Laboratory, Bar Harbor, ME, USA) that had been administered with C57B/6J to superovulate.

The primed female was placed with a C57B/6J male and examined the next morning for the presence of a vaginal plug. Pseudopregnant females used as recipients were selected for 8 oestrous cycles and placed with sterile, vasectomized CD-1 male mice to ensure CD-1.
Female mouse (Charles R'\'r Labora
tories, - Wilmington, Massachusetts, USA). The conjugate contained 5.1 g/l Na
were collected in BMOC-2 medium modified to contain C1 and 5 mg/al bovine serum albumin. Cumulus cells were grown at 60 IU/d in culture medium.
Hyaluronidase (Sigmatype I) diluted in
V, 300 in PBS containing 1% PVP 40T
It is removed by treatment with IU (Sigma). the zygote,
Wash twice in secondary medium to remove debris. Approximately 2p1
DNA solution was injected into the pronuclei of males so that about 50% survived. The injected zygote is anesthetized with tribromoethanol until it moves into the oviduct of the recipient female.
Incubated at 37 °C in air containing 5% C(h).

Palmiter, Ce1l,
29, 701 (1982), integration of genetic constructs is generally performed in a head-to-tail tandem arrangement.
occur as a single copy or multiple copies of array). A method for injecting genes into mouse embryos is described by Brinster et al., Proc. Natl.
, Acad, Sci, USA, vol. 82, 44
3B (1985). Increase the frequency of integration. The more important factor is the following: the concentration of injected DNA is the limiting factor for incorporation efficiency, with a DNA concentration of 1-2 ng/l or about 100-i
, ooo copies of DNA fragments appear to be optimal; linear DNA molecules are more effective when cut with endonucleases to non-blunt ends; and to obtain high efficiency. teeth.

Requires injection into the nucleus rather than into the protoplast.

Several other factors do not appear to affect the 2 incorporation rate. Injected DN by female and male pronuclear injections
A can be accommodated, giving equivalent integration efficiency.

Injection of DNA into the nucleus of a two-cell embryo results in a transgenic animal, so injection into the pronucleus is not essential to cause integration.

It has been suggested that the mouse strain used will influence the efficiency of integration. For example, C57X
The eggs of the S-hybrid have been proven to be more efficient than the inbred C57 strain. Efficiency comparable to the above hybrids has been obtained in the Kinbun CD-1 mouse (Charles River). Inbred C57BL/6J mice (Jackson Laboratories) can be used to isolate potential background modifier genes.
Follicle-stimulating hormone and luteinizing hormone were administered to female 6J black mice to induce superovulation.

Next, these mice are mated with male mice of the modern lineage.

The fertilized single-celled embryo is washed out of the oviduct and treated with hyaluronidase to extract the cumulus from the embryo.
phorus) cells are removed. Next, the target DNA
Microinject into the pronucleus of single-cell embryos. Approximately 100 copies of the tact-lacZ DNA construct are injected into each embryo and each embryo is then transferred to the oviduct of a pseudopregnant adoptive mother to complete the gestation period. These embryos can differentiate and develop into complete animals.

(C) Selection and Assay of Transgenic Mice for In Vivo Mutagenesis Model Transgenic mice carrying a single copy of the combined functional lacI-lacZ gene construct are screened and selected using the following method. Tail sections and/or liver lobes are used as a source to isolate genomic DN^.

The copy number and structure of the introduced gene are used as a probe. DNA from the lac repressor or reporter gene is analyzed by Southern blotting to determine whether rearrangements or modifications have occurred within the introduced gene during development of the transgenic mouse.

Only mice carrying a single copy of the complete 1acl-lacZ gene construct will be 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 in Northern blot assays, Western blot assays, and repressor function assays to detect the expression of the lac repressor gene in the tissues of transgenic mice. The reporter gene is active 1a
Since it should be repressed by the cl gene, expression of the reporter gene in one tissue is not expected unless the bifunctional lac repressor is completely absent or the inducer IPTG is added. Therefore.

Using the same promoter and enhancer in the structure of both repressor and reporter genes
“un-coupling” of the above two genes
)”. The presence of functional 1acI and 1acZ genes in the tissues of transgenic mice was also demonstrated in Example 1 using cells isolated from one tail tissue.
It can be assayed by the method described in [Japan].

Only gene transfer lines having a functional target gene at a rate of 1 copy per cell are selected and bred to create homozygous gene transfer lines as shown in FIG. Fl are bred together and the expected A homozygous transgenic line in F2 is identified by the strength of the signal in Southern analysis. Homozygosity is confirmed by progeny breeding tests and Southern analysis. Homozygous test animals for mutagenesis assays are produced by breeding homozygous offspring with two copies of the introduced gene and mating them with non-transgenic partners.

(d) localization and distribution of positive cells within major organs of both test control mice and mutagen-treated mice of transgenic animals exposed to the test compound;
Quantitative data can be obtained. Tissues obtained from the following organs were selected for morphometric analysis. The organs are the brain (set va collected from the cerebellum, midbrain and forebrain),
Liver, ll11! Viscera, kidneys, left ventricle of the heart, lungs, gonads, uterus, pancreas.

These are the fundus of the stomach, the 13 ↑ umbilical intestines, the ileum, the colon, and the sternum bone marrow. Following the fixation process, 1 Mi woven pro-nkus were cut for 8 micron cryostat sections or 30 micron pipratome sections to ensure random sampling.

A histochemical reaction for β-galactosidase is performed. Serial sections separated by 40 micron intervals are collected from each block. Positive cells were counted using a biological image analyzer system and a 2-pyoquant II (Bi
o-quant II) Convenience store tablogram (R
AM Biometrics, Tennessee, USA.

This is done by digitized morphometry using (Natschville). The system's hardware consists of an image projection camera attached to a standard Zeiss binocular microscope, an Apple microcomputer, and a digitizing tablet.

With this system, a sample is projected onto a computer screen and an operator selects and sketches the area to be analyzed. Exists in blood vessels and as lumens of hollow organs. An outline of the space from is drawn on the screen.The system is then instructed to subtract said area from the five areas to be analyzed.

Cells that were strongly stained blue were counted using a digitizer, and the number of positive cells per unit area of the nine tissue samples was automatically calculated. In this way.

The number of positive cells per unit area of a particular organ from untreated or treated animals can be counted, and the data obtained are statistically compared. Additional main benefits of this system are:

This means that specific cell lines that are particularly sensitive to a given mutagen can be identified and quantified.

Histochemical analysis is performed on tissues from treated and untreated male and female transgenic mice of the same strain. Morphometric assays provide quantitative data on the number of β-galactosidase-positive cells in the sampled organ and animal cells to give information about the test compound, the efficacy of the test method, and the nature of the transgenic animal. It can be used to check the frequency of background mutations. The expression of the lacZ gene in cells is as follows.

Assayed by β-galactosidase staining.

5 (]aM of Na, PO, were added. Soak one tissue in a cold 4% formaldehyde solution. Adjust the PTT of this mixture to 7.4. Fixed overnight at 4°C.Enzyme activity:
This fixation is not compromised.

The tissue is then rinsed in phosphate buffered saline (PBS) and 30 μm sections are cut on a vibratome or 8 μm sections are cut on a cryostat. Next, the obtained section was coated with 1 μ/g of X-gal, 5 m
M Potassium hexacyanoferrate(I) 51mM Potassium hexacyanoferrate(II), 2mM MgC1z
, 37 in PBS (pH 7,3) containing 0.02% NP-40 and 0.01% chlorate.
°(,??5% Cot atmosphere. The solution was brought to an alkaline pH, and M
The addition of g'' 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 either freely moving vibratome sections or cryostat sections fixed on gelatin-coated glass slides. Once the desired color intensity has been obtained, the vibratome sections are coated with 0.1% KCr.
It was placed on a glass slide coated with SOn and 1% gelatin, and dried by heating at 37°C for 0.5 hours. next,
Sections were dehydrated and washed in xylene for 2-3 minutes. Counterstain with methyl green and then place a coverslip over each section. Paraffin sections of whole tissue inclusions can also be stained.

This is because post-embedding of the sections does not compromise the blue positive staining. Thus, paraffin-embedded sections can be archived and stored indefinitely as a permanent record of each test method.

When SV40 large T antigen or other antigens are used as reporter genes, cytochemical immunoperoxidase methods [e.g. Hanahan, Natu
re.

Volume 315, page 115 (1985), O
rnitz) et al., 5science, vol. 238, 18
8 (1987), or Behr
inger), Proc, Natl, ^cad.

Sci, USA, vol. 85, p. 2648 (1988
) is used to detect the expression of reporter proteins.

Transgenic animal embryos for backyard group teratogen assay Transgenic animal embryos carrying the target gene and the binding reporter gene are utilized for teratogen screening. This transgenic embryo model allows for easy and rapid quantification of mutagenic events in a system similar to living human embryos, and allows for the detection of teratogenic compounds more accurately than currently used methods. You should be able to assess the risk.

Produced by the method detailed in Example 2.

Transgenic animals such as mice and pigs carrying the 1acl gene linked to the lacZ gene are used as test animals to generate transgenic offspring. Embryos isolated at various stages from female transgenic pregnant animals in which gene expression had been successfully performed were exposed to test reagents and reporter gene expression was analyzed.

For example, a 10 mm transgenic pig embryo, which develops during a 20-21 day gestation period and has an primordium with almost all adult structures, is exposed to a potentially teratogenic substance. Mutations caused by teratogens inactivate target genes and turn on reporter genes in certain cells/tissues of the embryo. These cells/tissues can be analyzed by the above-described histochemical analysis method, or by the allenzyme method, if the reporter protein is β-galactosidase or other suitable substance in place of the reporter protein.
Alien N, D, et al., “Tran
sgenes asprobes for active
e chromosomal domains in
m.

use development”, Nature,
It can be detected using the histochemical analysis method described in Vol. 333, pp. 852-855 (1988).

Detection methods and systems have a target gene linked to a reporter gene. It will be apparent to those skilled in the art from the foregoing detailed description of the invention to modify and modify transgenic animals and transgenic animal cells for testing mutagens and teratogens. Such modifications and variations are within the scope of the following claims.

SUMMARY OF THE INVENTION Rapid, sensitive and quantitative methods and analyzes are provided for rapidly screening compounds for mutagenic or teratogenic activity. in this way,
Transgenic animal cells, animals in which genes have been introduced into adults or embryos, are used. Each cell from a cell culture, differentiated tissue from an animal.

Or the animal embryo contains the target gene and the reporter gene. Both have animal promoters/enhancers, coding sequences and transcription termination signals. Therefore, they can be expressed in animal cells. The product of the target gene is produced by interaction with regulatory sequences in the reporter gene.

It is possible to regulate the expression of the J porter gene.

In a preferred embodiment, the target gene 1acl (l
ac repressor) is a reporter gene lacZ
(encodes β-galactosidase).

This is detected by cytochemical or histochemical methods. When an animal cell is exposed to a compound that causes a mutation that changes the target gene or changes the operator of the reporter gene, the reporter gene is expressed.

Figure 1 is a schematic diagram showing the structure of expression vectors pRSV-1-pRSV-Z. This expression vector is RS
contains the complete coding sequence of the lac repressor flanked by the V-LTR promoter and the SV40 polyadenylation site, which forms a functional transcription unit of the target gene); and the RSV-LTR promoter and the SV
It has the complete coding sequence of the β-galactosidase enzyme, which forms the functional transcription unit of the reporter gene, flanked by 40 polyadenylation sites.

FIG. 2 is a schematic diagram showing a method for constructing and testing transfected human embryonic or monkey epithelial cells containing pRSV-1-RSV-Z.

FIG. 3 is a schematic diagram showing the structure of an expression vector to be inserted into mouse fibroblasts or embryos.

This expression vector has two functional transcription sites physically linked to each other and each having a promoter, histone 3.2 and SV40 polyadenylation site. One promoter was inserted into the Xho 1 site of the final vector and the other promoter was used to express the Iacl gene.

To express the β-galactosidase gene.

It is used by inserting into the Sal 1 site of the final vector.

FIG. 4 is a schematic diagram showing a method for generating transgenic mice with one functional target gene and a reporter gene and performing in vivo mutagenesis assays.

FIG. 5 is a schematic diagram showing a method for breeding and producing transgenic mice for use in in vitro mutagenesis assays.

that's all

Claims (1)

[Claims] 1. A method for screening for compounds that cause mutations or changes in the expression of a nucleic acid sequence in eukaryotic cells, the method comprising providing a target gene and a reporter gene, and comprising: providing a target gene and a reporter gene; A screening method, wherein the product of the method can modulate the expression of the reporter gene. 2. The reporter gene comprises a eukaryotic promoter, a regulatory sequence, and a sequence encoding a reporter gene product;
2. The method of claim 1, wherein the product of expression of the target gene binds to a regulatory sequence of the reporter gene to regulate expression of a sequence encoding the reporter gene product. 3. The method according to claim 2, wherein the target gene has a eukaryotic promoter, a sequence encoding a regulatory molecule, and a transcription termination signal. 4. said reporter gene product is selected from the group consisting of biochemically detectable proteins and nucleic acid sequences;
The method according to claim 2. 5. The method of claim 2, wherein the regulatory sequence is an operator. 6. The method of claim 3, wherein 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. 7. The method of claim 6, wherein the product of the reporter gene is β-galactosidase and the regulatory molecule is a lac repressor protein. 8. The method of claim 1, further comprising inserting the target gene and reporter gene into a eukaryotic cell. 9. The method according to claim 8, wherein the eukaryotic cell is an animal embryo at an early stage of differentiation. 10. The method of claim 8, wherein the eukaryotic cell is in a well-differentiated animal tissue. 11. The method of claim 8, further comprising controlling the copy number of the target gene and reporter gene inserted into the animal cell. 12. The method according to claim 11, wherein the number of copies of the functional target gene is limited to one. 13. exposing said animal cell to a compound; and further comprising comparing the expression of a reporter gene product in said exposed cell to the expression of a reporter gene product in a control cell not exposed to said compound. 9. The method according to claim 8. 14. exposing an embryo to a compound; allowing the embryo to undergo cell division for a period of time; and comparing the expression of a reporter gene product in the embryo to the expression of a reporter gene product in an embryo that has not been exposed to the compound. 10. The method of claim 9, further comprising: 15. exposing said differentiated animal to a compound; causing said animal to metabolize said compound and undergo cell division; and controlling the expression of a reporter gene product in tissues from said exposed animal to said compound; 11. The method of claim 10, further comprising comparing the expression of the reporter gene product in tissues of the same strain of control animals that have not been tested. 16. The reporter gene encodes β-galactosidase, the method further comprises providing a substrate for β-galactosidase and measuring the expression of the reporter gene by staining with a ferricyanate-ferrocyanate solution. ,
The method according to claim 8. 17. The method according to claim 8, further comprising measuring the expression of the reporter gene using an enzyme-conjugated immunoglobulin and detecting the expression of the antigen by an immunochemical method. 18. A system for rapidly screening compounds that cause mutations or changes in the expression of nucleic acid sequences in eukaryotic cells, which includes a target gene and a reporter gene, and the product of expression of the target gene is the same as that of the reporter gene. A screening system that can modulate expression. 19. The reporter gene has a eukaryotic promoter;
It has a regulatory sequence, a sequence encoding a reporter gene product, and a transcription termination signal, and the product resulting from the expression of the target gene binds to the regulatory sequence of the reporter gene, resulting in the termination of the sequence encoding the reporter gene product. 19. The system of claim 18, wherein the system modulates expression. 20. The target gene has a eukaryotic promoter, a sequence encoding a regulatory molecule, and a transcription termination signal;
20. The system of claim 19. 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 antigen. 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 a lac repressor protein and the regulatory sequence is a 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 long 19. The system of claim 18, further comprising at least one promoter selected from the group consisting of terminal repeat portions. 26. The system of claim 18, further comprising a eukaryotic cell. 27. The system of claim 26, wherein the eukaryotic cell is an animal embryo at an early stage of differentiation. 28. The system of claim 26, wherein the animal cell is in a well-differentiated animal tissue.