JPH03502882A - Transgenic testing system for mutagens and carcinogens - Google Patents

Abstract

(57) [Abstract] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Transgenic testing system for mutagens and carcinogens 1. Introduction The present invention provides a transgenic animal test system for mutagens and carcinogens. related to music. Using recombinant DNA technology, mutations caused by mutagens or develops unique test genes and It creates expression regulation signals. This test gene was transferred to non-human transgenic cells. into genetic animals to develop unique bioassays for mutagens and carcinogens. Provide test panels.

2. Background of the invention 2.10 Testing for carcinogens and mutagens Can chemicals cause cancer in humans? It has been 200 years since it was recognized that it could be a causative agent or that there is a relationship. A while ago, in England, Lord Percival Pott gave a scrotum to a young chimney sweep. This was when it was described that the incidence of cancer was high. In 1918 Yamaka and Ichikawa ( 1918, J, Cancer Res, 3: 1-29) was obtained from the chimney. Cancer was developed in rabbit ears after prolonged topical application of coal tar.

In 1933, Cook, Hervett and Hieger (1933, J.

Chem, Soc,: 395-405) is the main carcinogenic component of coal tar. One of them was identified as 3,4-benzpyrene [benzo(a)pyrene]. in rapid succession , azo dye (1934) was shown to cause liver cancer in straw odonts, and beta Naphthylamine was found to cause bladder tumors in dogs (1938). child From their origin, animal tests on carcinogens began, and in 1941 they were used as insecticides. 2-acetylamino-fluorine, a compound intended for widespread use in the United States, is a powerful was shown to be a cancerous substance (Wilson et al., 1941 s, 1:595-608). Based on these early studies, various chemicals that it can cause cancer in humans and animals, and that it occurs in (1) a tissue-specific manner; (2) over long time frames; and (3) often with complex reaction mechanisms. It has become clear that this phenomenon requires multiple factors.

2.1.1. Bacterial and Cell Culture Test Systems How These Carcinogens Work T. Boveri (Boveri, 1941) ．． The Origin of Malignant Tumors, Jen Part A) was the first to treat it and suggested a genetic basis. This idea is based on somatic cell mutations in cancer. It became a source of controversy. In 1948, the benzpyrene series and ab dye compounds were introduced. Some of the carcinogens found in fruit flies, Osophila melanog It was clear that mutations could be caused in the aster (Demerec , 1984°Brlt, J. Cancer 2: 114-117). similar The result is the mold, Neurospora crassa (Barratt and Tatum, 1951゜Cancer Res, 11: 234-241 ) and mouse (Benzanthracene: Carr, 1947. Br1t, J , Caneer 1: 152-156; Methylcholanthrene: Stron g, 1949. Br1t, J, Cancer.

3:97-108) have been reported using other promising genetic systems.

These early observations suggest that they cannot be used as tests for mutagens and carcinogens. There were clearly limits. Some compounds are found in certain types of test specimens, such as bacteria and molds. It did not act as a mutagen in animals, but it did cause tumors and mutations in mice. It was. This led to the concept of metabolic activation (for example, Mil ler and Miller, 1953. Adv, CancerRes, 1: 339-396). Some chemicals (e.g. diethylnitroso) amines) in the liver (P450 oxidase system and hydroxylation system ) or are metabolized by enzymes in other tissues and are mutagens and carcinogens. This resulted in a new compound that is a quality.

Ames and coworkers (Ames et al., 1975. Mut, Res.

31: 347-364’) is a chemical substance containing Salmonella typh. These chemicals are mixed with liver oil extract (enzyme preparation) prior to exposing the imurium bacteria. We addressed this issue by incubating. This allows for sophisticated It has now become possible to test for mutagenic activity. In this study, grown under restrictive conditions, Increase the frequency of reversion of certain types of point mutations by assaying bacteria that can You can check the size. The nature of the mutation (the type of change at the DNA level) and frequency can be accurately quantified. This test is currently available as a rapid and inexpensive test for mutagens. It is widely used as an essay.

Other methods currently used to detect carcinogens or mutagens include culture Selection of mutations in mammalian cells under cultivation is included. chinese hummus Tar ovary cells (CHO cells) are treated with a mutagen and then the enzyme hyboxanthin Characteristics of the guanine phosphoribosyltransferase (HGPRT) gene locus Metagenesis can be identified by selection on appropriate media (Hsie et al. , 1979. fn Strategies for Short-Term T esting for Mutagens/Carcinogens.

Edited by Butterworth, CRC Press, PL) o This X-linked mutation occurs when male cells are haploid and detects single mutations caused by mutagens It is something. Resistance of cultured cells to the toxic drug ouabain is due to genetic ATPase caused by a single dominant mutation in the child locus, which also leads to abnormalities in cell culture. It is used to measure the frequency of natural mutations.

Bacteriological testing (Ames) even though there is no “gold standard method” for the field. It is clear that the cell culture assay (Hsie) has considerable shortcomings. Primarily , chemicals are often metabolized and changed in different ways by different tissues in the body . Blend test compounds in extracts of all body tissues before testing for mutagenesis. It is simply impossible to incubate. Second, each person has a different body. Body tissues exhibit different sensitivities to various chemicals. This happens very often (beta-naphthylamine and bladder cancer) bladder cancer; azo dyes and liver tumors; etc.).

Bacterial and cell culture tests do not address this issue.

Third, in response to metabolic activation of chemicals or to products made in situ. well-documented literature demonstrating genetically determined host variability exists (Banbury Report, 1984. #16: Gen eticVariability in Re5ponses to Chem ical Exposure.

Col. Spring Harbor Press, NY). This is possible Although degeneration will never be reproduced in any organism other than humans, it is evolutionarily indispensable. well-controlled genetic tests in mice or rats rather than in appropriate bacterial cells. is better approximated. Fourth, chemical carcinogenesis occurs prior to tumor formation. time (age of test host), number of events (promotion and initiation), It is a complex process that requires variables such as Ecker, 1968. Cancer Res, 28: 2338-2 349). Full animal models allow us to reconstruct these variables, but in detail This cannot be done with bacterial or cell culture tests.

2.1.2. Complete animal testing system Typical animal testing protocols for carcinogen detection include 50 animals per group. This includes the use of animals of both sexes from two species (e.g. mice and rats) that have Sontag et al., 1976, Guidelines for Carcin ogen Bio-assey in Small Rodents’, Ca rcin, Tech, Rep, Ser.

1, DHEW Pub, NrH76-81). [properly planned and executed] In a bioassay, a compound - or more - produces a new effect in a target organ. A compound is classified as a carcinogen if it statistically significantly increases the biological incidence. (Dunkel, 1985. in Mechanisms o f Toxicity of Chemical Carcinogens an d Mutagens, F1am and t, edited by orentzen, Pr1n ceton Sci, Pub, Co,, Pr1nceton.

NJ). To experimentally detect genetic mutations in vivo, two basic principles are needed: various tests have been developed; each test covers a single category, i.e. It is limited to the detection of either natural mutations or germline mutations. Imbi Detection of inherited mammalian gene mutations in mice is mouse-specific - locus test On the other hand, induced somatic mutations are limited to mouse coat spot assays. It can be detected using experiments. However, both tests are based on the same genetic system (For a review, see 5tiles and penman, 1985. Muta tionResearch 154: 183-204). Very recently, For use in detecting carcinogens or thought to confer protection against carcinogens. Transgenic organisms containing activated oncogenes are used to test substances that (European Patent Application No. 0169 by Leder and Stewart) 672).

Complete animal testing addresses some of the shortcomings of bacterial and cell culture systems However, these complete animal studies are generally incomplete for several reasons; (a) Statistically significant tumor incidence in the test group (50 individuals) (b) Assay detection level is not sensitive (at 5-10% incidence level); There is often well-documented variability between them, preventing clear conclusions. example For example, the National Cancer Institute Of the 203 bioassays of this type (Chu et al., 1981. OX, Environ, Health 8: 251-280), 15 assays (7%) were insufficient to evaluate the results; 25 assays (12, 5%) had only borderline or suggestive evidence of carcinogenicity; 53 assays (26,5%) were positive for only one of the two species, and 23 assays were positive for only one of the two species. (11,5%) were positive for only one sex of the species that showed tumors; (C ) Judgment of these in vivo assays is often difficult and depends on pathology, statistics and expertise in toxicology and toxicology must be combined, and the results must be independent based on individual judgments. (d) The time required to produce a tumor varies and negative termination The point is indeterminate. Some exams take 1.5 to 2 years to complete. negative result Waiting for the exam is a problem with exams that don't have a clear end point. currently being done Animal testing clearly has problems with that. Bacteria test (Ames test), mold There are often other tests available such as Drosophila, cultured CHO cells, etc. Recognizing that tobacco will be evaluated in comparison to a “standard” animal bioassay. If you do, the difficulty will be doubled. Judging or evaluating all trials at this time There is no fully or uniformly accepted standard that can be used.

Most experts in this field today use ``There is no general agreement regarding the most appropriate choice of test'' for (Dunkel, 1985. in Mechanisms of T Oxicity of Chemical Carcinogens and M utagens, edited by F1amm and Lorentzen, Pr1nceto n Sci, Pub, Co, Pr1nceton.

N.J., p. 69). The pharmaceutical and chemical industries produce thousands of compounds each year; The compounds are products with potential for human consumption or environmental release. some politics Government agencies require information on mutagen and carcinogen testing prior to approval of compounds. request or request. At this time, there are currently no officially articulated appropriate or approved There are no tests available. This clearly shows the absence of standards in this area. . reproducible, inexpensive, accurate, and comprehensive information on mutagens and carcinogens. Comprehensive testing is required.

2.21 How do carcinogens and mutagens act to cause cancer? 2.2.1. DNA mutation Ames and colleagues (McCann and Ames, 1976, Pro c.

Natl, Acad, Sci, USA 73:950-954) It has been shown that a significant proportion of cancerous substances are also mutagens.

These chemicals alter deoxyliponucleic acid (DNA), which is genetic information, and Causes heritable changes in genes and the signals that regulate them. various chemicals, and physical treatments such as irradiation cause various types of mutations in DNA molecules. (Chemical changes in DNA). Certain chemicals can reproduce the same type of mutation It is often caused in an effective manner. Certain chemicals react with DNA molecules and chemically change it. Other compounds are incorporated into the DNA strand and each generation Errors occur during DNA replication. The chemistry of such reactions follows the rules , because it is reproducible, the types of mutations that result are reproducible.

The four nucleotides that make up DNA (adenine (A), guanine (G) , thymidine (T), and cytosine (C); A and G are purine bases, and T and C are pyrimidine bases), and they are chemically Pairs according to the law of combination (A-T, G-C, or T-A, C- C base pair).

Some chemicals cause point mutations, which are single base pair changes in DNA. There is something. Two types of point mutations are well known: (1) transitions; Here one purine is replaced by another, i.e. instead of 01 Therefore, an A-T base pair changes to a G-C base pair, or one pyrimidine also changes. on the other hand, i.e. C1 instead of T, thus the T-A base pair becomes a C-C salt. and (2) transversion, where a purine changes to a pyrimidine. (C-G changes to A-T or GC) or vice versa. here Also, various carcinogens or mutagens undergo a high rate of transitions or transactivations. reproducibly induce either version, but often both. Not that. Therefore, various things caused by various chemicals A large number of tests need to be available to detect the phenomenon. carcinogen or physical A second type or class of mutations induced by agents are DNA rearrangements. . On a large scale, this is a chromosomal translocation or fusion of parts of two different chromosomes. Let's invite This is a common event in many types of cancer (Cairns, 　1981. Nature289: 353-357; Klein, 1 981. (See Nature 294: 313-318). On a smaller scale, deletions (missing base pairs), inversions (changes in the direction of the base sequence), insertions (added bases) pairs) or even amplification of DNA segments (increasing the number of copies of a series of base pairs) can lead to cancer. It has been reported as a related phenomenon (for a review, Cancer Cells 2 ．． Van de Woude.

Levine, Topp and Watson (MW), CSHPress, NY.

(see 1984). Finally, regulation of gene expression in specific tissues may be due to cytosine residues. Naturally modified by chemical changes in the base such as methylation (Doerfle et al. r, 1983. Ann.

Rev. Riochem, 52:93-124). a class of chemistry Substances change this pattern of methylation (5-azacytidine) and It also induces cell differentiation and transforms cells in culture.

In short, mutagens act in a wide variety of ways to alter genetic information, DNA. to become Mutagens work reproducibly to produce specific classes or types of mutations. cause. Good test systems detect each of these classes of mutations It is something that can be done.

2, 2.2. Oncogene activation Mutations in a cell's DNA differentiation do not necessarily cause cancer. It's obvious that there isn't. For example, certain mutations occur in genes essential for the life of a cell. There will be. If the modified product of this mutated gene is unable to function, The cells will die (lethal mutation). Multicellular organisms tolerate this well (some microorganisms (deficiency of follicles).

There are also mutations that can cause cancer. In fact, humans (and mice, rats, etc.) undergo cell division. , growth, wound healing, hematopoietic cell production (white blood cells, red blood cells, platelets, etc.) and other It has recently been discovered that species possess gene clusters that appear to be important in regulating developmental processes. It became clear. Such genes (approximately 50-100 populations per organism) are Mutations in genes and their regulatory signals can be involved in the cancer process. This is called the assigned gene (normal type). Activation of the assigned gene by mutation Oncogenes or genes that cause cancer are produced (for a review, see Cancer Ce1ls 2.1984. Van de Woude.

Levine, Topp and Watson, eds., Cold Spring H (see arborPress, NY). The specific target of a mutagen is that When a gene produces cancer, it is a set of genes called the attributable genes. for example, DNA extracted from cells derived from human bladder cancer (T24/EJ, P Arada et al., 1978, Nature 294: 474-478), H A specific oncogene called a-ras is responsible for transverging DNA from C to A. has a point mutation within the ras oncogenic molecule that encompasses the Glycine was changed to valine in amino acid 12 of protein. humans (and mice) Many B-cell lymphomas or plasmacytomas (such as 8-14.8-2.8- regulatory locus. Similarly, human chronic bone marrow Sexual leukemia (CML) is characterized by a translocation of chromosomes 9-22 and is associated with abl cancer. The original gene has been activated and mutated into an oncogene. Origin of myc cancer in chickens Inserting a mutation adjacent to the genus (within its regulatory region) causes mucosal lymphoma. arise. Mutagenic changes in viruses that give rise to numerous oncogenes It is captured and multiplied, which is then passed on to mice, cats, dogs, chickens, monkeys, etc. "Infectious cancer-causing agents. Many different types of mutations ( deletions, insertions, point mutations) can activate various oncogenes, resulting in cancer. . Oncogenes are associated with tissue-specific cancers, but oncogenes are associated with tissue-specific cancers. Can initiate or induce cancer.

Therefore, an estimated 50-100 homing genes are activated by various mutations and Changed genes (oncogenes) and changes that occur during the process of developing cancer produce a product (protein). These targets of mutagens have undergone a long evolutionary period. A set of genes that have been conserved throughout time. The human homing gene is D. Closely related to homologous genes of other animals in the nucleotide or base sequence of NA are doing. Yeast, mold, fruit flies, mice, rats, etc. are all humans. It contains genes associated with many (but not all) cancer genes.

Bacteria do not appear to have such homologs. Obviously, evolutionarily close to humans. The closer the animal is tested the mutagen for cancer-inducing effects. It will have a good representation of target assigned genes.

3. Summary of the invention The present invention relates to non-human transgenic animals with respect to mutagens and carcinogens. Regarding test systems.

In summary, using recombinant DNA technology, mutations caused by mutagens and Proprietary test genes and and generate regulatory signals. This test gene was transferred to a non-human transducer. for mutagens and carcinogens by introducing them into the germline of genetic animals. The test genes of the present invention provide a unique bioassay test panel. It can encode any of a wide variety of genes (and its expression depends on the transgenic It can be detected in animals. However, if the test gene is a mutagen or carcinogen, Therefore, if it is not activated, expression of the test gene product will be blocked or inhibited. The test gene is genetically engineered as described. For example, the test gene transfer of the test gene if not activated by a specific source that reverses the mutation. May contain mutations that prevent or inhibit transcription or translation. Or again, low density Genetically engineer the test gene to be highly methylated or highly methylated. It may also be produced chemically, in which case each test gene is amplified or demethylated. If the test gene is not activated by the carcinogen, its expression will be inhibited. Good morning. Transgenic test genes engineered in this way can be used in non-human transgenic animals. Introduce into the germline of something. The transgenic animal is then injected with the test gene. Exposure to activating mutagens or carcinogens can cause The test gene product is expressed and detected. For example, genetically engineered transgenic animals to mutagens that revert mutations created in Exposure to will result in expression of the test gene product. Similarly, test genes present at low concentrations expression that amplifies or demethylates highly methylated test genes. The substance will also induce expression of the test gene product.

Activated test genes produce transforming and tumorigenic products (e.g. oncogenes or or the assigned gene product), the product of which is a variant of the substance being tested. Generating tumors in transgenic animals as being pathogenic or carcinogenic . Alternatively, the activated test gene may be a non-tumorigenic assayable product. , e.g. its biological activity (e.g. toxins, hormones, enzymes, foreign antigens, etc.) substances that can be detected based on It can also be expressed by In one embodiment of the invention, a suitable mutagen Activation by substances or carcinogens appears to be tissue-specific in transgenic animals. Tissue-specific regulation of the test gene to result in expression of the test gene product in the expression Can be placed under the control of an element. Therefore, the activated test gene product Only specific tissues are needed to test for the presence of.

The assay system of the present invention = (a) increases the number of target genes per animal in the test; (b) increased sensitivity to current mutagen testing by Gene assembly and differential regulatory control regions for heterogeneous or multi-tissue mutations (C) using regulatory control regions specific for “expression time” in animals; Regarding the difference between fetal or adult activation of mutagens by (d) point mutations (transitions, transversions), deletions, insertions; detection of all classes of mutagens and carcinogens by detecting classify and detect; (e) take into account the different genetic backgrounds of the test animals; (f) the use of both mice and rats in the bioassay; (g) carry the same test system on the chromosome; using both male and female mice and rats; (h) changing within a limited time frame; Utilize test animals that respond to foreign substances, thereby ensuring that a negative result at the endpoint is not meaning that it is less than a certain number of mutations/genome/generation detected. (i) provide a time-dependent end point that has meaning; (i) Eliminate the current ambiguity arising from (sometimes contradictory) judgments; (j) - Ritsu has no uke. (k) reduce reliance on statistical methods that cannot be used; and (k) reduce reliance on statistical methods that cannot be used; Amplification systems that provide maximum sensitivity in bioassays - tumor type DNA synthesis or replication of viruses or cells.

3.1. Definition As used herein, the following terms have the meanings indicated below, whether singular or plural: It shall be.

Test gene: If the test gene is activated by a particular mutagen or carcinogen. If the expression of the gene product is not present, genetic engineering can be used to prevent or inhibit the expression of the gene product. A gene encoding a detectable product produced in Generally, test genes are of the test gene product if it is not activated by a mutagen that reverses the natural mutation. Mutations that prevent or inhibit transcription or translation (e.g. point mutations, deletions) , insertions, etc.). Alright Also test genes to be present in low concentrations or to be highly methylated. The offspring may be genetically engineered, if each test gene is amplified. If the test gene is not activated by the carcinogen that Expression is inhibited. The test gene of the present invention can be used in transgenic animals other than humans. Bioassay panels for germline mutagens and carcinogens I will provide a.

Activated test gene: The activated test gene is encodes and expresses a product that can be detected in a host animal. The test gene is e.g. By reversing genetically engineered mutations that inhibit the test genes by amplifying them or by demethylating them. The test gene is contained in a substance that derepresses or induces gene expression. It can be activated by exposing transgenic animals. activated The tested test gene expresses a transforming gene product, and the product is transgenic. produce a tumor in an animal (e.g., an oncogene or an assigned gene). Or again , the activated test gene gives the transgenic animal a specific function and/or can also express detectable gene products that exert biological effects. example For example, activation of a lethal test gene product results in death of the transgenic animal. activation of the hormonal test gene product may affect the metabolism and and/or homeostasis (e.g., growth hormone activity). activation of enzymatic test gene products will increase animal growth); Assay appropriate organs, tissues, or cells of the genetic animal for enzymatic activity. Activation of the antigenic test gene product can be detected by transfection. by immunoassaying appropriate organs, tissues, or cells of nicked animals. can be detected, etc.

Transgenic animal: introduced into its reproductive system and therefore transgenic Contains a foreign gene called a transgene, which is an inheritable characteristic of animals. non-human animals. The term transgenic animal used here is This refers only to non-human animals that have been genetically engineered; Includes genetic mice, rats, rabbits, guinea pigs, non-human primates, etc. However, it is not limited to this.

4. Explanation of drawings Figure 1 shows two nonsense (translation termination) protrusions generated in the v-ras coding region. Natural mutations and subsequent mutations that can reverse v-raS protein synthesis Diagrammatically represent an unusual event.

5. Detailed description of the invention The present invention relates to non-human transgenic animals with respect to mutagens and carcinogens. Regarding test systems.

Transgenic animals contain foreign genes called "transgenes" and this foreign gene was introduced into its germline; therefore, the transgene It is an inherited characteristic of transgenic animals. More specifically, the truck of the present invention The transgenic animal is treated with a suitable mutagen or generator. A test that expresses its gene product only in response to activation by exposure to cancerous substances. A non-human animal containing a transgene containing the test gene. Therefore , the mutagenic or carcinogenic power of a substance is determined by its ability to affect transgenic animals. can be evaluated based on its ability to activate the expression of the test gene product. any suitable Any suitable non-human animal can be used in accordance with the present invention, including mice. This includes, but is not limited to, rats, rabbits, guinea pigs, and non-human primates. It is not determined.

The assays of the invention can be used to detect specific types or classes of mutations or oncogenic activation. Test genes can be created by genetic engineering. e.g. to test for mutagenic activity For example, mutations that block or inhibit gene expression (e.g., transition and point mutations such as transversions (insertions, deletions, or inversions). It is possible to genetically engineer a test gene that encodes a structural gene with Good morning. For example, transcriptional regulators such as test gene promoters, enhancers, etc. Mutations located within the element can inhibit or prevent transcription. Ru. Mutations located within the coding region of structural genes that inhibit translation or can be prevented, thereby leading to early termination and non-transformation of genetic coding regions. a frameshift occurs that changes the ribosomal sequence; alternatively, this mutation Tests such as program binding regions and elements that affect the stability of transcribed messages It may also be present within the translational control elements of the gene. genetically engineered into the test gene test gene for mutagens that reverse certain types of mutations created in Exposure of transgenic animals containing resulting in the expression or increased expression of a test gene product in a transgenic animal It will be.

Alternatively, carcinogens that do not cause significant mutations but change chromosome ratios may be used. Detection is done using test genes that are genetically engineered to be present at low concentrations. expression of the gene product is undetectable or undetectable in the transgenic animal. or detected at low levels. A transgenic product containing such a test gene If an animal is exposed to a carcinogen that amplifies the test gene to a high copy number, , the expression of the test gene product in the transgenic animal is increased. moreover In addition, the detection of carcinogens that interfere with DNA methylation is based on the fact that their expression is due to methylation. may encode highly methylated structural genes that are inhibited or suppressed by It can be performed using a test gene created by genetic engineering. Such a trial A carcinogen that inhibits DNA methylation in transgenic animals containing the test gene. exposure to a transgenic animal that results in activation of the test gene, i.e. The test gene is expressed or has increased expression.

The test genes of the invention can be genetically engineered to encode any number of different genes. the activation and expression of the gene product, e.g. Can be assayed or detected based on biological activity. One implementation of the present invention In embodiments, the test gene is a non-human transgenic animal The transforming gene product is produced in the transgenic animal when exposed to substances that activate the Genetically engineered traits to be expressed and therefore tumors formed Can contain convertible genes. The resulting tumor carries the activated test gene It consists of millions of cells derived from the original cell (which is a clone of the cell).

Yes to using tumors as endpoints to detect characteristic type mutations There are several advantages. First, a single cell event is amplified (through cell division) Therefore, it is extremely sensitive. Second, the tumor tissue determines the source of DNA, i.e. Once the gene has been obtained, its DNA can be cloned from the genome and analyzed (e.g. e.g., by DNA sequencing), the mechanism of mutation was as expected ( By transition, transversion, deletion, addition, overexpression, methylation interference, etc.). This feature is not a necessary part of the invention is the verification of all test results performed. preferred practice In embodiments, the transforming test gene is activated and expressed in a tissue-specific manner. can be genetically engineered to be This means that specific tissue It is clear to “read” the test results because only one tumor needs to be investigated for tumor formation. It provides many advantages.

Second, it provides tissue-specific information, where the endpoint is determined by the pathologist. It is a clinically obvious tumor that requires little evaluation.

In another embodiment of the invention, the expression results in a non-human transgenic produce a biological activity or detectable function that can be detected in a genetic animal. A test gene can be genetically engineered to encode a gene that Ru. For example, for substances that activate test genes encoding toxins or lethal products. Exposure of the transgenic animal causes the death of the transgenic animal. Activation of the test gene encoding the hormone will result in transgenic influence the metabolism and/or homeostasis of the animal (e.g., growth hosts). Activation of the test gene encoding Lumon results in increased growth in transgenic animals. ). Alternatively, transgenic animals may have foreign structures. A test gene may be genetically engineered to encode a synthetic gene product ( e.g. prokaryotic gene products, or genes of another species, either animal or plant. product), in which case the test gene product is produced in the organs, tissues, or tissues of the transgenic animal. can be detected by immunologically assaying cells. Alternatively, enzyme (preferably foreign to the transgenic animal) Genes may be genetically engineered, and the enzymes may be grown in animal organs, tissues, or tissues. cells with appropriate enzymatic activity (e.g. bacterial enzymes, beta-galactosidase, alkaline enzymes). phosphatase, etc.). Such non- Tumorogenicity test genes can also be genetically engineered to be expressed in a tissue-specific manner. It can also be manufactured. For example, those encoding toxins, hormones, enzymes or foreign antigens. test genes to detect their biological effects (e.g. toxins or hormones). or blood samples (e.g. enzymes or antigens), germ cells or Products can be assayed in certain tissues or organs, such as body fluids Upon activation, they are required for protein secretion. The test gene product is expressed by a tissue with a unique cellular machinery, thus ensuring that the test gene product remains in circulation. You can make it happen.

Including molecular cloning with or without the use of synthetic oligonucleotides Constructing the test genes of the invention using recombinant DNA techniques well known to those skilled in the art. (e.g., Maniatis et al., 1982. “MolecularCl oning AT, laboratory Manual” Co1d Spr ing Harbor Laboratory). like this Molecular cloning techniques are used to isolate, clone, and If the test gene is not activated by a mutagen or carcinogen, genetically engineered or altered so that it is suppressed or blocked Can be done. These changes include specific sites for deletions, insertions, inversions, etc. It includes, but is not limited to, mutagenesis techniques such as mutagenesis. No; nucleotide sequences that result in methylation when used in vivo, e.g. Genetically engineer bacterial nucleotide sequences that promote cell synthesis and alter gene expression. Methylation of the test gene can be carried out by creating (for example, Ti Ighman and Levine, 1987. in Gene Trans fer, edited by Kucherlaputi, Plenum Pub, NY, p. (See p. 189-221). These test genes are then tested using the techniques described below. can be introduced into transgenic animals.

In practicing the present invention, transgenic activity related to mutagens or carcinogens may be The biological testing system can be used as follows: A group of transgenic animals other than the suspected mutagen or Exposure to carcinogens. Transgenic animals then mutate the test compound at specific endpoints Examine the expression of test gene products that are pathogenic or carcinogenic. activated test If the test gene expresses a transforming gene product, the transgenic animal is Kill and examine for the presence of tumors. activated and expressed in a tissue-specific manner If the transforming test gene is genetically engineered to Only certain tissues need to be examined for tumors.

If the activated test gene expresses a non-transforming product, it may be mutagenic or Detection of carcinogenicity depends on the nature of the test gene product. For example, Activation of a test gene product that is lethal to genetic animals cause death to biologically active animals; Activation of test gene products, e.g. hormones, indicates the presence of hormones. by testing transgenic animal tissue or to detect biological effects on animal metabolism and/or homeostasis. It can be detected by Activation of test gene products with enzymatic activity is performed in animal organs, Can be detected by testing tissues or individual cells for appropriate enzyme activity . Antigenic test gene products can be detected immunologically. Such test genes if it has been genetically engineered to be activated and expressed in a heterologous manner. , only relevant specific tissues need be tested for the presence of the test gene product.

For example, tissue samples obtained from appropriate organs at slaughter or by biopsy ( (e.g., tissue sections, blood samples, body fluids, cell specimens, etc.) for the presence of the test gene product. can be assayed for

Can transgenic mice carry multiple copies of a transgene in their chromosomes? Therefore, the transgenic animal test system of the present invention can be used for tests using normal mice. It would be more sensitive. Two stains each containing 80 copies of the test gene Mice with chromoplasts have 2 copies (l per chromosome) for the mutation It is very likely that they are 80 times more sensitive than normal mice. did Therefore, the target size is larger for mutagens or carcinogens. gene( The nucleotide sequences on either side of the chromosome) are mediated by mutation rates or mutagens. It is well known that this can affect the frequency of mutagenesis, so it is well known that can be optimized. This transgenic mouse system can be used in two ways. This variable can be exploited in the following way: (a) several independently induced triggers Transgenic mice contain test genes integrated at different sites or chromosomal locations. contains genes and thereby influences the mutagenic frequency of the gene location. Any other tests that can be examined or used will take this variable into account. and (b) the human test gene together with its surrounding human DNA sequence. Human D The influence of NA sequences can be evaluated.

Various aspects and components of the invention are described in more detail in the following subsections. Book description For purposes of clarity only, the present invention will be discussed in terms of (a) mutagens; or test genes activated in response to exposure to carcinogens (tumorigenic and non-tumorigenic); (b) results in tissue-specific expression of the activated test gene; regulatory elements; and (C) a transgenic gene of the invention containing the test gene. Creation of animals.

5.1. ) Regarding mutagens and carcinogens in transgenic animals tumor induction test gene system Genetically engineered and placed into the genetic background of a transgenic animal The transforming test gene of the present invention expresses a transforming gene product and therefore It induces tumors only in response to activation by mutagens or carcinogens. Book The transforming test gene of the invention includes the following transforming gene products. but are not limited to: (a) inhibiting the expression of a transforming oncogene product; Any cancer gene of viral or cellular origin that contains blocking or suppressing mutations. (b) when the expression of the oncogene product is amplified or when the assigned gene is mutated; Any assigned gene that transforms cells only when it becomes an oncogene; (C ) Highly methylated, which blocks or suppresses the expression of transforming gene products. any oncogene or attributable gene that is suppressed; (d) other genes upon reversion; Its expression is what can activate the tumor, which in turn induces tumor formation. Any gene that contains a mutation that blocks

More specifically, in one embodiment of the present invention, an oncogene having a mutation is Can be used as a test gene to detect foreign carcinogens . For example, the test gene may contain point mutations, insertions or insertions that block expression of the oncogene product. causes deletions; i.e., frameshifts, premature chain termination, etc. It may contain an oncogene that breeds mutations that cause it. Such test genetics The offspring can be placed into the genetic background of a transgenic animal, resulting in Transgenic with mutagens that revert point mutations, insertions or deletions, respectively. Exposure of the animal to the human body induces activation of oncogenes; i.e., the expression of transforming oncogene products. current, and thereby will give rise to the generation of tumors.

In another embodiment of the invention, the test gene comprising the assigned gene is transcribed. can be inserted into the genetic background of a genetic animal. This transgenic The test animal is not mutated, but rather the test gene (in this case, the attributed gene) is Exposure to carcinogens that amplify expression can result in transformation and tumor development. will bring. Alternatively, highly methylated attributed genes or cancer genes Inserting a test gene comprising a gene into the genetic background of a transgenic animal However, the gene product may be expressed at very low levels, if at all. cormorant. This transgenic animal is exposed to carcinogens that inhibit or disrupt methylation. exposure results in overexpression of the assigned gene or oncogene, resulting in trait causing transformation and tumor formation.

For clarity of discussion, various test gene systems are used for individual cancer genes. Although described in the following subsections, the invention is not limited to the exemplary oncogenes described. Not at all. In fact, whether it has already been described or has yet to be discovered. Any cancer gene, even if it is not, can be used in the assays described in the following subsections. can be done. Known oncogenes that are available include those derived from DNA tumor viruses. oncogenes (e.g., T antigen genes derived from SV40 or polyomaviruses, EIA and EIB genes derived from adenovirus and papillomavirus and It is known to have the potential to induce tumors derived from perpesvirus and perpesvirus. other genes; for a review, B15hop, 1985. Ce1l 42:23 -38) Oncogenes derived from niretrovirus (e.g. v-abl, v-f es, v-fps,, v-fgr.

v-src, v-erbA, v-erbB, v-fms, v-ros, v-yes, v-mos, v-ras, v-fos, v-my b, v-myc, v-ski, v-sis, v-rel, v-kit, v-jun, v-ets; as a review, B15hop, 1985. Ce1l 42:23-38) ; Derived from cellular oncogenes corresponding to viral oncogenes oncogenes, activated cellular oncogenes corresponding to viral oncogenes, and are cellular oncogenes for which viral counterparts have not yet been described (e.g. c-ne u; Hung et al., 1986. Proc, Natl, Acad, Sci .

USA 83: 261-264), but are limited to isn't it.

5, 1.1. Test gene system chain termination for mutagens Oncogenes can be constructed that carry oncogene mutations. Such a mutation Reversion mutations will give rise to tumors in transgenic animals carrying oncogenes. cormorant. Reversion events that can be tested with this method include (1) transitions; or transversions of both types of point mutations; and (2) deletions or Both types of insertions, frameshift mutations, are included.

For example, the normal myogenic gene ras is found in mice, rats, and humans. , functions in most cells to regulate cell division. This myogenic gene is A mutation that activates this gene and makes it an oncogene (amino acid 12 of the protein) changes), which can develop in many different types of tissue and give rise to vine tumors.

When the raS oncogene is placed in transgenic mice, the complete abnormal protein will give rise to tumors in almost any tissue that expresses it (fetal or is an adult). A second point mutation that prematurely terminates ras protein synthesis It is possible to prevent the production of ras oncogene protein by creating it within the oncogene. I can do it. This is called a “chain termination” mutation and is known as a “chain termination” mutation. Generates only protein fragments produced from mRNA encoded by genes (specific changes in the ras oncogene that lead to premature chain termination) (See Figure 1 for details). Such chain termination mutations are generation or transversion mutations at selected sites in the ras gene. In each case, the ras protein Only small fragments of the tumor are produced and no tumor is formed. Sudden change in chain termination Transgenic mice carrying the ras oncogene with differences in their normal The ras protein is made from its own mouse myogenic gene, and the ras protein Transgene fragment (ras oncogene with chain termination mutation) child). Sequence or signal of chain termination mutation Any mutational event (the first (see figure), cells carrying this mutation now produce the ras oncogene product. will be able to produce. This product (this is the normal ras proto-oncogene product) predominant), the mutagen undergoes a transition or transversion sudden change. May cause tumor formation in any tissue that acts to revert the mutation. I can do it. The efficiency of tumor formation is altered by the genetic background of transgenic animals. (For example, C3H mice are compared with 86 strains of mice, Demonstrates increased frequency of liver tumors that occur spontaneously and in response to carcinogens; B6 The /C3H system is described by Reynolds et al., 1987, 5science 237. (reviewed in: 1309-1316), or although it does not itself induce tumors. By introducing a second oncogene that promotes tumor formation in conjunction with other oncogenes. (For example, the adenovirus EIA gene enhances the oncogenic properties of the ras gene; Ruley, 1983. Nature304: 602-606), Can be adjusted.

In one embodiment of the invention, the normal human raS attributable gene (or one thereof) transgenics containing all other oncogenes tested in a series of bioassays. It is possible to generate genetic mice. If about 80 copies of this human gene one of them acquires a mutation at the precise site (amino acid 12 or 61) Unless otherwise, the mouse should be fine. The next mutation causes a tumor. make you feel the same Again, high copy number increases target size and sensitivity; human genes more closely resemble the human genome to test for carcinogens We provide a DNA environment with In this manner, more common oncogenes of human origin Some are tested for mutation frequency in transgenic mice. . It is sufficient that different oncogenes are selectively mutated by different carcinogens. This is possible in transgenic mouse systems. It will be possible to monitor the If this is correct, the mutagen or For testing carcinogens: (1) the use of human genes (human DNA context and (2) use of multiple myogenic genes; and (3) It will be necessary for tests to be performed in vivo for metabolic activation. The present invention Only transgenic mouse studies meet these criteria, and these issues It provides a unique testing system that can process

5.1.2. Test gene system for carcinogens that do not cause significant mutations Most carcinogens are also mutagens, but only weak mutagens at best. There are some clear examples of carcinogens. Diethylstilbestrol (DBS) ) is a synthetic steroid known to be carcinogenic in humans and rodents. It is a hormone. DES was not active in the Ames test, and its metabolic derivatives were also active. It's not gender. DBS is a cell culture medium for ouabain-resistant and HGPRT-negative cells. It is also inactive in the nutritional test. DBS in cell culture, rodents and humans Known effects include altering chromosome ratios in dysplastic lesions or in cells treated with this hormone. (generate aneuploid cells). in aneuploidy or abnormal ratios An abnormal number of chromosomes increases the concentration of some genes or myogenic genes and increases the concentration of others. This is thought to reduce the

This results in altered cell growth control and tumor formation. High copy of myogenic genes Mice carrying it in number or on many chromosomes are susceptible to the effects of DES or related drugs. Expected to be very sensitive. of myogenic genes rather than generating mutations. Remove any chemicals that act by changing the ratio of can be tested in mice or rats whose ratios have already been changed depending on the condition. child Tests like DES detect low levels of drugs like DES and test positive for carcinogenicity. recorded, while Ames test and others are negative.

Another class of carcinogens promotes translocation or amplification of selected DNA segments be able to. What this mechanism has in common is that myogenic genes are produced in abnormally large amounts. The fact is that it is possible to produce gene products. Insertional mutations (from viral preparations) ) and translocations adjacent to oncogenes often result in very high levels of gene expression and and lead to overproduction of normal products. Amplification of gene segments yields the same result . Overexpression of myogenic gene products can generate tumors. drugs like this To test for various numbers of myogenic genes (e.g., 3.10.1.5. 20.30.50.80 copies) to determine the tumor-producing levels. It should be possible to determine with a copy number slightly below the threshold for tumor induction By using mice, any duplication or amplification events can generate tumors. cormorant. This test is more sensitive than tests using normal mice. Furthermore, myogenic genes Mice carry genes on multiple chromosomes that can activate genes and cause tumors. increases the target size for possible translocations. As the sensitivity of this type of test increases, and provide a quantitative basis.

5.1.3. Test gene system for DNA methylation 5-Azacytidine-like Although such drugs have weak mutagenicity in cultured mammalian cells (Landolph and and Jones.

1982, Cancer Res, 42: 817-823) Culture odor (Benedict et al., 1977, Canc erRes, 37:2202-2208), this is a neoplasia-like event. be. One of the properties of 5-azacytidine is that it can add a methyl group to cytosine in DNA. It is by inhibiting the enzyme that causes As a result, D NA is poorly methylated. DNA methylation reduces gene expression and Therefore, insufficient methylation of DNA can result in the expression of normally inactive genes. .

In this way, myogenic genes are overexpressed and produce abnormally high levels of normal proteins. It may be produced in the body and cause cancer. Therefore, chemicals that affect the methylation mode Quality testing can be useful.

These animals carry viral oncogenes that can give rise to tumors. Transgenic mice were generated (van Dyke et al., 1985.C5 H3QE 50: 671-678). Some of these mice have viruses The tumor expresses a sexual oncogene and develops a tumor as expected. Other strains of mice have the same virus This oncogene is highly methylated at its cytosine residues. and these strains are capable of expressing viral oncogene products. Incapable of producing scars or tumors. In one rare case, the master of this strain A tumor was detected in a mouse, and T'INA from this tumor was detected as a viral oncogene protein. It was poorly methylated compared to other tissues that do not express protein. this strains of mice are useful for screening chemicals and altered methylation patterns. be. Moloney retrovirus in the chromosome, highly concentrated in its cytosine residues It is known that there are similar strains of mice that are methylated and do not express the virus. There is. Any drug that backmutates this methylation pattern activates virus production. do. Viral replication within susceptible hosts is similar to the previously described clonal tumor cells. It is similar to a cell amplification system. Transient amplification is performed and easily assayed.

5.2. Regarding mutagens and carcinogens in transgenic animals Non-tumorigenic test gene system mutagens or carcinogens in the transgenic animal test system of the present invention; Genetically engineer a large number of non-transforming test genes to detect quality be able to. Therefore, in response to activation by appropriate mutagens or carcinogens, Any structural element that encodes an assayable product, such that it is expressed only when The gene was genetically engineered in the same manner as described above for the transforming test gene. can be manufactured. For example, activation by a mutagen that reverts this mutation such that they contain mutations that inhibit or block expression unless sexualized. Test genes can be genetically engineered. Or, if the test results if not activated by carcinogens that amplify or demethylate the gene, respectively. Is such a test gene present at low concentrations such that expression is inhibited or prevented? or can be genetically engineered to be highly methylated .

Non-tumorigenic test genes are 1. as described in more detail below. toxins, hormones any of a number of products including, but not limited to, enzymes, antibodies, etc. It may be coded as follows. Such gene production in transgenic animals Activation and expression of the gene product is based on the biological effect of the gene product on the animal. , or by assaying appropriate animal tissue for the test gene product. Can be detected.

Use of non-transforming test genes in the transgenic test system of the present invention One limitation that one skilled in the art may encounter in its use is that The possibility that one cell does not make enough product to be detected in the transgenic animal There is. This limitation can be overcome by the following assay system: cterophage T7 DNA-dependent RNA polymerase gene in tissue-specific (e.g. liver albumin control region) Wear. This gene has a nonsense mutation. Oncogenes (e.g. activated r as) Viral genes present on the cell surface and known to be immunogenic The product (e.g. influenza M2 protein or VSVG protein) Each is inserted behind a bacteriophage F-specific control region. three legacies All genes will be introduced into transgenic mice and rats. carcinogen Active T7 RN was created by reversing the nonsense mutation in the T7 polymerase gene by A polymerase will be produced. This polymerase then If Tsage is stable in the cap structure, oncogenes and cell surface proteins Activates both expressions simultaneously. Oncogenes are amplified in cells where an activation event has occurred start. When a sufficient number of cells accumulate foreign antigen on their cell surface, an antibody response occurs. answers are elicited. Serological testing of antibody responses to test gene antigens It becomes ssei. For example, antibodies may be present in a blood sample before a palpable tumor is evident. should be present and detectable and therefore the endpoint of transgenic animal testing systems. can be accelerated. However, when using this system, the following Potential problems should be kept in mind: (1) The T7 polymerase gene is It was expressed on the vaccinia chromosome in the cytoplasm of human cells, but it was rarely expressed in the nucleus. It's not.

Nuclear localization to increase the efficiency of bacteriophage polymerase reaching the nucleus The signal can be fused with a polymerase gene (e.g., Kalde ron et al., 1984, Ce1l 39: 499-509; Welsh et al., 1986. Mo1. Ce1l.

Biol, 6: 2207-2212). (2) Mold by T7 polymerase Metsenger RNA produced in the mammalian nucleus has a cap structure (5° modified end). It may or may not be decorated. This means that Metsenger RNA would destabilize the polymerase system, necessitating the search for alternative polymerase systems. ( 3) Foreign antigens tend to increase rejection of tumor cells by the host. (4) Natural Tsuorusu infection may occur and therefore a second antibody against a second viral antigen (e.g. For example, it is necessary to screen for production of influenza HA protein). Might happen. A second viral antigen if the antibody response results from tumor induction There are no antibodies against the virus, but it would be detected if an infection occurred. That cost Alternatively, non-viral antigens can be used as test genes (e.g. human cell surface antigen).

Non-tumorigenic test genes that can be used according to this embodiment of the invention include This will be discussed in more detail in the following subsections.

5.2.1. Lethal test genetic system Toxic proteins, such as snakes and vertebrates, as well as some plants (plants toxins) and those produced by toxic strains of bacteria (e.g. botulinum toxin) The gene encoding the can be genetically engineered as a test gene; This can be inserted into transgenic non-human animals according to the invention. test Activating the gene to express the test gene product into the circulatory system of the transgenic animal Exposure of such transgenic animals to substances that cause secretion and Transgenic animals will die.

Toxic proteins such as scales used in accordance with the present invention include snake venom toxins; These include, but are not limited to, plant toxins, bacterial toxins, and the like.

For example, known snake venom toxins are highly toxic substances that cause paralysis and death in victims. They are highly antigenic polypeptides and proteins, and depending on their mode of action, they can cause damage to the heart muscle. Cardiotoxicity that causes irreversible depolarization of cell membranes of nerve cells and nerve cells; Blocks receptors for transmitters at synapses and at the motor terminals of skeletal muscles. neurotoxins that interfere with neuromuscular transmission by inhibiting neuromuscular transmission; and acetylcholinesterase and protease inhibitors that inhibit similar enzymes involved in neurotransmission be done. Procardial and protease inhibitors consist of 60 amino acid residues ( Molecular weight (MW): 6900 Daltons), -Potency is also a well-studied neurotoxin Bula) are long chain (71-74 amino acids, MW 8000 Daltons) and short chain (6 It is further divided into molecules of 0-62 amino acids, MW 7000 Daltre). Chain Neurotoxins isolated from snakes have a high molecular weight (e.g. Singh, MW 30.000 Daltons). The most powerful snake venom, typhoxin, has two It consists of different polypeptide chains. Neurotoxicity is distinguished by the presence of disulfide bridges. It is extremely stable (4 pieces for MW7000 Dalton, 5 pieces for MW8QOO Dalton, 7 pieces with MW 13,5000 Daltons). Scorpion venom is similar to cobra toxin , MW 6800-7200 Daltons, 4 disulfide bridges and known sequence There are 63-64 amino acids.

Known plant toxins that may be used in accordance with the present invention include, but are not limited to: but not limited to: (a) having antihypertensive activity and causing a delay in heartbeat; The homologous biscotoxin (MW 4840 daltons, 46 amino acids of known sequence, 3 disulfide bridges); and (b) Toxic albumins such as ricin and abrin that inhibit protein biosynthesis hmm. Ricin (MW66,000 Da) derived from the seeds of castor bean (Rieinus) luton and 493 amino acid residues) are linked by a disulfide bridge W32.000 Daltons) and B-* (MW34.000 Daltons) Become. Toxicity is carried by the 8-chain, but the B-chain connects the toxin to the cell surface. Join. Abrin with similar action and structure is Abrus Bretorius ( Derived from the red seeds of Abrus precatorius.

The most well-studied of bacterial toxins are those secreted by Gram-positive bacteria. exotoxins, including but not limited to: No: (a) acidic single-chain protein with high toxicity (fatal at 1 μg/kg body weight) Corynebacterium diphtheriae, which is a proteinaceous protein (MW 62.000 Daltons) (Coryne-bacterium diphtheriae) phtheria toxin, which works by promoting the binding of ADP-ribose to the enzyme. Inactivates peptidyltransferase in nuclear cells; (b) is a powerful neurotoxin Derived from a certain Clostridium Tetani Tetanus toxin (MW 150.000 Daltons), which comes in two forms: 2 subunits The filtrate toxin, consisting of and as a single-chain cytotoxin (0.01 ng/k in mice). (C) Clostridium botulinum (fatal); and (C) Clostridium botulinum Free toxic botulinum toxin (type A MW1) derived from A. umbotulinum 50,000 daltons, type B MW 167,000 daltons), which has neurotoxic activity. requires the presence of one free SH-group for (0.03n in mouse g/kg (fatal).

A number of toxic proteins that can be used in accordance with the present invention are listed and specified in Table 1 below. gender, but is not intended to be limiting.

Table 1 toxic protein Botulinum toxin Clostridium botulinum 3X10 −'Break Wind Poison Element Clostridium Tetani lX l0-'Diphtheria toxin Corynebacterium diphtheriae 0.3 animal toxins Cobra Poison Cobra 0.3 Crotalus toxin Rattlesnake 0.2 As shown in Table 1, Many of these proteins are highly toxic and therefore cannot be used in activated test products. The gene does not need to be expressed at high levels to produce a lethal dose. In this case, the book A T7 amplification system is not required to practice the invention. However, this The test gene allows the gene to transgenic the toxic product upon activation. so that it is expressed in tissues (e.g. liver, pancreas, etc.) that can be secreted into the animal's bloodstream , conveniently genetically engineered so that the toxin has no biological effect on animals. It can be made so that it can have an effect. In the necessary steps in the method of the invention However, animals can be necropsied to confirm the cause of death.

5, 2.2. Hormone test gene system according to the present invention protein or peptide as a test gene inserted into a genetic animal. Genes encoding hormones can be genetically engineered. Trance A test gene that results in the expression and secretion of a test gene product into the circulatory system of a genetic animal. Exposure of these transgenic animals to substances that activate the gene Detection of hormone-induced biological effects on transgenic animals (e.g. appropriate metabolic reactions), or by immunoassay for hormones. Detection can be based on any of the following: Hormone activity is generally species-specific. Because it is not heterogeneous, and very low concentrations of the hormone usually produce a large metabolic response Therefore, amplification of test genes using, for example, the previously described T7 amplification system is It is not necessary for the practice of this method of the invention. However, hormone gene production Amplification may be necessary if detection of the substance is performed using an immunoassay.

In any case, the test gene encoding the hormone secretes the gene product into the circulation. The gene product is expressed by tissues that have cellular machinery that allows it, such as the liver and pancreas. It is preferable to produce it by genetic engineering so that it can be used.

Any hormone that induces a measurable metabolic effect can be used in accordance with the present invention can do. These hormones include growth hormone, releasing hormone, These include, but are not limited to, surin and the like. The test gene is , can properly process the gene product to produce the mature, active form of the hormone molecule. As such, these hormones include the [pro] and [pre-pro J] forms. stomach.

5, 2.3. Enzyme or Antigen Test Gene System The gene encoding the enzyme is Test gene inserted into a non-human transgenic animal according to the invention It can be produced by genetic engineering. Test genetics by activating test genes Exposure of such transgenic animals to substances that lead to progeny product expression can be detected by assaying the animal for the presence of. The gene product is tissue specific. When genetically engineering a test gene to be expressed in a different manner, Only the specific tissues involved need be tested for enzymatic activity. This is a related organization autopsy or biopsy, and tissue enzyme activity testing, e.g. Tissue sections can be treated with appropriate substrates to detect the presence of enzymatic activity. Wear. To enable detection of the enzyme in the blood, the gene product must be introduced into the circulatory system. Genetically engineer the test gene so that it is expressed in cells that can secrete It should be made. According to this embodiment of the invention, expressed and secreted into the blood test genetics using e.g. the T7 amplification system described above to increase the concentration of the enzyme. Sometimes it is necessary to amplify the child.

Any enzyme that catalyzes the production of a detectable product can be used as a test gene according to the invention. Can be used. Enzymes that catalyze the production of colorimetrically quantifiable products, as well as from other species Enzymes that target transgenic animals to suspected mutagens or carcinogens Particularly useful due to the ease of detecting test gene activation and expression in response to exposure. It is for use. For example, it contains beta-galactosidase, alkaline phosphatase, etc. Enzymes including, but not limited to, can be used.

5, 2.4. P450 test system for non-genotoxic carcinogens 2, 3. 7. 8-tetrachlorodibenzo-p-dioxin (diox Certain compounds, such as halogenated aromatic hydrocarbons) and similar other halogenated aromatic hydrocarbons, It is a non-denotoxic carcinogen, and it affects the transcription rate of the cytochrome P-450 gene. can be induced. Chemicals that can activate P-450 are carcinogens Assuming that, an assay to screen for activation in transgenic mice We can come up with something. Sequence elements of the P-450 gene required for transcriptional activation have not been identified. (JOneS et al., 1986. Proc, Natl. Acad, Sci, USA83: 2802-2806; Johes et al., 198 6. J. Biol. Chem. 261:6647-6650). Daioki The control of P-450 by syn is not an all-or-nothing, but rather a regulatory This is a powerful effect. Therefore, the product produced must be quantified. beta moth Enzymes such as lactosidase are useful test genes, and this It is under the control of the hepatocytes and, when activated, can exert activity on a per hepatocyte basis. Expresses an assayable product that is easily quantified.

5.3. ) Expression of activated test genes in transgenic animals Tissue-specific regulation Transgenic animals with foreign genes carry the foreign genes throughout the animal's life. Information can be expressed in a tissue-specific or time-dependent manner. various fruits (for a review, see Ti Ighman and Levine, 1987. Gen eTransfer, Kucherlapati, Ed, Plenum Pub, NY, pp.

189-221), a DNA sequence existing around or near a certain gene is due to both the time of expression (fetal or adult) and the tissue pattern of gene expression. It is shown that something is true. fetal or adult liver, breast tissue, brain tissue, immune system cells, Red blood cells, pancreatic cells (both beta and pig cell types), lens of the eye, muscle cells and Regulatory signals that drive gene expression in connective tissue and connective tissue are mapped or isolated. Other gene regulatory signals have been identified that give rise to a wide range of tissue-related expression. ing.

This is used to generate tumors in specific tissues using oncogenes, and Correcting the underlying defects by supplementing with appropriate hormones, altering the immune system and We tracked the regulatory circuits that control the epidemic. Therefore, tissue- and time-specific regulation By placing a signal in front of a foreign gene (which is not normally expressed in that tissue), A transgene that expresses a given gene (which makes a protein) in that tissue at a specific time. It is clear that nicked mice can be produced.

Expression of activated test genes in the transgenic animal system of the present invention Currently, it is possible to place one or more specific regulatory signals adjacent to the test gene. can be made tissue-specific. For example, the liver undergoes mutations here. Particularly suitable as a site for monitoring This is because hepatocytes contain enzymes that are known to be activated. Tiger Fetal liver (alphafetus) is placed in front of the test gene inserted into the bipedal animal. regulatory signals for adult liver (albumin gene) or adult liver (albumin gene) Carcinogens can be transferred to fetal and adult liver tissue by genetically engineering can be tested for mutations. In transgenic animals The exact anatomical site to be monitored will be known, thereby allowing It becomes easier to ``decipher'' ugly test animals. Various different types of organizations as appropriate A transducer into which a test gene carrying control sequences that mediate expression in genic animals can be created. Therefore, animal libraries are It can be used to test the effects of suspected mutagens on organs and tissues.

Tests can be standardized and expressed quantitatively: for example, if the activated test gene When expressing tumor-inducing products, known mutagens and carcinogens can be Can be used with transgenic mouse lines: dose and tumor detection Quantitative representation of the number of independent tumors (in the liver) as a function of time for mutagenesis frequency can be calculated; Variables such as age, tissue growth, and tissue regeneration can be quantified; A set of standards can be created; a negative result within a certain time frame indicates that mutagenic Indicates that a level or frequency of occurrence could be detected but was not actually found. ; relatively low mutagenesis frequency levels can be applied;

5.3.1. Regulatory elements for tissue-specific expression of activated test genes Any transcriptional control region, whether previously described or not yet described. can be used to regulate the transcription of a test gene. Already described transducer Examples of tissue-specific transcriptional control regions used in genetic animals include: Includes: elastase I gene regulatory region (S swift et al., 1984. Ce1l 38: 639-646; 0rni tz et al., 1986゜Co1d Spring Harbor Symp, Qu ant, Biol, 50:399-409; MacDonald, 1987 ．． Hepatology 7: 42s-51s); in pancreatic beta cells active insulin gene control region (Hanahan, 1985. re 315: 115-122); Immunog active in lymphoid cells Robulin gene control region (Grosschedl et al., 1984. Ce1l 3 8: 647-658 HAdams et al., 1985, Nature 318: 533-538; Alexander et al., 1987 Mo1. Ce1 1. Biol, 7: 1436-1444); fruit, breast, lymphatic system Mouse mammary tumor virus control region active in and mast cells (Leder et al. , 1986. Ce1l 45: 485-495); active in the liver Albumin gene control region (Pinkert et al., 1987. nd Devel, 1: 268-276); Farfetoprotein gene control region (Krumlauf et al., 1985. o1. Ce11. Biol, 5:1639-1648; Hammer et al. , 1987. 5science 235:53-58); Active in the liver Alpha1-antitrypsin gene control region (Kelsey et al., 1987. Genes and Devel, 1:161-171); in bone marrow cells and the active beta-globin gene control region (Magram et al., 1985. ture 315: 338-340; Kollias et al., 1986. C. e1l　46　:89-94); basic protein gene control region (Readhead et al., 1987. Ce1 l48nia03-712); myosin light chain-2 gene active in skeletal muscle Control region (Sha, ni, 1985. Nature 314:283-28 6); and gonadotropin-releasing hormone gene regulation active in the hypothalamus. domain (Mason et al., 1986.5science 234:1372-13 78).

5.4. Test gene systems for germline targets In another embodiment of the invention The aim is to focus on the male germline and identify carcinogens that affect germline cells. An assay can be carried out to detect the following: carrying a nonsense mutation. test genes that function in all tissues (e.g. liposomal L32 protein). regulation of protein regulatory regions) or transcriptional control regions that function specifically during spermatogenesis. Introduce it to the original. Sperm were obtained from adult males by dissection (approximately 5xlO”/animal). , reversion of nonsense mutations is achieved using fluorescently labeled antibodies against the product and fluorescently activated cells. Sorting sperm for activation and expression of test gene products using a sorter Evaluate by. The test gene is detected by an antibody labeled with a fluorescent reporter group. Can be detected. Even though it encodes a membrane protein that is not normally expressed on sperm cells. good. Alternatively, the activated test gene product is beta-galactosidase. This may be evaluated by cleavage of its substrate x-gal. mutagenesis A single male mouse exposed to a sexual carcinogen could, in theory, develop a statistically significant mutation. Triggered frequency. If gene expression is altered (e.g., dioxin is p -450 transcription) in germline cells in this generalized assay. Select non-genotoxic (i.e. non-mutagenic) carcinogens also with regard to Can be done. Although the number of cells obtained from each individual animal will be small, this test The scope of application can be expanded to lineage cells.

5.5. Creation of transgenic animals used in the bioassay of the present invention Preparation of animals for which a foreign gene is introduced into the animal's germ line (inherited traits) Utilizes recently developed technology. A foreign substance called a transgene in its germ line Animals carrying the DNA fragment are called transgenic animals. Created test animals One method for creating transgenic mice is described below.

In general, the schemes currently used to create transgenic mice are: This is as follows: Male and female mice with a clear progeny genetic background were mated late at night. let After 12 hours, females are sacrificed and fertilized eggs are removed from the utero-oviduct. at this point , the pronuclei have not yet fused and you can see them with a light microscope. then outside Microinject the foreign DNA into the pronucleus (1oo-iooo per egg).

molecule). Fusion of the pronuclei occurs shortly thereafter, and in some cases foreign DNA is transferred to the fertilized egg. or into one chromosome of the zygote (usually). Then the zygote is pseudopregnant implanted into female mice (previously mated with vasectomized males) where the embryos were incubated at 20-21 days. Develops over the entire gestation period. The surrogate mother gave birth to these mice and kept them for up to 4 weeks. Wean the baby mouse from its mother. These mice were examined for the presence of foreign DNA. In order to survive, a part of the tail (an organ that is optional) is removed and DNA is extracted. D.N. A-DNA hybridization (Todd plot, slot plot, or southern plot) Determine whether the mouse has foreign DNA using . 10% of injected eggs develop correctly and give rise to mice. Mau born A quarter (25%) of the population are transgenic and therefore The efficiency is 2.5%. Once these mice are bred, normal (Mendelian) The mouse receives a foreign gene attached to its chromosome in a regular manner. Transdi Breeding two homozygous mice with genetic DNA produces 10 offspring. 0% means carrying the Nicoby transgene.

When this is done, the mouse repeats tandemly at one chromosomal location or site. They usually have copies of the foreign gene (3 to 80 copies).

The present invention is not limited in scope to the embodiments disclosed herein, but is The embodiments are intended merely as illustrative of various aspects of the invention. functionally equivalent Any embodiment of is within the scope of this invention. In fact, what is shown and described here In addition, various modifications of the invention will be apparent to those skilled in the art from the foregoing description. cormorant. It is intended that such modifications come within the scope of the appended claims.

Nonsense mutation I in VRAS Nonsense mutation 2 in VRAS international search report

Claims (1)

[Claims] 1. Transgenic animals are exposed to mutagens or substances that activate expression of the test gene product. test genetics in the transgenic animal unless exposed to carcinogens or carcinogens. A detectable genetically engineered product such that expression of the child product is prevented or inhibited. Contains in its germline a transgene containing a test gene encoding a product Transplants for testing mutagens and carcinogens consisting of non-human animals that genetic animals. 2. The test gene comprises a transforming gene encoding a tumorigenic product. Transgenic animals according to the first item of the scope of the request. 3. The transfection of claim 2, wherein the transforming gene comprises an oncogene. nick animal. 4. The transfection of claim 2, wherein the transforming gene comprises an oncogene. nick animal. 5. Claim 3, wherein the oncogene comprises a DNA tumor virus oncogene. transgenic animals. 6. The transformer of claim 3, wherein the oncogene comprises a retroviral oncogene. genetic animals. 7. The transgenic gene according to claim 3, wherein the oncogene comprises a cellular oncogene. animal. 8. The test gene contains a non-transforming gene encoding a detectable gene product. The transgenic animal according to claim 1, comprising: 9. The transfection of claim 8, wherein the detectable gene product comprises a toxin. nick animal. 10. The transducer of claim 8, wherein the detectable gene product comprises a hormone. genetic animals. 11. The transducer of claim 8, wherein the detectable gene product comprises an enzyme. genic animal. 12. The transducer of claim 8, wherein the detectable gene product comprises an antigen. genic animal. 13. If the test gene is mutated, the transgenic animal reverts the mutation. The test gene product in the transgenic animal if not exposed to the source material. It encodes a structural gene that has a mutation that prevents or inhibits the expression of The transgenic animal according to claim 1, 2, or 3. 14. The transfection of claim 13, wherein the mutation inhibits transcription. nick animal. 15. The transfection of claim 13, wherein the mutation inhibits translation. nick animal. 16. The transgenic gene of claim 13, wherein the mutation comprises a point mutation. animal. 17. The transgenic activity of claim 13, wherein the mutation comprises an insertion. thing. 18. The transgenic animal of claim 13, wherein the mutation comprises a deletion. thing. 19. The transgenic movement of claim 13, wherein the mutation comprises an inversion. thing. 20. Transgenic animals are not exposed to carcinogens that demethylate DNA Claim 1, wherein the test gene is methylated so that expression is inhibited as long as possible. Transgenic animal of item 2 or 3. 21. Transgenic animals are not exposed to carcinogens that amplify the test gene As long as the test gene is present at a low concentration such that expression of the test gene is inhibited. A transgenic animal according to certain claims 1, 2, or 3. 22. (a) Transgenic non-human animals treated with mutagens or carcinogens; The transgenic animal is then exposed to a substance suspected of being Transgenic animals are exposed to mutagens or agents that activate expression of the test gene product. Expression of test gene products in transgenic animals unless exposed to cancerous substances Coating a detectable product that has been genetically engineered to prevent or inhibit containing in its germline a transgene comprising a test gene for And (b) the transgenic animal or a suitable organ or organ of the transgenic animal; examines the tissue for expression of the test gene product, where the transgenic The expression of a test gene product in a substance is an indicator of the mutagenicity or carcinogenicity of the substance being tested. shall be a mark; A test method for mutagens and carcinogens, comprising: 23. The test gene does not contain a transforming gene encoding a tumor-inducing product. and its expression is shown by tumor formation in transgenic animals. 23. The method of claim 22. 24. 24. The method of claim 23, wherein the transforming gene comprises an oncogene. 25. 24. The method of claim 23, wherein the transforming gene comprises an oncogene. 26. Claim 24, wherein the oncogene comprises a DNA tumor virus oncogene. the method of. 27. Claim 24, wherein the oncogene comprises a retrovirus oncogene. Law. 28. 25. The method of claim 24, wherein the oncogene comprises a cellular oncogene. 29. The test gene contains a non-transforming gene encoding a detectable gene product. 23. The method of claim 22, comprising: 30. 30. The method of claim 29, wherein the detectable gene product comprises a toxin. 31. 29. The method of claim 29, wherein the detectable gene product comprises a hormone. . 32. 30. The method of claim 29, wherein the detectable gene product comprises an enzyme. 33. 30. The method of claim 29, wherein the detectable gene product comprises an antigen. 34. If the test gene is mutated, the transgenic animal reverts the mutation. The test gene product in the transgenic animal if not exposed to the source material. It encodes a structural gene that has a mutation that prevents or inhibits the expression of 25. The method of claim 22, 23, or 24. 35. 35. The method of claim 34, wherein the mutation inhibits transcription. 36. The method of claim 34, wherein the mutation inhibits translation. 37. 35. The method of claim 34, wherein the mutation comprises a point mutation. 38. 35. The method of claim 34, wherein the mutation comprises an insertion. 39. 35. The method of claim 34, wherein the mutation comprises a deletion. 40. 35. The method of claim 34, wherein the mutation comprises an inversion. 41. Transgenic animals are not exposed to carcinogens that demethylate DNA Claim 22, wherein the test gene is methylated so that expression is inhibited as long as possible. The method of item 23 or 24. 42. As long as the transgenic animal is not exposed to carcinogens that amplify the test gene. The test gene is present at low concentrations such that expression of the test gene is inhibited. 25. The method of certain claims 22, 23, or 24.