TRANSGENIC TESTING SYSTEMS FOR MUTAGENS AND CARCINOGENS

Abstract: Non-human transgenic animal testing systems for mutagens and carcinogens are described. Recombinant DNA techniques are used to create unique test genes and regulation signals for detecting increased frequencies of mutations produced by mutagens and cancers induced by carcinogens. The test genes are introduced into the germ line of non-human transgenic animals to provide a unique bioassay panel for mutagens and carcinogens. The test genes of the invention may encode any of a number of different genes, the expression of which could be detected in a transgenic animal. However, the test gene is engineered so that the expression of the test gene product is either prevented or inhibited unless the gene is activated by a mutagen or carcinogen.

Description: TRANSGENIC TESTING SYSTEMS FOR MUTAGENS AND CARCINOGENS . 1. INTRODUCTION The present invention relates to a transgenic animal testing system for mutagens and carcinogens. Recombinant DNA techniques are used to create unique test genes and expression regulating signals for detecting increased frequencies of mutations produced by mutagens or the induction of cancer by carcinogens. These test genes are introduced into non-human transgenic animals to provide a unique bioassay test panel for mutagens and carcinogens. 2. BACKGROUND OF THE INVENTION 2.1. TESTS FOR CARCINOGENS AND MUTAGENS The first recognition that chemical compounds could be the causative agent of or be associated with cancer in humans came over 200 years ago when Sir Percival Pott described a high incidence of scrotal cancers in young chimney sweeps in England. In 1918, Yamcigiwa and Itchi awa (1918, J. Cancer Res. 3:1-29) produced carcinomas on the ears of rabbits after prolonged topical applications of coal tar from chimneys. By 1933, Cook, Hervett and Hieger (1933, J. Chem. Soc: 395-405) identified one of the major carcinogenic components of coal tar as 3,4-benzpyrene [benzo(a)pyrene]. In rapid succession the azo dyes (1934) were shown to cause liver carcinomas in rodents and beta- naphthylamine proved to induce bladder tumors in dogs (1938) . From these origins animal testing for carcinogens had begun and in 1941, 2-acetylamino-fluorine, a chemical intended for wide use in the United States as a pesticide, was shown to be a potent carcinogen (Wilson et al., 1941, Cancer Res. 1:595-608). Based upon these early studies it was clear that a variety of chemicals could induce cancers in humans and animals, and do so (1) in a tissue specific fashion, (2) over prolonged time frames, and (3) often with complex kinetics and multiple factors required. 2.1.1. BACTERIAL AND CELL CULTURE TEST SYSTEMS The question of how these carcinogens acted to cause cancer was addressed early by T. Boveri (Boveri, 1914, The Origin of Malignant Tumors, ed. Jena) who suggested a 5 genetic basis. This idea was the predecessor of the somatic cell mutation theory of cancer. By 1948 it was clear that some carcinogens, such as the benzpyrene series and the azo dye compounds, could cause mutations in the fruit fly, Drosophila melanogaster (Demerec, 1948, Brit. J. Cancer ■ j ø 2:114-117). Similar results were reported using other favorable genetic systems such as the mold, Neurospora crassa (Barratt and Tatum, 1951, Cancer Res. 11:234-241) , and mice (benzanthracene: Carr, 1947, Brit. J. Cancer 1:152-156; methylcholanthrene: Strong, 1949, Brit. J. 15 Cancer, 3:97-108). Clearly these early observations had their limitations in use as tests for mutagens and carcinogens. Some compounds failed to act as mutagens in some test organisms such as bacteria and fungi, yet caused tumors and mutations in mice. This led to the concept of 20 metabolic activation (see, for example. Miller and Miller, 1953, Adv. Cancer Res. 1:339-396). Some chemicals (for example, diethylnitrosoamine) were metabolically altered by enzymes in the liver (the P450 oxidase system and hydroxylation systems) or other tissues, creating new compounds that were both mutagens and carcinogens. Ames and 25 his coworkers (Ames et al., 1975, Mut. Res. 31:347-364) addressed this problem by incubating chemical compounds with liver extracts (enzyme preparations) prior to exposure of Salmonella typhimurium bacteria to these products. This permitted a more sophisticated test for mutagenic activity. 30 In this test, specific types of point mutations can be tested for an increased frequency of reversion by assaying for bacteria that can grow under restrictive conditions. The nature of the mutation (type of change at the DNA level) and its frequency can be quantitated accurately. This test 35 is commonly employed today as a rapid, inexpensive assay for mutagens. Other tests presently employed to detect carcinogens or mutagens include the selection for mutations in mammalian cells in culture. Chinese hamster ovary cells (CHO cells) can be treated with a mutagen, after which a mutagenic event at the genetic locus for the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT) can be identified by selection in the appropriate media (Hsie et al., 1979, _in Strategies for Short-Term Testing for Mutagens/Carcinogens, Butterworth, Ed., CRC Press, FL) . This X-linked mutation occurs in a haploid condition in male cells and detects a single hit by a mutagen. Resistance of cells in culture to the toxic drug ouabain can occur by a single dominant mutation at the genetic locus for an ATPase, and this has also been employed to measure mutation frequencies in cell culture. Even without a "gold standard* for the field, it is clear that the bacterial tests (Ames) and the cell culture tests (Hsie) have considerable drawbacks. First, chemical compounds are often metabolically altered in different ways by different tissues of the body. It is simply not possible to preincubate a test compound in extracts of all body tissues prior to testing for mutagenesis. Second, different body tissues show distinct susceptibilities to different chemical compounds. This results in the tissue-specific cancers seen with so many chemicals (beta-naphthylamine and bladder carcinomas; azo dyes and liver tumors; etc.). Bacterial and cell culture tests do not address this issue. Third, there is well documented literature (see Banbury Report, 1984, #16: Genetic Variability in Responses to Chemical Exposure, Cold Spring Harbor Press, NY) showing a genetically determined variability of the host in metabolic activation of chemicals or in response to products made in situ. While this variability can never be reproduced in any organism but the human, it is approximated by well- controlled genetic tests in mice or rats better than the evolutionarily irrelevant bacterial cell. Fourth, chemical carcinogenesis is a complex process often requiring variables such as time (age of the test host) , multiple events (termed promotion and initiation) and multiple stimuli prior to tumor formation (Hecker, 1968, Cancer Res. 28:2338-2349). These variables can be reconstructed in the whole animal models, but not in the bacterial or cell 0 culture tests. 2.1.2. WHOLE ANIMAL TEST SYSTEMS A typical animal test protocol for the detection of a carcinogen involves using male and female animals from two •J5 species fe.g. , mouse and rat) with 50 animals per group (Sontag et al. , 1976, "Guidelines for Carcinogen Bioassay in Small Rodents", Carcin. Tech. Rep. Ser. 1, DHEW Pub., NIH 76-81) . A chemical is then defined as a carcinogen if *in a well designed and conducted bioassay, it produces a 20 statistically significant increase in the incidence of neoplasms in one or more target organs* (Dunkel, 1985, in, Mechanisms of Toxicity of Chemical Carcinogens and Mutagens, Flamm and Lorentzen Ed., Princeton Sci. Pub. Co., Princeton, NJ) . Two basic tests have been developed for the 25 experimental detection of gene mutation in vivo; each test is confined to a single category, i.e. , the detection of either somatic or germ cell mutations. The detection of heritable mammalian gene mutation in vivo is limited to the mouse specific-locus test, whereas induced somatic mutation 30 can be detected using the mouse coat spot test. Both tests, however are based on the same genetic system (for a review, see Styles and Penman, 1985, Mutation Research 154:183-204). Very recently, transgenic animals containing an activated oncogene were proposed for use in detecting carcinogens or -_ for testing material thought to confer protection against 35 carcinogens (see European Patent Application 0169672 by Leder and Stewart) . Although the whole animal tests address some of the drawbacks of the bacterial and cell culture systems, the whole animal tests are generally inadequate for several reasons: (a) the detection level of statistically significant tumor incidences in the test group (50 animals) is not sensitive (at the 5-10% incidence level) ; (b) there is often well-documented variability between assays where no definite conclusion can be made. For example, of 203 bioassays of this type carried out by the National Cancer Institute (see Chu et al., 1981, J. Tox. Environ. Health 8:251-280), 15 assays (7%) were inadequate for evaluation of the results, 25 assays (12.5%) had only borderline or suggestive evidence for carcinogenesis, 53 assays (26.5%) were positive for only one of the two species, and 23 assays (11.5%) were positive for only one sex of the species which exhibited tumors; (c) the interpretation of these in vivo assays is often difficult requiring a mixture of expertise in pathology, statistics and toxicology, lending the results to subjective criteria which rest upon individual interpretations; (d) the time it takes for a tumor to arise is variable and negative endpoints are arbitrary. Some tests take up to 1.5 to 2 years to complete. Waiting for a negative result is a problem in a test with no defined endpoint. Clearly the whole animal tests currently in use have problems associated with them. Difficulties are compounded when one realizes that the other tests in use, such as the bacterial test (Ames test), fungal, Drosophila, cultured CHO cells, etc., are often evaluated against this 'standard* whole animal bioassay. At present, there is no adequately or uniformly accepted standard by which all tests can be judged or measured. Most experts in the field today agree, *there is no general agreement as to the most appropriate selection of tests* for measuring carcinogenesis or mutagenesis (Dunkel, 1985, in Mechanisms of Toxicity of Chemical Carcinogens and Mutagenr , Flamm and Lorentzen, Ed., Princeton Sci. Pub. Co., Princeton, NJ, p.69). The pharmaceutical and chemical industries generate thousands of compounds each year that are potential products for human consumption or environmental release. Several government agencies request . or require information related to mutagen and carcinogen testing prior to approval of a compound. At present, there is no official definition of an appropriate or approved test. This underscores the absence of a standard in the field. There is a need for a reproducible, inexpensive, accurate and encompassing test for mutagens and carcinogens. 2.2. HOW CARCINOGENS AND MUTAGENS ACT TO PRODUCE CANCER 2.2.1. DNA MUTATIONS The work of Ames and his colleagues (McCann and Ames, 1976, Proc. Natl. Acad. Sci. USA 73:950-954) has shown that a substantial proportion of carcinogens are also mutagens. These chemicals alter the genetic information, deoxyribonucleic acid (DNA) , producing an inherited change in a gene or a signal that regulates a gene. Different chemical, or physical treatments such as radiation, produce different types of mutations in the DNA molecule (chemical changes in the DNA) . It is often the case that a particular chemical will cause the same class of mutation reproducibly. Some chemicals react with the DNA molecule and alter it chemically. Other compounds are incorporated into the DNA chain and force mistakes to be made during duplication of the DNA at each generation. Because the chemistry of these reactions follows rules and is reproducible, the resultant type or class of mutation is reproducible. There are four nucleotides which make up DNA (adenine (A) , guanine (G) , thymidine (T) , and cytosine (C) ; with A and G being purine bases, and T and C pyrimidine bases) which pair in a double helix (A-T, G-C, or T-A, C-G base pairs) according to the rules of chemical bonding. Some chemicals produce point mutations which are single base-pair changes in the DNA. Two classes of point mutations are well-known: (1) transitions, where one purine is substituted with another purine, i.e., G for A so an A-T base pair changes to a G-C base pair, or one pyrimidine is substituted for another, i.e. , C for T so a T-A base pair is changed to a C-G base pair; and (2) transversions, where a purine replaces a pyrimidine (C-G changes to A-T or G-C) or vice versa. Again, different carcinogens or mutagens reproducibly induce higher rates of either transitions or transversions but in many cases not both. Thus, multiple tests must be available to detect the different events produced by different chemicals. The second type or class of mutation induced by carcinogens or physical agents is a DNA rearrangement. On a large scale, this could result in a chromosome translocation or the fusion of portions of two different chromosomes. In many types of cancers, this is a common event (see Cairns, 1981, Nature 289:353-357; Klein, 1981, Nature 294:313-318). On a smaller scale, deletions (loss of base pairs) , inversions (change in the orientation of a sequence of bases) , insertions (additional base pairs) or even amplifications of DNA segments (increase in copy number of a series of base pairs) have been reported as events associated with cancers (for a review, see Cancer Cells 2, van de Woude, Levine, Topp and Watson (Ed.), CSH Press, NY, 1984) . Finally, the regulation of gene expression in a specific tissue may well be modified by chemical alterations of bases such as methylation of cytosine residues (see Doerfler, 1983, Ann. Rev. Biochem. 52:93-124). A class of chemicals alters these patterns of methylation (5-azaσytidine) and also induces cell differentiation or transforms cells in culture. In sum, mutagens act in a wide variety of ways to alter the genetic information, DNA. They function in a reproducible manner to rause specific classes or types of mutations. A good test system is one that can detect each of these classes of mutation. 2.2.2. ACTIVATION OF ONCOGENES Clearly, a mutation by itself anywhere in the DNA molecule of a cell will not necessarily result in a cancer. For example, some mutations will occur in a gene that is essential for the life of a cell. If the altered product of this mutated gene fails to function, the cell will die (lethal mutation) . A multicellular organism tolerates this well (the loss of a few cells) . Other mutations will lead to cancer. Indeed, it has recently become clear that humans (and mice, rats, etc.) have a collection of genes which appear to be important for regulating cell division, growth, healing of wounds, production of hematopoietic cells (white blood cells, red blood cells, platelets, etc.) and some developmental processes. These genes (a collection of some 50-100 per organism) are termed proto-oncogenes (normal form) because they can, upon mutation of the gene or its regulatory signal, be involved in a cancerous process. Mutational activation of a proto-oncogene produces an oncogene or cancer causing gene (for a review see Cancer Cells 2, 1984, van de Woude, Levine, Topp and Watson, Ed., Cold Spring Harbor Press, NY) . The specific target of a mutagen, when it results in a cancer, is the set of genes termed proto- oncogenes. For example, when the DNA was extracted from cells derived from a human bladder carcinoma (T24/EJ, Parada et al., 1978, Nature 294:474-478) a specific oncogene, called Ha-ras, carried a point mutation within the ras proto-oncogene involving a C to A transversion in the DNA, resulting in a glycine to valine change at amino acid 12 in the protein. Many B-cell lymphomas or plasmoσytomas of humans (and mice) have a specific chromosome translocation (involving chromosomes 8-14, 8-2 8-22) in which the myc proto-oncogene is fused to a regulatory locus for immunoglobulins which normally directs high levels of expression. Similarly, human chronic myeloid leukemia (CML) is characterized by a translocation of chromosomes 9-22 with the abl proto-oncogene being activated and mutated to the oncogene. Insertion mutations adjacent to the myc proto- oncogene (in its regulatory region) in chickens result in bursal lymphomas. Mutagenic alterations which form a large number of oncogenes have been captured and propagated in viruses that then become 'infectious* cancer-causing agents in mice, cats, dogs, chickens, monkeys, etc. Many different classes of mutations (deletions, insertions, point mutations) can activate different oncogenes and result in a cancer. In some cases, oncogenes are associated with tissue-specific cancers although others are able to initiate or induce cancers in many tissue types. Thus, the estimated 50-100 proto-oncogenes are activated by a variety of mutations to produce an altered gene (oncogene) and an altered product (a protein) which is clearly on the pathway to produce a cancer. These targets of mutagens are a set of genes which have been conserved throughout substantial period of evolution. The proto- oncogenes of humans are closely related, in their nucleotide or base sequences in the DNA, to the homologous genes of other animals. Yeast, molds, fruit flies, mice, rats, etc. all contain related genes to many (but not all) of the human oncogenes. Bacteria do not appear to have such homologs. Clearly animals that are evolutionarily closer to humans will have a better representation of target proto-oncogenes when testing mutagens for cancer-causing effects. 3. SUMMARY OF THE INVENTION The invention is directed to a non-human transgenic animal testing system for mutagens and carcinogens. Briefly described, recombinant DNA techniques are used to create unique test genes and regulation signals for detecting increased frequencies of mutations produced by mutagens and cancers induced by carcinogens. The test genes are introduced into the germ line of non-human transgenic animals to provide a unique bioassay panel for mutagens and careinogens. The test genes of the invention may encode any of a number of different genes, the expression of which could be detected in a transgenic animal. However, the test gene is engineered so that the expression of the test gene product is either prevented or inhibited unless the gene is activated by a mutagen or carcinogen. For example, the test gene may contain a mutation that prevents or inhibits transcription or translation of the test gene unless activated by a mutagen that reverses the mutation. Alternatively, the test gene may be engineered so that it is present in a low concentration or so that it is heavily methylated, in which case expression would be inhibited unless the test gene is activated by a carcinogen that amplifies or demethylates, respectively, the test gene. A test gene so engineered is introduced into the germ line of a non-human transgenic animal. Subsequent exposure of the transgenic animals to a mutagen or carcinogen which activates the the test gene will result in the expression of the test gene product and its detection in the transgenic animal. For example, exposure of the transgenic animal to a mutagen that reverses a mutation engineered into the test gene would permit expression of the test gene product. Similarly, a carcinogen which amplifies test genes present in low concentrations or which demethylates heavily methylated test genes will similarly induce expression of the test gene product. The activated test gene may express a transforming and tumorigenic product (e.g. , an oncogene or proto-oncogene product) which produces a tumor in the transgenic animal as an indication of the mutagenicity or carcinogenicity of the substance being tested. Alternatively, the activated test gene may express a nontumorigenic assayable product, for example one which is capable of being detected based upon its biological activity (e.g. , a toxin, hormone, enzyme, foreign antigen, etc.) as an indication of the mutagenicity or carcinogenicity of the substance tested. In one embodiment of the invention, the test gene may be placed under the control of a tissue-specific regulatory element so that activation by appropriate mutagens or carcinogens will result in the expression of the test gene product in a tissue specific fashion within the transgenic animal. Thus only the specific tissue need be examined for the presence of the activated test gene product. The assay system of the present invention will: (a) increase the sensitivity to present-day mutagen testing by increasing the number of target genes per animal in a test; (b) test for tissue-specific mutations or multi-tissue mutations by employing tissue-specific regulatory control regions of genes; (c) test for differences between fetal life activation of mutagens or adult activation by utilizing regulatory control regions specific for *time of expression" in animals; (d) detect point mutations (transitions, transversions) , deletions, insertions, amplifications, methylation, etc., allowing classification and detection of all classes of mutagens and carcinogens; (e) take into account, and impose, different genetic backgrounds of the test animals; (f) address the two species requirement for bioassays through use of both mice and rats; (g) employ both male and female mice and rats that carry identical test systems in their chromosomes; (h) utilize a test animal that will respond to a mutagen in a defined time frame, providing a time-dependent endpoint at which a negative result will mean something, i.e. , less than a certain number of mutations detected/genome/generation; (i) eliminate present ambiguities that result from an interpretation (sometimes conflicting) of a pathologist or toxicologist; (j) reduce the dependence upon statistical methods which are not uniformly accepted; and (k) use, in certain embodiments, an amplification system - the production of a tumor or replication of a virus or cell - that provides a maximal sensitivity in the bioassay. 3.1. DEFINITIONS The following terms as used herein, whether in the singular or plural, shall have the meanings indicated: Test Gene: a gene that encodes a detectable product engineered so that the expression of the test gene product is prevented or inhibited unless the gene is activated by a particular mutagen or carcinogen. In general, the test gene may be engineered to contain a mutation (e.g. , a point mutation, deletion, insertion, etc.) that prevents or inhibits transcription or translation of the test gene product unless activated by a mutagen that reverses the mutation. Alternatively, the test gene may be engineered so that it is present at a low concentration or is heavily methylated so that expression is inhibited unless activated by a carcinogen that amplifies or demethylates, respectively, the test gene. Test genes of the invention are introduced into the germline of non-human transgenic animals to provide a bioassay panel for mutagens and carcinogens. Activated 5 Test Gene: an activated test gene encodes and expresses a product that can be detected in a non-human transgenic animal. Test genes may be activated by exposure of the transgenic animal containing the test gene to substance that derepresses or • J O induces expression of the test gene, e.g. , by reversing the engineered mutation that inhibits expression, by amplifying the test gene, or by demethylation. Activated test genes may express transforming gene products which result in 15 tumor-formation in the transgenic animal (e.g. , an oncogene or proto-oncogene) . Alternatively, activated test genes may express detectable gene products that have a particular function and/or biological effect on the transgenic animal. For 2Q example, the activation of lethal test gene products may result in death of the transgenic animal; the activation of hormone test gene products may affect the metabolism and/or homeostasis of the trangenic animal, (e.g. , „ activation of growth hormone may increase growth of the animal) ; the activation of enzymatic test gene products can be detected by assaying appropriate organs, tissues or cells of the transgenic animals for the enzymatic activity; „ the activation of antigenic test gene products may be detected by assaying appropriate organs, tissues or cells of the transgenic animals immunologically, etc. 35 Transgenic Animal: a non-human animal containing a foreign gene, called a transgene, which was introduced into its germ line, and therefore, is an inheritable trait of the transgenic animal. As used herein, the term transgenic animal refers only to non-human animals so engineered, which may include but are not limited to transgenic mice, rats, rabbits, guinea pigs, non-human primates, etc. 4. DESCRIPTION OF THE FIGURES FIG. 1 diagrammatically represents two nonsense (stop translation) mutations that can be generated in the v-ras coding region and the mutational events which can then restore synthesis of the v-ras protein. 5. DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to a non-human transgenic animal testing system for mutagens and carcinogens. A transgenic animal contains a foreign gene called a "transgene* which was introduced into its germ line; therefore, the transgene is an inheritable trait of the transgenic animal. More specifically, a transgenic animal of the present invention is a non-human animal that contains a transgene comprising a test gene that expresses its gene product only in response to activation by exposure of the transgenic animal to an appropriate mutagen or carcinogen. Thus, the mutagenicity or carcinogenicity of a substance can be assessed based upon its ability to activate the expression of the test gene product in the transgenic animal. Any appropriate non-human animal may be used in accordance with the invention, including but not limited to mice, rats, rabbits, guinea pigs, non-human primates, etc. The test genes of the invention may be engineered to detect a particular type or class of mutation or carcinogenic activation. For example, in order to test for mutagenic activity, a test gene could be engineered to encode a structural gene containing a mutation (e.g. , a point mutation such as a transition or transversion; an insertion, deletion, or inversion, etc.) that prevents or inhibits gene expression. For example, transcription may be inhibited or prevented by a mutation located within the transcription regulatory elements of the test gene such as the promoter, enhancer, etc. Translation may be inhibited or prevented by a mutation located within the coding region of the structural gene which results in early chain termination or a frameshift that converts the gene coding region to a nonsense sequence; alternatively the mutation can be located within translational control elements of the test gene, such as the ribosome binding site, or elements which affect stability of the transcribed message. Exposure of a transgenic animal containing such a test gene to a mutagen which reverses the particular type of mutation engineered into the test gene will result in "activation" of the test gene, i.e. , expression or increased expression of the test gene product in the transgenic animal. Alternatively, the detection of carcinogens which do not produce significant mutations but which alter chromosome ratios can be accomplished using a test gene engineered to be present at a low concentration so that expression of the gene product either escapes detection or is detected at low levels in the transgenic animal. Exposure of a transgenic animal containing such a test gene to a carcinogen that amplifies the test gene to high copy numbers will result in an increase in the expression of the test gene product in the transgenic animal. In yet another alternative, the detection of carcinogens that interfere with methylation of DNA can be accomplished using a test gene engineered to encode a heavily methylated structural gene, the expression of which is inhibited or repressed by the methylation. Exposure of transgenic animals containing such a test gene to a carcinogen which inhibits DNA methylation will result in "activation" of the test gene, i.e. , expression or increased expression of the test gene product in the transgenic animal. The test genes of the invention may be engineered to encode any of a number of different genes, the activation and expression of which can be assayed or detected, for example, based upon the biological activity of the gene products. In one embodiment of the invention, the test gene may comprise a transforming gene engineered so that exposure of the non-human transgenic animal to a substance that activates the test gene will result in the expression of the transforming gene product and, therefore, tumor formation in the transgenic animal. The resulting tumor is composed of millions of cells derived from the original cell (it is a clone of cells) that carry the activated test gene. Using a tumor as an endpoint to detect a specific type of mutation has several advantages. First, it has great sensitivity due to the amplification (via cell division) of a single cell event. Second, the tumor tissue provides a source of the DNA, i.e. , the transgene, which could be cloned from the genome and analyzed to prove (e.g. , by DNA sequencing techniques) that the mechanism for the mutation was as predicted (transition, transversion, deletion, addition, overexpression, interference with methylation, etc.). This feature, while not a necessary part of the invention, provides a check on the result to any test being carried out. In a preferred embodiment, the transforming test gene can be engineered so that it is activated and expressed in a tissue-specific fashion. This provides a distinct advantage for-"reading" the results of the test because only the specific tissue need be examined for tumor formation. Second, it provides tissue-specific information where the endpoint is a frank tumor with little need for interpretation by a pathologist. In another embodiment of the invention, the test gene may be engineered to encode a gene whose expression results in a biological activity or assayable function that can be detected in the non-human transgenic animal. For example, exposure of a transgenic animal to a substance that activates test genes encoding toxins or lethal products will result in the death of the transgenic animal; the activation of a test gene encoding a hormone will affect the metabolism and/or homeostasis of the transgenic animal (e.g. , the activation of a test gene encoding growth hormone will result in the increased growth of the transgenic animal) . Alternatively, the test gene may be engineered to encode a structural gene product that is foreign to the transgenic animal (e.g. , a procaryotic gene product, or a gene product of another species either animal or plant) so that the test gene product can be detected by assaying the transgenic animal organs, tissues, or cells immunologically. In another alternative, the test gene may be engineered to encode an enzyme (preferably one which is foreign to the transgenic animal) that can be detected by assaying the animal organs, tissues, or cells for the appropriate enzymatic activity (e.g. , bacterial enzymes, beta- galactosidase, alkaline phosphatase, etc.). Such non- tumorigenic test genes may also be engineered so that they are expressed in a tissue-specific fashion. For example, test genes encoding toxins, hormones, enzymes or foreign antigens could be engineered so that upon activation they are expressed by tissues that have the necessary cellular apparatus for protein secretion, thus ensuring the circulation of the test gene product in order to allow for the detection of its biological effect (e.g. , toxins or hormones) or to assay for the product in certain tissues or organs, such as blood samples (e.g. , enzymes or antigens) , germ cells or body fluids. The test genes of the invention may be constructed using i-acombinant DNA techniques well known to the skilled artisan, including molecular cloning with or without the use of synthetic oligonucleotides (see, for example, the techniques described in Maniatis et al., 1982, "Molecular Cloning A Laboratory Manual" Cold Spring Harbor Laboratory) . Using such molecular cloning techniques, structural genes can be isolated, cloned and engineered or altered so that expression of the test gene is repressed or blocked unless activated by a mutagen or carcinogen. Such alterations include but are not limited to mutagenizing techniques such as site directed mutagenesis, to accomplish deletions, insertions, inversions, etc.; methylation of test genes may be accomplished by engineering nucleotide sequences that provide for methylation in vivo using, for example, bacterial nucleotide sequences that promote methylation and alter gene expression (e.g. , see Tilghman and Levine, 1987, in Gene Transfer, Kucherlaputi, Ed. Plenum Pub., NY, pp. 189-221) . These test genes may then be incorporated into transgenic animals using techniques described infra. In practicing the invention, the transgenic animal testing system for mutagens or carcinogens may be used as follows: a group of non-human transgenic animals containing the test gene is exposed to the suspected mutagen or carcinogen to be tested. The transgenic animals are then examined at a particular end-point for the expression of the test gene product which would indicate the mutagenicity or carcinogenicity of the compound being tested. Where the activated test gene expresses a transforming gene product, the transgenic animals are sacrificed and examined for the presence of tumors. Where the transforming test gene was engineered to be activated and expressed in a tissue- specific fashion, only the particular tissue involved need be examined for tumors. Where the activated test gene expresses a non-transforming product, detection of mutagenicity or carcinogenicity depends upon the nature of the test gene product. For example, activation of test gene products which are lethal to the transgenic animal will result in the death of the transgenic animal; activation of test gene products that have a biological activity or exert . a biological effect, such as hormones, can be detected either by testing the transgenic animal tissues for the presence of the hormone or by detecting the biological effect on the metabolism and/or homeostasis of the transgenic animal. Activation of test gene products that have enzymatic activity can be detected by testing the animal organs, tissues or individual cells for the appropriate enzymatic activity. Antigenic test gene products may be detected immunologically. Where such test genes are engineered to be activated and expressed in a tissue specific fashion, only the particular tissue involved need be examined for the presence of the test gene product. For example, tissue samples obtained from the appropriate organ (e.g. , histological sections, blood samples, fluids, cytological specimens, etc.) either at sacrifice or by biopsy may be assayed for the presence of the test gene product. The transgenic animal test system of the invention will be more sensitive than tests which utilize normal mice because transgenic mice can carry multiple copies of the transgene in their chromosomes. A mouse with two chromosomes, each containing 80 copies of the test gene is very likely to be 80 times more sensitive to a mutation than a normal mouse with two copies (one per chromosome) . Therefore, the target size is greater for mutagens or carcinogens. Sensitivity can be further optimized because it is well-known that the nucleotide sequences on either side of a gene (in a chromosome) can affect the mutation rate or mutagen-mediated mutation frequency, The transgenic mouse system can exploit this variable in two ways: (a) several independently derived transgenic mice will have the test gene integrated at " different sites or chromosomal locations which will permit one to examine the effect of gene location upon the mutation frequency - no other test available can take this variable into consideration; and (b) by using a human test gene with its surrounding human DNA sequences in a transgenic animal, the impact of human DNA sequences upon mutation frequency can be assessed. The different aspects and components of the invention are described. in more detail in the subsections below. For purposes of clarity in description only, the invention is discussed in terms of: (a) the test genes (tumorigenic and nontumorigenic) which are activated in response to exposure to a mutagen or carcinogen; (b) the regulatory elements that provide for tissue specific expression of activated test genes; and (c) the creation of the transgenic animals of the invention containing the test gene. 5.1. TUMORIGENIC TEST GENE SYSTEMS FOR MUTAGENS AND CARCINOGENS IN TRANSGENIC ANIMALS The transforming test genes of the invention which are engineered and placed into the genetic background of a transgenic animal express a transforming gene product, and thus induce tumors, only in response to activation by an appropriate mutagen or carcinogen. Transforming test genes of the invention include, but are not limited to transforming gene products such as: (a) any oncogene of viral or cellular origin containing a mutation which blocks or suppresses the expression of the transforming oncogene product; (b) any proto-oncogene which transforms cells only when the expression of the proto-oncogene product is amplified or the proto-oncogene is mutated to an oncogene; (c) any oncogene or proto-oncogene which is heavily methylated so that the expression of the transforming gene product is blocked or suppressed; and (d) any gene containing a mutation blocking its expression which upon reversion can activate other genes that in turn induce formation of a tumor. More specifically, in one embodiment of the invention, an oncogene containing a mutation could be used as a test gene for the detection of carcinogens which are mutagens. For example, a test gene may comprise an oncogene containing a point mutation, insertion or deletion which blocks the expression of the oncogene product; i.e. , a mutation that results in a frameshift, early chain termination, etc. Such a test gene could be placed into the genetic background of the transgenic animal so that exposure of the transgenic animal to a mutagen which reverses the point mutation, insertion, or deletion, respectively, would result in activation of the oncogene; i.e. , expression of the transforming oncogene product and, therefore, production of a tumor. In another embodiment of the invention, a test gene comprising a proto-oncogene may be inserted into the genetic background of a transgenic animal. Exposure of this transgenic animal to a carcinogen which does not cause mutations but, rather, amplifies the expression of the test gene (herein proto-oncogene) would result in transformation and tumor development. Alternatively, a test gene comprising a highly methylated proto-oncogene or oncogene may be inserted into the genetic background of a transgenic animal where the gene products would be expressed at very low levels if at all. Exposure of this transgenic animal to a carcinogen that inhibits or interferes with methylation will lead to over-expression of the proto-oncogene or oncogene, resulting transformation and tumor formation. For clarity in discussion, the various test gene systems are described in the subsections below with reference to particular oncogenes, however the invention is not meant in any way to be limited to the exemplary oncogenes described. In fact, any oncogene whether previously described or yet to be discovered could potentially be utilized in the assays described in the subsections below. Known oncogenes which could be utilized include but are not limited to those derived from DNA tumor viruses (e.g. , T antigen genes from SV40 or polyoma viruses, E1A and E1B genes from adenoviruses, and other genes known to have tumorigenic potential derived from papillomaviruses and herpes viruses; reviewed in Bishop, 1985, Cell 42:23- 38) ; retroviruses (e.g. , v-abl, v-fes, v-fps, v.-fgr, v-src, v-erbA, v-erbB, v-fms, v-ros, v-yes, v-mos, v-ras, v-fos, v-myb, v-myc, v-ski, v-sis, v-rel, v-kit, v-jun, v-ets; reviewed in Bishop, 1985, Cell 42:23-38) ; cellular proto- oncogenes corresponding to the viral oncogenes, activated cellular oncogenes corresponding to viral oncogenes, or cellular oncogenes for which no viral counterpart has yet been described (e.g. , c-neu. Hung et al., 1986, Proc. Natl. Acad. Sci. USA 83:261-264) . 5.1.1. TEST GENE SYSTEMS FOR MUTAGENS Oncogenes can be constructed which carry chain termination mutations. Reversion of such a mutation would result in a tumor in a transgenic animal carrying the oncogene. Reversion events that can be tested for in this way include: (1) point mutations of both transition or transversion classes; and (2) frameshift mutations which are of both the deletion or insertion type. For example, the normal proto-oncogene, ras, can be found in mice, rats, and humans and functions in most cells to regulate cell division. When it carries the mutation that activates this gene to become an oncogene (a change at amino acid 12 in the protein) it causes a tumor which can occur in many different tissue types. If one were to place the ras oncogene in a transgenic mouse it would produce a tumor in almost any -tissue that expressed the complete abnormal protein (fetal or adult) . It is possible to prevent the production of the ras oncogene protein by creating a second point mutation in the ras oncogene that prematurely terminates the synthesis of the ras protein. This is called a "chain termination" mutation and it results in only a fragment of the protein being produced from the RNA coded by the ras oncogene (see FIG. 1 for the specific alterations of the ras oncogene that result in premature chain termination) . Such chain termination mutations can be accomplished by placing a transition or transversion mutation in selected sites in the ras gene; in each case, only a small fragment of the ras protein is produced and no tumor results. A transgenic mouse carrying the ras oncogene with a chain termination mutation would make its normal ras protein from its own murine proto-oncogene and a fragment of the ras protein from the transgene (the ras oncogene with a chain termination mutation) . Any mutagenic event that reverts the chain termination sequence or signal (called a codon) in the ras gene (see FIG. 1) , would now permit the cell carrying the mutation to produce the ras oncogene product. This product (which is dominant over the normal ras proto-oncogene product) could cause a tumor to form in any tissue where the mutagen acted to revert the transition or transversion mutation. The efficiency of tumor production can be modulated by varying the genetic background of the transgenic animal (e.g. , C3H mice, by contrast to the B6 strain of mice, display an enhanced frequency of liver tumors both arising spontaneously and in response to carcinogens; the B6/C3H systems are reviewed in Reynolds et al., 1987, Science 237:1309-1316) or by introducing a second oncogene which by itself does not induce tumors but can cooperate with other oncogenes to enhance tumor production (e.g. , the adenovirus E1A gene enhances the oncogenicity of the ras gene; Ruley, 1983, Nature 304:602-606). In one embodiment of the invention, a transgenic mouse containing the normal human ras proto-oncogene may be created (or any other oncogene to be tested in this battery of bioassays) . The mouse should be normal unless any one of the approximately 80 copies of this human gene acquires a mutation at the correct sites (amino acid 12 or 61) . A mutation would then result in a tumor. Once again, the high copy number increases target size and sensitivity and the human gene provides a DNA environment more like the human genome to test for a carcinogen. In this format, several of the more common oncogenes from humans would be tested for mutation frequency in transgenic mice. It may well be the case that different oncogenes are preferentially mutated by different carcinogens and this could be monitored in the transgenic mouse system. If this is correct, then mutagen or carcinogen testing would require: (1) the use of human genes (a human DNA context or environment might be important) ; (2) the use of multiple proto-oncogenes; and (3) that testing be performed in vivo because of metabolic activation. Only the transgenic mouse test of the present invention fulfills these criteria and provides a unique test system able to address these questions. 5.1.2. TEST GENE SYSTEMS FOR CARCINOGENS THAT DO NOT PRODUCE SIGNIFICANT MUTATIONS While most carcinogens are also mutagens, there are several clear-cut examples of carcinogens that are, at best, poor mutagens. Diethylstilbestrol (DES) is a synthetic steroid hormone known to be carcinogenic in humans and rodents. DES is not active in the Ames test nor are its metabolic derivatives. DES is also inactive in the cell culture tests for ouabain resistance and HGPRT negative cells. What DES is known to do in cell culture, rodents, and in humans, is alter the chromosome ratios (produce anueploid cells) in dysplastic lesions or cells treated with this hormone. Anueploidy, or abnormal numbers of chromosomes in abnormal ratios, is thought to increase the concentrations of some genes or proto-oncogenes and decrease others. This could result in altered growth control of cells and tumor formation. Mice carrying proto-oncogenes in high copy numbers or on many chromosomes would be expected to be very sensitive to the effects of DES or related agents. Any chemical that acts by altering the ratio of proto-oncogenes, rather than producing a mutation, could be tested for in mice or rats that already have altered ratios because of the extra transgenes. Such tests may detect lower levels of agents like DES and score positive for carcinogenesis, while the Ames test and others would be negative. Another class of carcinogens may promote translocations or amplifications of selected DNA segments. Common to those mechanisms is the fact that proto-oncogenes can produce their gene products in abnormally large amounts. Insertion mutations (from viral agents) and translocations adjacent to an oncogene often result in very high levels of gene expression and an overproduction of a normal product. Amplification of a gene segment has the same result. Over- expression of a proto-oncogene product can result in a tumor. To test for such agents, it should be possible to obtain mice that are carrying various numbers of a proto- oncogene (for example, 3, 10, 15, 20, 30, 50, 80 copies) and determine the levels that produce tumors. By using the mice that have copy numbers just below the threshold for tumorigenesis, any duplication or amplification event will produce a tumor. This test is more sensitive than tests using a normal mouse. In addition, mice carrying proto- oncogenes on multiple chromosomes (introduced by transgenic technology and subsequent matings) increase the target size for possible translocations which activate the genes and result in tumors. This class of tests increases sensitivity, and provides a quantitative base. 5.1.3. TEST GENE SYSTEMS FOR DNA METHYLATION A drug such as 5-azacytidine is poorly mutagenic in mammalian cells in culture (Landolph and Jones, 1982, Cancer Res. 42:817-823) but can transform cells in culture (Benedict et al., 1977, Cancer Res. 37:2202-2208) which is a neoplastic-like event. One of the properties of 5- azacytidine is to inhibit the enzymes that add methyl groups to cytosine in DNA. The result is that DNA is undermethylated in cells treated with this drug. It is thought that methylation of DNA may decrease gene expression and, therefore, undermethylation of DNA may result in the expression of genes that are normally inactive. In this way proto-oncogenes could be overexpressed, producing normal proteins at abnormally high levels and causing a cancer. Thus, tests for chemicals that affect methylation patterns may be useful. Transgenic mice carrying viral oncogenes capable of producing tumors in these animals have been created (van Dyke et al., 1985, CSHSQB 50:671-678). Some of these mice express the viral oncogene and produce tumors on a regular schedule. Other strains of mice carry the same viral oncogene, which is heavily methylated at its cytosine residues, and these strains fail to express the viral oncogene product and fail to get tumors. In one rare case, a tumor was detected in this strain of mice and DNA from the tumor was undermethylated when compared to other tissues not expressing the viral oncogene protein. This strain of mice may be useful in screening chemicals and alter methylation patterns. Similar strains of mice carrying the Moloney retrovirus in a chromosome, heavily methylated at its cytosine residues and not expressing virus, are known to exist. Any agent that reverses this methylation pattern will activate the virus production. Virus replication in a susceptible host is similar to the clonal tumor cell amplification system described previously. A tremendous amplification is achieved, and it is easily assayed. 5.2. NON TUMORIGENIC TEST GENE SYSTEMS FOR MUTAGENS AND CARCINOGENS IN TRANSGENIC ANIMALS A number of non-transforming test genes can be engineered in the transgenic animal testing system of the invention for the detection of mutagens and carcinogens. Accordingly, any structural gene that encodes an assayable product can be engineered so that it is expressed only in response to activation by an appropriate mutagen or carcinogen much in the same manner as described supra for transforming test genes. For example, such test genes can be engineered to contain a mutation that inhibits or blocks expression unless activated by a mutagen that reverses the mutation. Alternatively, such test genes can be engineered so that they are present in low concentations or heavily methylated so that expression is inhibited or prevented unless activated by a carcinogen that amplifies or demethylates, respectively, the test gene. Nontumorigenic test genes may encode any of a number of products, described in more detail below, including but not limited to toxins, hormones, enzymes, antigens, etc. The activation and expression of such gene products in the transgenic animal could be detected based upon the biological effect the gene product has on the animal or by assaying appropriate animal tissues for the test gene product. One limitation that skilled artisan may encounter when using non-transforming test genes in the transgenic testing system of the present invention is that a single cell expressing a secreted product may not make enough product for detection in the transgenic animal. This limitation may be overcome by the following assay system: the bacteriophage T7 DNA-dependent RNA polymerase gene may be inserted behind a tissue-specific (e.g. , albumin control region for liver) transcriptional control region. This gene will carry a nonsense mutation. An oncogene (e.g. , activated ras) plus a viral gene product known to reside on the cell surface and to be immunogenic (e.g. , the influenza M2 protein or VSV G protein) will each be inserted behind a bacteriophage T7-specific control region. All three genes would be introduced into a transgenic mouse or rat. Reversion of the nonsense mutation in the T7 polymerase gene by a carcinogen would generate active T7 RNA polymerase, that will, in turn, simultaneously activate expression of both the oncogene and the cell surface protein, provided the messages are capped and stable. The oncogene will cause the cell in which the activation event occurred to begin to multiply. When a sufficient number of cells accumulate with the foreign antigen on their cell surface an antibody response will be induced. A serological test for an antibody response to the test gene antigen will become the assay for carcinogenesis. For example, antibodies should be present and detectable in a blood sample before a palpable tumor is evident, thus accelerating the end point of the transgenic animal testing system. When using this system, however, one should be aware of the following potential problems: (1) The T7 polymerase gene has been expressed in the cytoplasm of mammalian cells on board the vaccinia chromosome but not in the nucleus as yet. To increase the efficiency with which the bacteriophage polymerase reaches the nucleus, a nuclear localization signal may be fused to the polymerase gene (e.g. , Kalderon et al., 1984, Cell 39:499-509; Welsh et al., 1986, Mol. Cell. Biol. 6:2207- 2212) . (2) Messenger RNAs produced in mammalian nuclei by the T7 polymerase may not be capped (modified at their 5' end) . This could lead to unstable messenger RNAs and the need to search for an alternative polymerase system. (3) The foreign antigen will likely enhance rejection of the tumor cells by the host. (4) A natural viral infection might occur, thus it may be necessary to screen for production of a second antibody to a second viral antigen (e.g. , influenza HA protein) . Antibody to a second viral antigen will not be present if the antibody response results from tumor induction but it will be detected if an infection has occurred. Alternatively, a non-viral antigen could be employed as the test gene (e.g. , human cell surface antigen) . The nontumorigenic test genes which may be used in accordance with this embodiment of the invention are discussed in more detail in the subsections below. 5.2.1. LETHAL TEST GENE SYSTEMS Genes which encode toxic proteins such as those produced by snakes and invertebrate animals as well as some plants (phytotoxins) and virulent strains of bacteria (e.g. , botulinus toxins) may be engineered as test genes which are inserted into the non-human transgenic animals in accordance with the present invention. Exposure of such transgenic animals to a substance that activates the test gene leading to expression and secretion of the test gene product into the circulatory system of the transgenic animal will result in the death of the transgenic animal. Such toxic proteins which may be used in accordance with the invention include but are not limited to snake venom toxins, phytotoxins, bacterial toxins, etc. For example, known snake venom toxins are highly toxic, antigenic polypeptides and proteins that cause paralysis and death of the prey and are classified according to their mode of action: cardiotoxins which cause irreversible depolarization of the cell membranes of heart muscle and nerve cells; neurotoxins which prevent neuromuscular transmission by blocking the receptors for the transmitters at the synapses of autonomic nerve endings and at motor end plate of skeletal muscle; and protease inhibitors which inhibit acetylcholine esterase and similar enzymes involved in nerve transmission. The cardiotoxins and protease inhibitors consist of 60 amino acid residues (molecular weight (MW) 6900 daltons) whereas the best studied neurotoxins (cobra) can be subdivided into long chain (71-74 amino acids, MW 8000 daltons) and short chain (60-62 amino acids, MW 7000 daltons) molecules. Neurotoxins isolated from vipers have high molecular weights (e.g. , rattlesnake crotoxin, MW 30,000 daltons). The most potent snake venom, taipoxin, consists of two nonidentical polypeptide chains. Neurotoxins are extremely stable due to the presence of disulfide bridges (4 at MW 7000, 5 at MW 8000 and 7 at MW 13,500 daltons). Scorpion venoms are similar to cobra toxins having an MW 6800-7200 daltons, four disulfide bridges and 63-64 amino acids of known sequence. Known phytotoxins which could be used in accordance with the invention include but are not limited to: (a) the homologous viscotoxins (MW 4840, 46 amino acids of known sequence, 3 disulfide bridges) from leaves and branches of the European mistletoe which have a hypotensive activity and cause a slowing of the heartbeat; and (b) the toxalbumins such as Ricin and abrin which inhibit protein biosynthesis. Ricin, derived from Ricinus seeds (MW 66,000 daltons and 493 amino acid residues) consists of an A-chain (MW 32,000 daltons) and a B-chain (MW 34,000 daltons) joined by disulfide bridges. Toxic activity is carried by the A-chain whereas the B-chain is responsible for binding the toxin to the cell surface. Abrin, of similar action and structure is derived from the red seeds of Abrus precatorius. The best studied of the bacterial toxins are the exotoxins secreted by gram positive bacteria and include but are not limited to: (a) Diphtheria toxin from Corynebacteriui,. diphtheriae, an acidic single chain protein (MW 62,000 daltons) of high toxicity (1 ug/kg body weight is fatal) , which inactivates peptidyl transferase II in eucaryotic cells by promoting the attachment of ADP-ribose to the enzyme; (b) Tetanus toxin (MW 150,000 daltons) from Clostridium tetani, a potent neurotoxin which occurs in two forms: filtrate toxin, consisting of two subunits (MW 95,000 and 55,000 daltons), and cell toxin consisting of one chain (0.01 ng/kg in the mouse is fatal); and (c) the free toxic Botulinus toxins (Type A MW 150,000 daltons, Type B MW 167,000 daltons) from Clostridium botulinum, which require the presence of one free SH-group for neurotoxic activity (0.03 ng/kg in the mouse is fatal). Table I below lists and characterizes a number of toxic proteins which may be used in accordance with the invention, but is in no way intended as a limitation. As shown in Table I, the toxicity of many of these proteins is very great, thus the activated test gene need not be expressed at high levels in order to produce lethal doses. In this instance, the T7 amplification system may not be required in order to practice the invention. However, such test genes would be advantageously engineered so that upon activation the gene is express.ed in tissues capable of secreting the toxic product into the bloodstream of the transgenic animal (e.g. , the liver, pancreas, etc.) so that the toxin may exert its biological effect on the animal. The animals may be autopsied to verify the cause of death, though this is not a necessary step in the method of the invention. 5.2.2. HORMONAL TEST GENE SYSTEMS Genes which encode protein or peptide hormones can be engineereed as test genes inserted into the non-human transgenic animals in accordant; with the present invention. Exposure of such transgenic animals to a substance that activates the test gene leading to expression and secretion of the test gene product into the circulatory system of the transgenic animal can be detected based upon either the detection of a hormonally-induced biological effect on the transgenic animal (e.g. , an appropriate metabolic response) or by an immunoassay for the hormone. Because the activity of hormones is not, as a rule, species specific, and since very low concentrations of hormones usually produce a large metabolic response, amplification of the test gene, e.g. , using the T7 amplification system previously described, may not be required to practice this mode of the invention. However, amplification may be required if detection of the hormone gene product is to be accomplished using an immunoassay. In any case, test genes encoding hormones are preferably engineered so that the gene product is expressed by tissues that have the cellular machinery capable of secreting the gene products into the circulatory system, e.g. , liver, pancreas, etc. Any hormone which induces a measurable metabolic effect may be used in accordance with the invention. Such hormones include, but are not limited to, growth hormone, releasing hormones, insulin, etc. Test genes may encompass the "pro* and "pre-pro" forms of these hormones in order to allow for proper processing of the gene product to the mature active form of the hormone molecule. 5.2.3. ENZYMATIC OR ANTIGENIC TEST GENE SYSTEMS Genes which encode enzymes can be engineered as test genes which are inserted into the non-human transgenic animals in accordance with the present invention. Exposure of such transgenic animals to a substance that activates the test gene leading to expression of the test gene product can be detected by assaying the animal for the presence of the enzyme. If the test gene is engineered so that the gene product is expressed in a tissue-specific manner, then only the particular tissue involved need be examined for enzymatic activity. This may be accomplished at autopsy or by biopsy of the tissue involved, and examination of the tissue for enzymatic activity, e.g., histological sections may be treated with an appropriate substrate in order to detect the presence of the enzyme. To enable detection of the enzyme in blood, the test gene should be engineered so that it is expressed in cells capable of secreting the gene product into the circulatory system. According to this embodiment of the invention, it may be necessary to amplify the test gene, for example, using the T7 amplification system previously described in order to increase the concentration of enzyme expressed and secreted into the blood. Any enzyme that catalyzes the production of a detectable product can be used as a test gene in accordance with the invention. Enzymes which catalyze the formation of colorimetric products as well as enzymes derived from other species may be particularly useful for ease in detecting activation and expression of the test gene in response to exposure of the transgenic animal to the suspected mutagen or carcinogen. For example, enzymes including but not limited to beta-galactosidase, alkaline phosphatase, etc. may be used. 5.2.4. P450 TEST SYSTEM FOR NON-GENOTOXIC CARCINOGENS Certain compounds, such as 2,3,7,8- tetrachlorodibenzo-p-dioxin (dioxin) and likely other halogenated aromatic hydrocarbons are non-genotoxic carcinogens that are able to induce transcription rate of the cytoσhrome P-450 gene. Given the assumption that chemical compounds able to activate the P-450 gene could be carcinogens, a assay could be devised in transcrenic mice to screen for activation. The sequence elements in the P-450 gene required for transcriptional activation have been identified (Jones et al., 1986, Proc. Natl. Acad. Sci. USA 83:2802-2806; Jones et al., 1986, J. Bicl. Chem. 261:6647- 6650) . The control of P-450 by dioxin is not all or nothing, but rather a odulatory effect. Thus, the product produced must be quantitated. An enzyme such as beta- galactosidase could be a useful test gene to be placed under the control of the P450 regulatory signals and which, when activated, expresses an assayable product whose activity on a per-liver-cell-basis could be readily quantitated. 5.3. TISSUE SPECIFIC REGULATION OF EXPRESSION OF THE ACTIVATED TEST GENE IN THE TRANSGENIC ANIMAL Transgenic animals, carrying a foreign gene, may express the foreign genetic information in a tissue-specific or time-dependent fashion over the lifetime of the animals. A variety of experiments (reviewed by Tilghman and Levine, 1987, in Gene Transfer, Kucherlapati, Ed., Plenum Pub., NY, pp. 189-221) have shown that DNA sequences localized around or near a gene are responsible for both the time of expression (fetal or adult life) and the tissue pattern of expression of a gene. Regulatory signals have been mapped or isolated which permit genes to be expressed in fetal or adult liver, breast tissue, brain tissue, immune system cells, red blood cells, pancreatic cells (both beta or acinar cell types) , eye lens, muscle cells, and connective tissue. Other gene regulator signals have been identified that give a broad spectrum of tissue-related expression. This has been employed to produce tumors in a specific tissue using oncogenes, to correct genetic defects in a mouse by supplying the appropriate hormones, to alter the immune system, and to follow regulatory circuits that control immunity. Thus, it is now clear that one can place a tissue and time-specific regulatory signal in front of a foreign gene (not normally expressed in that tissue) and create a transgenic mouse that expresses that foreign gene (makes a protein) in the tissue at a specified time. The expression of activated test genes in the transgenic animal system of the invention can be made tissue specific by the placement of a particular regulatory signal or signals adjacent to the test gene. For example, the liver is especially appropriate as a site in which to monitor mutations since its cells contain enzymes known to metabolically activate a variety of carcinogens. By engineering the regulatory signals for fetal liver (alpha- fetoprotein gene) or adult liver (albumin gene) in front of the test gene inserted into the transgenic animal, carcinogens can be tested for mutations in fetal or adult liver tissue. The precise anatomical site to be monitored in the transgenic animals will be known, simplifying "reading" many thousands of test animals. As appropriate, transgenic animals can be generated whose inserted test gene will carry control sequences that mediate expression in a variety of different tissue types. Thus, a library of animals will be available to test effects of suspected mutagens on different organs and tissues. The test may be standardized and quantitated: for example, where the activated test gene exrpresses a tumorigenic product known mutagens and carcinogens can be employed with a standard transgenic mouse strain; the number of independent tumors (in a liver) as a function of dose and the time for tumor detection can be quantitated; a mutation frequency can be calculated; variables such as age of the animal during exposure, growth of the tissue, and regeneration of the tissue can be quantified; a set of standards may be generated; a negative result in a certain time frame may indicate a level or frequency of mutation, which could have been detected, was not found; a lower level for the mutation frequency can be assigned. 5.3.1. REGULATORY ELEMENTS FOR TISSUE SPECIFIC EXPRESSION OF ACTIVATED TEST GENES Any transcriptional control region whether previously described or yet to be described could be used to regulate transcription of the test gene. Examples of transcriptional control regions that exhibit tissue specificity which have been described and utilized in transgenic animals include: Elastase I gene control region which is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639 ^ -646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:42S-51S); insulin gene control region which is active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-122) ; immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adams et al., 1985, Nature 318:533- 538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444); mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495); albumin gene control region which is active in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276); alpha-fetoprotein gene control region which is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 235:53-58); alpha 1-antitrypsin gene control region which is active in the liver (Kelsey et al., 1987, Genes and Devel. 1:161-171); beta-globin gene control region which is active in myeloid cells (Magram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94); myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-712); myosin light chain-2 gene control region which is active in skeletal muscle (Shani, 1985, Nature 314:283-286); and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378). 5.4. TEST GENE SYSTEMS FOR GERM LINE TARGETS In another embodiment of the invention an assay which specifically detects carcinogens that exert their effect in germline cells focusing on the male germline may be accomplished as follows: a test gene carrying a nonsense mutation, is introduced under regulation of a transcriptional control region that functions in all tissues (e.g., ribosomal L32 protein control region) or specifically during spermatogenesis. Sperm will be dissected from mature males (approximately 5 x 10 /animal) , and reversion of the nonsense mutation scored by sorting sperm for activation and expression of the test gene product using a fluorescent labeled antibody directed against the product and a fluorescence-activated cell sorter. The test gene may encode a membrane protein that is not normally expressed on sperm cells which could be detected by an antibody tagged with a fluorescent reporter group. Alternatively, the activated test gene product could be beta-galactosidase which could be scored by cleavage of its substrate X-gal. One male mouse subjected to a mutagenic carcinogen could, in theory, yield a statistically significant mutation frequency. Non-genotoxic (i.e. , non-mutagenic) carcinogens could also be screened for effects on germline cells in this general type of assay if gene expression is altered (e.g. , dioxin increases p-450 transcription) . This test could be extended to female germline cells, although the number of cells obtained from each animal would be less. 5.5. CREATION OF THE TRANSGENIC ANIMALS The preparation of animals used in the bioassay of the invention will utilize the recently developed technology of introducing foreign genes into the germ line (inheritable trait) of an animal. The animal carrying in its germ line a foreign piece of DNA, termed a transgene, is called a transgenic animal. One method for the creation of transgenic mice for producing test animals is described below. In general, the scheme presently employed to produce transgenic mice involves the following: male and female mice, from a defined inbred genetic background, are mated at midnight. Twelve hours later, the female is sacrificed and the fertilized eggs are removed from the uterine tubes. At this time, the pronuclei have not yet fused and it is possible to visualize them in the light microscope. Foreign DNA is then microinjected (100-1000 molecules per egg) into the pronucleus. Shortly thereafter fusion of the pronuclei occurs and in some cases and foreign DNA inserts into (usually) one chromosome of the fertilized egg or zygote. The zygote is then implanted into a pseudo-pregnant female mouse (previously mated with a vasecto ized male) when the embryo develops for the full gestation period of 20-21 days. The surrogate mother delivers these mice and by four weeks the pups are weaned from the mother. To test these mice for the presence of foreign DNA, a portion of the tail (a dispensible organ) is removed and the DNA extracted. DNA- DNA hybridization (in a dot blot, slot blot or Southern blot test) is employed to determine whether the mice carry the foreign DNA. Of the eggs injected, 10% develop properly and produce mice. Of the mice born, one in four (25%) are transgenic for an overall efficiency of 2.5%. Once these mice are bred they pass along the foreign gene in a normal (Mendelian) fashion linked to a mouse chromosome. Mating two homozygous mice with the transgenic DNA means 100% of the offspring carry two copies of the transgene. When this is done it is common that the mice carry tandemly repeated copies of the foreign gene (from 3-80 copies) at one chromosomal location or site. The present invention is not to be limited in scope by the embodiments disclosed herein which are intended as single illustrations of the different aspects of the invention. Any embodiment which is functionally equivalent is within the scope of this invention. Indeed, various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

Claims: WHAT IS CLAIMED IS: 1. A transgenic animal for testing mutagens and carcinogens, comprising a non-human animal containing in its germ line a transgene comprising a test gene which encodes a detectable product engineered so that the expression of the test gene product in the transgenic animal is prevented or inhibited unless the transgenic animal is exposed to a mutagen or carcinogen that activates the expression of the test gene product. 2. The transgenic animal of claim 1 in which the test gene comprises a transforming gene encoding a tumorigenic product. 3. The transgenic animal of claim 2 in which the transforming gene comprises an oncogene. 4. The transgenic animal of claim 2 in which the transforming gene comprises a proto-oncogene. 5. The transgenic animal of claim 3 in which the oncogene comprises a DNA tumor virus oncogene. 6. The transgenic animal of claim 3 in which the oncogene comprises a retrovirus oncogene. 7. The transgenic animal of claim 3 in which the oncogene comprises a cellular oncogene. 8. The transgenic animal of claim 1 in which the test gene comprises a non-transforming gene encoding a detectable gene product. 9. The transgenic animal of claim 8 in which the detectable gene product comprises a toxin. O. The transgenic animal of claim 8 in which the 5 detectable gene product comprises a hormone. 11. The transgenic animal of claim 8 in which the detectable gene product comprises an enzyme. 0 12. The transgenic animal of claim 8 in which the detectable gene product comprises an antigen. 13. The transgenic animal of claim 1, 2 or 3 in which the test gene encodes a structural gene with a mutation that 5 prevents or inhibits expression of the test gene product in the transgenic animal unless the transgenic animal is exposed to a mutagen that reverses the mutation. 14. The transgenic animal of claim 13 in which the 0 mutation inhibits transcription. 15. The transgenic animal of claim 13 in which the mutation inhibits translation. __ 16. The transgenic animal of claim 13 in which the 25 mutation comprises a point mutation. 17. The transgenic animal of claim 13 in which the mutation comprises an insertion. 30 18. The transgenic animal of claim 13 in which the mutation comprises a deletion. 19. The transgenic animal of claim 13 in which the mutation comprises an inversion. 35 20. The transgenic animal of claim 1, 2 or 3 in which the test gene is methylated so that expression is inhibited unless the transgenic animal is exposed to a carcinogen that demethylates E A. 21. The transgenic animal of claim 1, 2 or 3 in which the test gene is present at a low concentration, so that expression of the test gene is inhibited unless the transgenic animal is exposed to a carcinogen that amplifies the test gene. 22. A method for testing mutagens and carcinogens, comprising: (a) exposing a non-human transgenic animal to a substance suspected of being a mutagen or carcinogen in which the transgenic animal contains in its germ line a transgene comprising a test gene which encodes a detectable product engineered so that the expression of the test gene product in the transgenic animal is prevented or inhibited unless the transgenic animal is exposed to a mutagen or carcinogen that activates the expression of the test gene product; and (b) inspecting the transgenic animal or an appropriate organ or tissue of the transgenic animal for the expression of the test gene product, in which the expression of the test gene product in the transgenic animal is an indication of the mutagenicity or carcinogenicity of the substance being tested. 23. The method of claim 22 in which the test gene comprises a transforming gene encoding a tumorigenic product the expression of which is indicated by the formation of a tumor in the transgenic animal. 24. The method of claim 23 in which the transforming gene comprises an oncogene. 25. The method of claim 23 in which the transforming gene comprises a proto-oncogene. 26. The method of claim 24 in which the oncogene comprises a DNA tumor virus oncogene. 27. The method of claim 24 in which the oncogene comprises a retrovirus oncogene. 28. The method of claim 24 in which the oncogene comprises a cellular oncogene. 29. The method of claim 22 in which the test gene comprises a non-transforming gene encoding a detectable gene product. 30. The method of claim 29 in which the detectable gene product comprises a toxin. 31. The method of claim 29 in which the detectable gene product comprises a hormone. 32. The method of claim 29 in which the detectable gene product comprises an enzyme. 33. The method of claim 29 in which the detectable gene product comprises an antigen. 34. The method of claim 22, 23 or 24 in which the test gene encodes a structural gene with a mutation that prevents or inhibits expression of the test gene product in the transgenic animal unless the transgenic animal is exposed to a mutagen that reverses the mutation. 35. The method of claim 34 in which the mutation inhibits transcription. 36. The method of claim 34 in which the mutation inhibits translation. 37. The method of claim 34 in which the mutation comprises a point mutation. 38. The method of claim 34 in which the mutation comprises an insertion. 39. The method of claim 34 in which the mutation comprises a deletion. 40. The method of claim 34 in which the mutation comprises an inversion. 41. The method of claim 22, 23 or 24 in which the test gene is methylated so that expression is inhibited unless the transgenic animal is exposed to a carcinogen that demethylates DNA. 42. The method of claim 22, 23 or 24 in which the test gene is present at a low concentration, so that expression of the test gene is inhibited unless the transgenic animal is exposed to a carcinogen that amplifies the test gene.

Inventors: LEVINE ARNOLD J, SHENK THOMAS E, LEVINE, ARNOLD, J., SHENK, THOMAS, E.
Applicants: UNIV PRINCETON, THE TRUSTEES OF PRINCETON UNIVERSITY
Application year: 1988
Main classification: C12Q1
IPC codes: A01K67, C12N15, C12Q1, G01N33