Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
1999-07-29
2001-05-15
Crouch, Deborah (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S003000
Reexamination Certificate
active
06232524
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to novel transgenic animals for detecting mutagenic agents and characterizing the nature of the mutations thereby induced in vivo. The present invention further relates to novel constructs, cell lines and chimeric animals for producing the transgenic animals. The present invention further relates to novel methods for detecting and characterizing forward and reverse mutations in vivo.
BACKGROUND
The impact of environmental chemicals on human health has been clearly recognized and extensively reviewed. See, for example, Fishbein, L. pp. 329-363. In D. B. Walters, Ed.
Safe Handling of Chemical Carcinogens, Mutagens, Teratogens and Highly Toxic Substances.
Vol. I, Ann Arbor, Mich.: Ann Arbor Science (1980); and
Identifying and Estimating the Genetic Impact of Chemical Mutagens,
National Academy Press, Washington, D.C. (1983). There are more than 70,000 synthetic chemicals in current commercial use, including pharmaceuticals, food additives, industrial chemicals, and pesticides.
Identifying and Estimating the Genetic Impact of Chemical Mutagens,
National Academy Press, Washington, D.C. (1983) and Hollstein, M. et al.:
Mutat. Res.,
65:133-226 (1979). About a quarter of these are believed to be produced in abundance, with additional new chemicals introduced at a rate of about 1,000 per year. These numbers represent an alarming statistic when one considers the strong correlation between somatic cell mutagenesis and carcinogenesis, and between germ cell mutagenesis and heritable disease. McCann, J. et al.:
Proc. Nat. Acad. Sci. USA,
72:5135-5139 (1975). Exposure to many of these compounds is believed to pose a significant environmental health risk. In particular, somatic mutation, incurred as a consequence of exposure to environmental mutagens, is currently thought to produce an increased risk for the development of cancer.
Assessment of the mutagenicity of compounds or environments is extremely important for establishing a rational basis for reducing human exposure to those compounds that prove mutagenic. To this end, numerous short-term mutagenicity assays have been devised. See, for example, Waters, M. D. pp. 449-467. In A. W. Hsie, P. J. O'Neil and U. K. McElheny, Eds.
Mammalian Cell Mutagenesis: The Maturation of Test Systems.
Banbury Report 2. New York: Cold Spring Harbor Laboratory (1979). For example, the Salmonella/liver microsome test which was pioneered by Ames and his colleagues, has the ability to detect some mutagens. See, for example, McCann, J. et al.:
Proc. Nat. Acad. Sci. USA,
72:5135-5139 (1975), Waters, M. D. pp. 449-467. In A. W. Hsie, P. J. O'Neil and U. K. McElheny, Eds.
Mammalian Cell Mutagenesis: The Maturation of Test Systems.
Banbury Report 2. New York: Cold Spring Harbor Laboratory (1979), and Ames, B. N. et al.:
Science,
176:47-48 (1972); Maron, D. M. and Ames, B. N.:
Mut. Res.,
113:173-215 (1983); Ashby, J. pp. 1-33. Mutagenicity: New Horizons in Toxicology. Ed. J. A. Heddle, N.Y., Academic Press (1982); and McCann, J. and Ames, B. N.:
Proc. Nat. Acad. Sci. USA,
73:950-954 (1976). In addition to the Ames bacterial test, there are short-term tests that utilize fungi, cultured mammalian cells, Drosophila and mice. While many of these short-term tests measure mutation at one or more genetic loci, others exploit end-point criteria such as clastogenesis, aneuploidy, DNA repair, micronucleus production, mitotic recombination, sister chromatid exchange or the formation of DNA adducts.
Unfortunately, the short-term mutagenicity assays are not without certain limitations and drawbacks. One major problem with the Ames bacterial test is believed to be its inability to recognize a significant number of known carcinogens. Another major problem with the existing short-term mutagenicity assays stems from tissue-specific differences in the ability to metabolize various chemicals. See, for example,
Identifying and Estimating the Genetic Impact of Chemical Mutagens,
National Academy Press, Washington, D.C. (1983). For instance, some mutagens are direct-acting and are active in their parental (nonmetabolized) forms; however, most require metabolic conversion by one or more P450 enzymatic activities. There are numerous P450 activities, which constitute a large subset of monooxygenases, and many appear to have overlapping substrate specificities. See, for example,
Identifying and Estimating the Genetic Impact of Chemical Mutagens,
National Academy Press, Washington, D.C. (1983); and Lu, A. Y. H. and Est, S. B.:
Pharmacol. Rev.,
31:277-295. The genes and cDNAs for some have been cloned and characterized. See, for instance, Gonzalez, F. J. et al.:
Mutation Research,
247:113-127 (1991). While subcellular fractions, freshly prepared cells or long-term cell cultures may retain several P450 activities, many are lost. See, for example,
Identifying and Estimating the Genetic Impact of Chemical Mutagens,
National Academy Press, Washington, D.C. (1983). Because of these problems, current in vitro mutagenicity assays are believed to be unable to precisely reproduce the spectrum of complex metabolic activities found in intact animals, tissues or differentiated cells and, as a consequence, rely upon compromises. Also, data from in vitro mutagenicity assays are difficult to correlate with carcinogenic potency in whole animals as measured by the incidence of tumors and the required dose of carcinogen.
In addition to these in vitro short-term mutagenicity assays, there are, for example, two in vivo assays that rely upon transgenic mice as mutagen detectors, which are marketed by Strategene and Hazelton. Both have adopted a similar approach. Their basic strategy has been to incorporate a bacterial reporter gene (lacZ or lacI) into a bacteriophage lambda, and to render mice transgenic for these constructs by pronuclear injection. The recombinant lambda prophage DNA integrates into the host genome as a tandem array, and can be rescued as particles infectious for
E. coli
by incubation with an extract that provides lambda phage capsid and tail proteins. In carrying out these in vivo assays, the mice are exposed to mutagens/carcinogens, and two or three days later (or longer) they are sacrificed. Individual organs (e.g. brain, liver, kidney, etc.) are recovered and DNA is extracted. The purified DNA is incubated with the lambda phage packaging extract, and infectious particles containing the packaged reporter gene are added to
E. coli.
If the lacZ gene is the reporter gene, wild-type lacZ will produce blue colored plaques when stained for beta-galactosidase activity. Conversely, mutant lacZ will produce colorless plagues. When lacI is used as the reporter gene, the color scheme is reversed. Wild-type lacI will produce colorless plagues and mutant lacI will produce blue plaques in the appropriate
E. coli
host. By counting plaques with mutant reporter genes, both groups, Strategene and Hazelton, estimate the relative mutagenicity of each compound for different organs.
Like the in vitro short-term mutagenic assays, these two in vivo assays are not without drawbacks. For example, it is difficult to separate mutation frequency from contributions by mitotic activity. In other words, if a cell with a mutant reporter gene is stimulated to proliferate, one would observe multiple mutant plagues as a consequence of a single mutagenic event. As a further drawback, the animals must be sacrificed and dissected for analysis, and their DNAs must be extracted and packaged before infecting the reporter
E. coli.
This requirement of dissection restricts the inherent power of the system to resolve which cell types or specific tissues are susceptible to mutagenesis. As a further disadvantage, the need to destroy the animals for detection of mutagenesis obviates the ability to follow the fates of mutagenized cells through the life cycle of the animals. Moreover, the possibility of correlating mutagenesis with carcinogenesis in the same animal is obviated.
In yet another drawback, the above in vivo transgenic systems re
Stambrook Peter J.
Tischfield Jay A.
Crouch Deborah
Jenkens & Gilchrist a Professional Corporation
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