Bacteriophage-based transgenic fish for mutation detection

Details Bacteriophage-based transgenic fish for mutation detection Bacteriophage-based transgenic fish for mutation detection

1999-05-28

2001-10-23

Clark, Deborah J. R. (Department: 1633)

Multicellular living organisms and unmodified parts thereof and

Method of using a transgenic nonhuman animal in an in vivo...

C800S020000, C800S021000, C800S025000, C536S023100, C536S023700

Reexamination Certificate

active

06307121

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to a transgenic fish carrying a bacteriophage-derived transgene construct, and in particular relates to a transgenic fish for use in evaluating the effect of a potential mutagenic agent or event. The transgenic fish is exposed to the mutagenic agent or event, and mutagenesis is detected by assaying for a mutation target nucleic acid sequence present as a genomically integrated transgene in the transgenic fish.
BACKGROUND OF THE INVENTION
Thousands of chemicals are currently in commercial use in the USA, some of which pose significant health risk to humans. Among these toxicants are mutagens to which exposure is likely to cause genetic changes that lead to somatic or inherited diseases. In particular, cancer has been shown to result from a series of mutations in specific oncogenes and tumor suppressor genes (B. Vogelstein et al.,
N. Engl. J. Med.
319: 525-532 (1988)). Despite the recognition of the role of chemically-induced mutation as an important event leading to disease, few methods are available for the assessment of genetic hazard, or focus on the study of gene mutations as they occur at the DNA level in vivo. As a result, there is an immediate need to develop sensitive and biologically relevant methods that can be applied to the study of the mechanisms of mutagenesis and hazard assessment.
Until recently, progress in the analysis of gene mutations directly at the DNA level was limited by the standard molecular techniques and the available endogenous genes. During past years, the most relevant assays for induction of transmissible mutations have been based on the appearance of visible or biochemical mutations among the offspring of exposed mice (L. B. Russell et al.,
Mutation Res.
86: 329-354 (1981); L. R. Valcovic et al.,
Environ. Health Perspect.
6:201-205 (1973); S. E. Lewis et al.,
Prog. Clin. Biol. Res.
209B. 359-365 1986)). These tests cannot be practically applied to large numbers of compounds because they require extensive resources and very large numbers of animals. The tests also fail to provide information regarding somatic mutagenesis or clustering of mutations, which may be important in the understanding of the development of various diseases.
In order to circumvent some of the problems inherent in rodent assays, short-term mutagenicity tests were developed, based on the assumption that many of the chemicals toxic to rodents would also be genotoxic to bacteria. However, an analysis by the National Toxicology Program (R. W. Tennant et al.,
Science
236:933-941 1987)) revealed significant differences in results between rodent and bacterial tests. This failure of predictive correlation may be related to: 1) a lack of understanding of the roles mutation plays in cell transformation, and 2) differences between animals and bacterial cells in terms of exposure, biological milieu, metabolism, replication and repair. While comparisons between animals and animal cells in culture provide appropriate genomic similarity, there are few known biological markers for mutation of cells in culture. The biological markers that have been identified are restricted to specific cell types and therefore are of limited use for in vivo comparisons.
There thus remains a need to combine the simplicity of short-term in vitro assays with in vivo studies. Ultimately, reliable and realistic hazard assessment and informative mechanistic studies of mutagenesis require the development of practical methods for evaluating somatic and genetic events in whole animals exposed to environmental agents. New approaches that use recombinant DNA and gene transfer techniques to develop transgenic animal models offer significant promise for in vivo studies of mutagenesis, cancer, birth defects and other diseases (T. L. Goldsworthy et al.,
Fund. Appl. Toxicol.
22:8-19 (1994)). To be effective, the transgenic approach as applied to mutagenesis should include the following components: 1) unique genes with known sequences; 2) a capacity to observe changes at the single copy level; 3) an easily attainable sample population of sufficient size to allow measurement of low frequency events; and 4) the ability to determine the exact nature of the mutation, independent of the host phenotype.
Transgenic animal models have been developed. Typically, transgenic animals are produced by the transfer of novel DNA sequences into the animal's genome followed by transmission of the sequence to subsequent generations. The use of transgenic rodents that carry genes specifically designed for the quantitation of spontaneous and induced mutations is a major advancement in rapidly analyzing tissue-specific mutations in a whole organism following mutagenic agent exposure (J. C. Mirsalis et al.,
Ann. Rev. Pharmacol. Toxicol.
35:145-164 1995)).
Mutagenesis assay systems that utilize transgenic animals typically rely on bacteriophage or plasmid shuttle vectors to carry the mutation target. The basic principle in this approach is that a recombinant gene carrying a mutation target is introduced into the genome of a host animal using the shuttle vector. Following exposure to a mutagen, the target gene is recovered from the transgenic animal and serves as an indicator of mutagenesis (reviewed by R. B. Dubridge et al.,
Mutagenesis
3(1):1-9 (1988)). Two forms of bacteriophage shuttle vectors are most commonly in use. One is known as the &psgr;X174 integrated shuttle vector. This vector is recovered from the transgenic host, transfected into a suitable
E. coli
host, and mutations at specific locations in the phage sequence are identified by suppressor-mediated selection on permissive and non-permissive
E. coli
(H. V. Malling et al.,
Mutation Res.
212:11-21 (1989); R. N. Winn et al.,
Marine Environ. Res.
40(3):247-265 (1995)).
Another useful mutagenesis detection system is based on a lambda (&lgr;) phage-based recombinant vector which combines cos site packaging for recovery of the phage sequence from the host DNA with the use of the lacI or lacZ target gene for mutation detection (J. S. Lebkowski et al.,
Proc. Natl. Acad. Sci.
82:8606-8610 (1985); J. A. Gossen et al.,
Proc. Natl. Acad. Sci.
86:7971-7975 (1989)).
Mutation-induced inactivation of the lac genes is detected after recovery of the shuttle sequence from the transgenic host, typically via complementation assay in
E. coli.
See U.S. Pat. No. 5,589,155 (Sorge et al., Dec. 31, 1996); U.S. Pat. No. 5,347,075 (Sorge, Sep. 13, 1994); and U.S. Pat. No. 5,510,099 (Short et al., Apr. 23, 1996), the texts of which are incorporated by reference, in their entireties, as if fully set forth herein. A mutation detection system based on the lacI gene as the mutation target, known by the tradename BIG BLUE, is commercially available from Stratagene Inc. (La Jolla, Calif.). The vector used in BIG BLUE mutagenesis detection system is known as &lgr;LIZ, and the genetic map of this vector is shown in FIG.
1
.
The &lgr;LIZ vector contains an additional mutagenesis target in the form of the cII region (see FIG.
1
). Mutations in the cII gene and at certain related locations in the cII region can be detected by evaluating whether an
E. coli
host that has been infected with the shuttle sequence recovered from the transgenic host can multiply through the lytic or the lysogenic cycle (J. L. Jakubczak et al.,
Proc. Natl. Acad. Sci. U.S.A.,
93:9073-9078 (1996)). The commitment to either lysis or lysogeny made by lambda phage upon infection of an
E. coli
cell is regulated by a group of proteins, one of which is the product of the cII gene. Mutagenesis detection packaging and selection systems based upon the cII region as the mutation target sequence are commercially available from Stratagene Inc., La Jolla, Calif. (available under tradename &lgr; SELECT-cII) and Epicentre Technologies, Madison, Wis. (available under the tradename MutaPlax cII-Select Packaging and Selection Kit).
To date, the lambda (&lgr;) phage-based recombinant vectors disclosed in U.S. Pat. Nos. 5,589,155, 5,347,075, and 5,510,099; European Patent No. 028912

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