Plasmid-based mutation detection system in transgenic fish

Details Plasmid-based mutation detection system in transgenic fish Plasmid-based mutation detection system in transgenic fish

1999-10-26

2002-10-29

Nguyen, Dave T. (Department: 1632)

Multicellular living organisms and unmodified parts thereof and

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

C800S020000, C800S021000, C800S025000

Reexamination Certificate

active

06472583

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to a transgenic fish carrying a plasmid-based marker, and in particular relates to a transgenic fish for use in evaluating the effect of a potential mutagen. The transgenic fish is exposed to the mutagen, 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
The health risk posed by exposure to mutagenic agents in the environment remains an important concern as it is known that induction of mutations may lead to various somatic or inherited diseases. In particular, cancer has been shown to result from a series of mutations in specific oncogenes and tumor suppressor genes (Vogelstein et al.,
N. Engl. J. Med
. 319: 525-532 (1988)). Despite the recognition of the role of induced mutation as an important event leading to disease, there are few methods 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.
There are two practical requirements common to any study of mutagenesis: 1) the specific loci to be examined should be sensitive to mutation induction, and 2) the mutants must be recovered in sufficient numbers. 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 volts, 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)). Transgenic rodents that carry genes specifically designed for the quantitation of spontaneous and induced mutations are currently available and represent a major advance in the study of mutagenesis by allowing rapid analysis of tissue-specific mutations in a whole organism following mutagenic agent exposure (J. C. Mirsalis et al.,
Ann. Rev. Pharmacol. Toxicol
. 35:145-164 1995)).
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 gene 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 mutagenesis assay systems based on this approach rely on bacteriophage or plasmid shuttle vectors to carry a mutation target. The basic principle in this approach is that a recombinant gene which carries a mutation target (shuttle vector) is introduced into a host genome. Following exposure to a mutagen, the target gene is recovered to serve as an indicator of mutagenesis (reviewed by R. B. Dubridge et al.,
Mutagenesis
3(1):1-9 (1988)).
Shuttle vectors currently in use include both bacteriophage-based and plasmid-based vectors. For example, the lambda (&lgr;) bacteriophage-based recombinant vector combines cos site packaging for recovery of the phage sequence from the host DNA and uses the lacI, lacZ or cII genes as the target gene (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); J. L. Jakubczak et al.,
Proc. Natl. Acad. Sci. U.S.A
., 93:9073-9078 (1996)). Another system is based on the pUR288 plasmid vector which contains the lacZ sequence as the mutation target (M. Boerrigter et al.,
Nature
377:657-659 (1995); M. Dollé et al.,
Mutagenesis
11:111-118 (1996)). In both the &lgr; and plasmid-based assays, mutation-induced inactivation of the lac genes are then detected histologically in
E. coli
. Another system is based on the bacteriophage &phgr;X174 integrated shuttle vector in which the vector is recovered by transfection. 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)).
A fundamental limitation of the bacteriophage-based mutation detection systems is their apparent inability to detect large-scale DNA deletions characteristically induced by clastogenic agents such as ionizing radiation (K. Tao et al.,
Proc. Nat'l. Acad. Sci, U.S.A
., 90:10681-10685 (1993)). Most deletions reported thus far in the abased systems have only been 1-23 base pairs in length. Deletions in the range of hundreds of base pairs are rarely reported using bacteriophage-based mutagenesis detection assays (G. Douglas et al.,
Mutagenesis
9:451-458 (1994)). Current estimates, depending upon the particular test system, are that up to 90% of radiation-induced mutations are thought to be DNA deletions. The bacteriophage shuttle-vector systems seem to have an inherent bias against detecting certain types of deletions primarily due to restrictive packaging and recovery requirements. It is speculated that since two intact cos-sites are required for the packaging of a single &lgr; vector, any deletions that extend into regions adjacent to a transgene concatamer may prevent vector recovery.
A plasmid-based process for detecting mutations in whole animals is described in Gossen et al. (U.S. Pat. No. 5,602,300), but is limited to use in transgenic mammals, namely rodents. The plasmid pUR288, which contains a pBR322 Ori for replication, the ampicillin gene, and the whole lacZ gene including the lacZ operator sequence, was inserted into a bacteriophage lambda vector and transferred to the germ line of a mouse by means of

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