The development of unique animal models offers significant promise to improve methods for evaluation of the genetic risk posed to human health through exposure to chemical contaminants in the environment. The overall goal of the proposed research is to develop a sensitive and environmentally realistic transgenic fish model for in vivo quantification of spontaneous and induced mutations. The applicants propose to use a recoverable mutation target and assay system developed for rodents, based on the lambda LIZ shuttle vector, for use in fish. LIZ, which carries the lacI mutation target gene, is inserted into the genome of a transgenic animal. Following exposure to a mutagen, the target construct is recovered from animal tissues and analyzed for mutations. The transgenic fish model proposed will combine the benefits of an in vivo mutation assay, such as the ability to detect gene mutations directly at the DNA level and in a variety of tissues, with the known advantages offered by fish as animal models.
Specific Aim 1 of this study proposes to transfer LIZ into the genome of the medaka (Oryzias latipes) by microinjection of one-cell embryos. The applicants will test the hypothesis that multiple copies of the bacteriophage sequence can be integrated in the fish genome and subsequently be transmitted through the germ line.
Specific Aim 2 proposes to characterize the recovery of LIZ and assess the spontaneous mutation frequency of the lacI mutation target in transgenic fish. The applicants will evaluate the utility of lacI as a viable mutation target gene by testing the hypotheses that intact LIZ can be efficiently recovered from selected fish tissue, that there is a low background system to recover the shuttle vector by in vitro packaging, and that the system can detect mutations in lacI after infecting Escherichia coli with the phage.
Specific Aim 3 proposes to characterize the response of the mutation target in fish following mutagen exposure. The applicants will test the hypothesis that the lacI gene is a sensitive and reliable marker of chemically induced mutation in transgenic fish through a series of mutagen exposure tests using two mutagens, N-ethyl-N-nitrosourea (ENU) and dimethylnitrosamine (DMN). These studies will establish transgenic fish as a valuable in vivo mutation detection model for comparative mutation studies and improved assessments of contaminant risks in aquatic systems.
| Norris, Michelle B; Winn, Richard N (2010) Isolated spermatozoa as indicators of mutations transmitted to progeny. Mutat Res 688:36-40 |
| Broussard, Gregory W; Norris, Michelle B; Schwindt, Adam R et al. (2009) Chronic Mycobacterium marinum infection acts as a tumor promoter in Japanese Medaka (Oryzias latipes). Comp Biochem Physiol C Toxicol Pharmacol 149:152-60 |
| Winn, Richard N; Majeske, Audrey J; Jagoe, Charles H et al. (2008) Transgenic lambda medaka as a new model for germ cell mutagenesis. Environ Mol Mutagen 49:173-84 |
| Winn, R N (2001) Trangenic fish as models in environmental toxicology. ILAR J 42:322-9 |
| Winn, R N; Norris, M B; Brayer, K J et al. (2000) Detection of mutations in transgenic fish carrying a bacteriophage lambda cII transgene target. Proc Natl Acad Sci U S A 97:12655-60 |
