Transgenic mutation detection systems have a heightened capability to detect low frequency events and promise to provide important information for elucidating mutational mechanisms in male gametogenesis and reproduction. This proposal is targeted at the study of germinal mutation in male mice, utilizing transgenic technology for the analysis of germinal mutation in males. The primary advantages of this system is that it can detect mutations in individual male germ cells, which can number in the millions, rather than the small number of progeny that can be recovered in the progeny from breeding each male. The system chosen for these studies is the lacI transgenic shuttle-vector system, which employs an inbred mouse strain (C57 BL/6) that has integrated into its genome multiple copies of a lambda shuttle vector that contains both the lacI and lacZ genes. The lacI gene is the mutational target which, when inactivated by mutation, permits expression of the lacZ gene that is detectable by X-gal staining of plaques with mutations in the target gene. Thr primary need in establishing the worthiness of shuttle-vector systems for the study of germinal mutation is the lack of information regarding the transmissibility of mutations induced in transgenes. Thus, the first specific aim of this proposal is to demonstrate and characterize the heritability of transgenes in breeding studies. In order to maximize the probability of detecting mutation transmission, ethylnitrosourea (ENU), the most effective mutagen in inducing mutations in both transgenes and endogenous genes, will be used. A breeding scheme has been devised that will permit the concurrent study of mutations in a well-validated system the 7-locus recessive visible test as an internal, positive control. The second specific aim will be to study the stage-specific effects of three known germinal mutagens, ENU, methylnitrosourea (MNU), and propylnitrosourea (PNU). In order to explore stage-specific mutation induction, mature sperm will be sampled at weekly intervals from mutagen-treated males in order to recover gametes that were in different stages of spermatogenesis at the time of treatment. Finally, the third specific aim will be to define the molecular lesion responsible for mutations for transgenes. The mutational spectra of both induced and spontaneous mutations will be determined by sequencing the mutant genes recovered in the assay. All studies of mutation in this system will assess and take into account the reproductive function as potentially impacted by the administration of mutagens. With these studies, it is hoped to enhance our understanding of mutational mechanisms in male germinal tissue and thus help to prevent increases in the human genetic load from chemical mutagens.
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