IN VIVO MUTAGENESIS STUDIES USING TRANSGENIC MICE

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SUMMARY OF WORK: While specific cellular mutational mechanistic pathways are often most conveniently studied in vitro, differences in tissue response, in chemical deposition or in the response of different cell types to mutagen/carcinogen exposure can only be defined in vivo. Big Blue carries 30-40 copies of lambda phage each with a single copy of lacl which is used as a mutational target. More recently, the lambda cll gene has also been defined as a useful mutational target gene; thus providing an alternative to lacl for in vivo mutagenesis studies. While lacl has been a useful marker for mutagenesis studies, the cll assay provides quantitative data similar to those generated with lacl, allows for studies to be performed with fewer manipulations and is less costly per data point generated. With the development transgenic animals useful for the study of in vivo mutations, the tissue specific response to a mutagen/carcinogen can now be determined. We have defined the nature of spontaneous mutations in the lacl transgene of Big Blue mice was determined in selected tissues. In all tissues, the predominant class of mutations was G:C AE A:T transitions, most of which occurred at 5'-CpG-3' dinucleotide sequences. The second most common class of mutations was G:C AE T:A transversions. These data have been used to establish a database of spontaneous mutations that arise spontaneously in Big Blue mice, thus serving as a reference against which mutations recovered after treatment can be compared. In short the timing of treatment and of partial hapatectomy were shown to be critical in these studies. We have studied the effects of cellular proliferation on the frequency of mutations arising in the liver of male C57B1/6 Big Blue transgenic mice by subjecting the mice to partial hepatectomy following treatment with benzo(a)pyrene (B[a]P). We have also demonstrated that the carcinogenic flame retardant tris(2,3-dibromopropyl)phosphate (TDBP) is mutagenic in the kidney which is the same tissue for which tumors were observed in male and female rats and in male mice. We intend to continue to study the effects of specific defects in DNA metabolism on the process of mutagenesis in vivo. Specifically, cells form lacl/pol b -knockout mice are available for study. By evaluating mutations in either the lacl or the cll genes, we will gain insight regarding the role of the b- polymerase in both normal and repair replication. Furthermore, we will begin to coordinate the in vitro and in vivo mutagenesis studies that are presently on-going in the lab by focusing on studies using mismatch repair (MMR) defective transgenic mice and cell lines derived from these mice.