USE OF RETROVIRAL VECTORS IN THE ANALYSIS OF MUTATIONS IN MAMMALIAN CELLS

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DNA sequence analysis of mutations induced in mammalian cells has been limited by the lack of systems available for the convenient analysis of isolated mutants. Our approach has been to construct cell lines designed to simplify the isolation and characterization of mutations induced in mammalian cells. To generate mammalian cell lines useful for mutagenesis studies, we have constructed several retroviral vectors that carry gpt and other selectable markers. These vectors can be used to infect a variety of mammalian cell types. The integration of retroviral sequences occurs randomly and usually in single copy in the genome. Therefore, it is unlikely that any two isolated cell clones will carry the gpt gene inserted at precisely the same genomic location. We can, therefore, study the effect of genomic position on mutagenesis using the same target gene (gpt) integrated at several sites. We have data with the AS52 cell line that suggests deletion mutations are influenced by the genomic position of the target gene; however, there are no data available to assess the influence of genomic position on either spontaneous or induced point mutations. We will utilize retroviral vectors to isolate human and mouse repair proficient cell clones with single copy gpt integrations and will generate both spontaneous and induced point mutations at the gpt locus. Mutant DNAs will be isolated and sequenced to generate mutant spectra. Comparison of the spectra generated will allow an assessment of the influence of genomic position on the occurrence and type of point mutations generated in mammalian cells. These data should provide a foundation for the use of human repair deficient cell lines, e.g. Xeroderma pigmentosum, Ataxia telangiectasia, Bloom's Syndrome, etc., derived using retroviral vectors in future studies of mutational mechanisms in mammalian cells.