We are using retroviral vectors to construct cell lines useful fOr the study of mutational mechanisms in human cells. Under appropriate infection conditions one can isolate single copy genomic integrations of retroviral vectors, a requirement for mutagenesis studies. The retroviral vectors used in these studies carry and express both the bacterial gpt and neo genes. An appropriate integration of the gpt gene and the linked neo gene provides cellular resistance to both mycophenolic acid and the aminoglycoside, G418. We have evaluated several vector constructions to determine requirements for the optimal expression of both the at and neo genes. We find optimal expression in constructions in which the gpt gene is under the regulatory control of the SV40 promoter and the neo gene is under the regulatory control of the 5'-LTR. We are also constructing vectors that allow differential regulation of the gpt gene using the metallothionein promoter. Using these vectors, we are in the process of generating single copy gpt integrations into the genome of the human cell line, HT1080. HT1080 cells are derived from a human fibrosarcoma and have a particularly stable karyotype, another important requirement for mutagenesis studies. Our goal is to determine the effect of genomic position on both spontaneous and induced mutations at a chromosomally integrated gpt locus in human HT1080 cells. Mutant spectra will be generated for comparative analysis from various HT1080 derived independent gpt integrations. Such an approach provides a unique opportunity to evaluate """"""""position effects"""""""" on mutagenesis using the same target gene (i.e., gpt) integrated at various sites in the genome. Long term, these studies should provide a data base for the use of retroviral vectors in the construction of human repair-deficient cell lines useful for mechanistic studies of mutations.
