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

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We are interested in the effect of chromosomal position on the frequency and types of mutations observed using a target gene (gpt) integrated at different chromosomal sites in human cells. For these studies, we are using retroviral vectors that carry and express both the bacterial gpt and neo genes to construct human HT1080 cell lines with single copy integrations. A limitation to the general use of retroviral vectors in gene transfer studies is that some cells are resistant to retroviral infection. Therefore, to facilitate the application of retroviral vector technology to a variety of cell lines, we have devised a system that utilizes cationic liposomes (Lipofectin) for the delivery retroviral capsids. We have demonstrated that this approach allows one to bypass the normal receptor mediated route of infection allowing gene transfer to cell lines that either lack appropriate receptors or carry endogenous retroviruses that block the surface membrane receptors. For our mutagenesis studies, we have chosen a retroviral vector which allows the regulation of gpt gene expression using the human metallothionein (MTII) promoter. Evidence is accumulating to suggest that DNA repair may be more rapid in transcriptionally active regions of the genome and that the transcribed DNA strand may be repaired more rapidly than the nontranscribed strand. Thus, we hope to assess both the influence of DNA repair using a transcriptionally active or inactive target gene (gpt) as well as the effects of chromatin structure on mutagenesis. These studies are intended to provide a data base for using retroviral vectors to assess the role of human DNA repair pathways on mutational spectra generated in human repair deficient cell lines.