UBC Theses and Dissertations
Applications of a retroviral integrase towards substrate DNA in vivo Heale, John-Paule
This study explored the efficacy of using retroviral integrase expressed transiently in vivo to mediate the recombination of exogenous DNA into host cell chromatin. Stable recombination of a substrate DNA into the genome of Baby Hamster Kidney (BHK) cells was achieved by co-transfecting them with a circular plasmid encoding Rous Sarcoma Virus (RSV) integrase (pIN) and a linearized plasmid (pNR) serving as a substrate for the integrase. Ndel linearized pNR (substrate DNA) mimicked the wild type RSV viral DNA in being a linear, double-stranded DNA molecule possessing terminal sequences recognizable by the integrase. Either a dihydrofolate reductase or neomycin cassette engineered into pNR served as a marker for recombination by conferring resistance to methotrexate or G418 selection, respectively. After 10 days growth in media supplemented with either 500µM methotrexate or 575µM G418, a small number of discrete colonies had formed on control plates containing cells which had been transfected only with substrate DNA. However, plates containing cells which had been co-transfected with both substrate DNA and pIN showed a ten-fold or higher increase in colony numbers over control plates. Sequence analysis of co-transfected BHK genomes identified many clonal recombinants; however none was a conserved, full length substrate DNA molecule expected from RSV IN mediated integration. Southern blot analysis of genomic DNA from co-transfected cells indicated that multiple copies of pNR had recombined with the BHK. Hence, in vivo, integrase increases the frequency of recombination of a recognizable substrate DNA molecule. However RSV IN mediated integration of substrate DNA was not observed.