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Applications of a retroviral integrase towards substrate DNA in vivo

University of British Columbia

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.

Thesis/Dissertation

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2009-06-02

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http://hdl.handle.net/2429/8583

Biochemistry and Molecular Biology

University of British Columbia

UBCV

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