Enrichment for gene targeting in mammalian cells by inhibition of poly(ADP-ribosylation)

Abstract

Inhibition of poly(ADP-ribosylation) reduces random genomic integration of transfected DNA and mildly stimulates intrachromosomal homologous recombination in mammalian cells. We investigated the effect of inhibition of poly(ADP-ribosylation) on the efficiency of gene targeting in Chinese hamster ovary (CHO) cell line ATS-49tg. This cell line is hemizygous for a defective adenine phosphoribosyltransferase (aprt) gene and is hypoxanthine phosphoribosyltransferase (hprt) deficient. Plasmid pAG100 contains a portion of the CHO aprt gene sufficient to correct the defect in ATS-49tg cells via gene targeting; pAG100 also contains an Escherichia coli guanine phosphoribosyltransferase (gpt) gene. Following transfection of ATS-49tg cells with pAG100, selection for gpt-positive transfectants allowed recovery of cells that had randomly integrated pAG100 while selection for aprt-positive cells allowed recovery of cells that had undergone gene targeting at the endogenous aprt locus. Treatment of cells with 3 mM 3-methoxybenzamide (3-MB), an inhibitor of poly(ADP-ribose) polymerase, decreased random integration and gene targeting of electroporated pAG100 about 5-fold. In contrast, treatment with 3 mM 3-MB during calcium phosphate transfection could reduce random integration more than 150-fold while reducing gene targeting less than two-fold. Therefore, as much as a 100-fold enrichment for gene targeting was achieved with calcium phosphate transfection.