ENRICHMENT FOR GENE TARGETING IN MAMMALIAN-CELLS BY INHIBITION OF POLY(ADP-RIBOSYLATION)

Inhibition of poly(ADP-ribosylation) reduces random genomic integratio n of transfected DNA and mildly stimulates intrachromosomal homologous recombination in mammalian cells. We investigated the effect of inhib ition of poly(ADP-ribosylation) on the efficiency of gene targeting in Chinese hamster ovary (CHO) cell line ATS-49tg. This cell line is hem izygous for a defective adenine phosphoribosyltransferase (aprt) gene and is hypoxanthine phosphoribosyltransferase (hprt) deficient. Plasmi d pAG100 contains a portion of the CHO apa gene sufficient to correct the defect in ATS-49tg cells via gene targeting; pAG100 also contains an Escherichia coli guanine phosphoribosyltransferase (gpt) gene. Foll owing transfection of ATS-49tg cells with pAG100, selection for gpt-po sitive transfectants allowed recovery of cells that had randomly integ rated pAG100 while selection for apa-positive cells allowed recovery o f 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, treatme nt with 3 mM 3-MB during calcium phosphate transfection could reduce r andom integration more than 150-fold while reducing gene targeting les s than two-fold. Therefore, as much as a 100-fold enrichment for gene targeting was achieved with calcium phosphate transfection.