title: 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.
authors.affiliation: Department of Biological Sciences, University of South Carolina, Columbia 29208, USA. awaldman@biol.scarolina.edu
authors.forename: B C
authors.initials: BC
authors.lastname: Waldman
authors.forename: J R
authors.initials: JR
authors.lastname: O'Quinn
authors.forename: A S
authors.initials: AS
authors.lastname: Waldman
mesh_terms: D000228:Adenine Phosphoribosyltransferase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001549:Benzamides / Q000494:pharmacology; D015139:Blotting, Southern; D016466:CHO Cells; D002130:Calcium Phosphates / Q000494:pharmacology; D002455:Cell Division; D006224:Cricetinae; D018274:Electroporation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation; D018390:Gene Targeting / Q000379:methods*; D017930:Genes, Reporter; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D011064:Poly Adenosine Diphosphate Ribose / Q000378:metabolism*; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D001549:Benzamides; D002130:Calcium Phosphates; D004791:Enzyme Inhibitors; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; D011064:Poly Adenosine Diphosphate Ribose; C485838:calcium phosphate, monobasic, anhydrous; C020243:calcium phosphate; D000228:Adenine Phosphoribosyltransferase; D007041:Hypoxanthine Phosphoribosyltransferase; D001616:beta-Galactosidase; C485829:calcium phosphate, dibasic, anhydrous; C025159:3-methoxybenzamide