Efficient recruitment of transfected DNA to a homologous chromosomal target in mammalian cells.
A Chinese hamster ovary cell line hemizygous for a defective adenine phosphoribosyltransferase (aprt) gene was transfected with a plasmid, pAG100, capable of correcting the endogenous aprt mutation by targeted homologous recombination. In some experiments, pAG100 was transfected in combination with one of two 'competitor' plasmids. Competitor pCOMP-A was identical to pAG100 except that the aprt sequence on pCOMP-A had the same mutation as the endogenous aprt gene. Competitor pCOMP-B was identical to pAG100 except for a 763 bp deletion in the aprt sequence encompassing the site of mutation in the endogenous gene. Neither pCOMP-A nor pCOMP-B was capable of correcting the defect in the endogenous aprt gene via gene targeting. We asked whether cotransfection of a 4-fold excess of either competitor DNA molecule with pAG100 would reduce the efficiency of targeted correction of the endogenous aprt gene. We report that while plasmid pCOMP-B did not influence the efficiency of gene targeting by pAG100, plasmid pCOMP-A reduced the number of gene targeting events about 5-fold. These observations indicate that the initial homologous interaction between transfected DNA and a genomic target sequence occurs rapidly and that targeting efficiency is limited by a step subsequent to homologous pairing.
Lukacsovich
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Department of Biological Sciences, University of South Carolina, 700 Sumter Street, Columbia, SC 29208, USA.
Waldman
B C
BC
Waldman
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AS
D000228:Adenine Phosphoribosyltransferase / Q000172:deficiency / Q000235:genetics*; D000818:Animals; D015139:Blotting, Southern; D016466:CHO Cells; D006224:Cricetinae; D018390:Gene Targeting; D010957:Plasmids; D014162:Transfection
D000228:Adenine Phosphoribosyltransferase