Targeted homologous recombination at the endogenous adenine phosphoribosyltransferase locus in Chinese hamster cells.
We have developed a system that permits analysis of targeted homologous recombination at an endogenous, chromosomal gene locus in cultured mammalian cells. Using a hemizygous, adenine phosphoribosyltransferase (APRT)-deficient, Chinese hamster ovary (CHO) cell mutant as a transfection recipient, we have demonstrated correction of a nonrevertible deletion mutation by targeted homologous recombination. Transfection with a plasmid carrying a fragment of the APRT gene yielded APRT+ recombinants at a frequency of approximately 4.1 x 10(-7). The ratio of targeted recombination to nontargeted integrations of plasmid sequences was approximately 1:4000. Analysis of 31 independent APRT+ recombinants revealed conversions of the endogenous APRT gene, targeted integration at the APRT locus, and a third class of events in which the plasmid donor APRT fragment was converted to a full-length, functional gene.
Adair
G M
GM
University of Texas M. D. Anderson Cancer Center, Science Park-Research Division, Smithville 78957.
Nairn
R S
RS
Wilson
J H
JH
Seidman
M M
MM
Brotherman
K A
KA
MacKinnon
C
C
Scheerer
J B
JB
D000228:Adenine Phosphoribosyltransferase / Q000172:deficiency / Q000235:genetics*; D000818:Animals; D015139:Blotting, Southern; D002460:Cell Line; D002874:Chromosome Mapping; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D005796:Genes; D005822:Genetic Vectors; D009693:Nucleic Acid Hybridization; D010053:Ovary; D010430:Pentosyltransferases / Q000235:genetics*; D011995:Recombination, Genetic; D014162:Transfection
D010430:Pentosyltransferases; D000228:Adenine Phosphoribosyltransferase