Targeted gene replacement at the endogenous APRT locus in CHO cells.
We demonstrate the feasibility of targeted gene replacement at an endogenous, chromosomal gene locus in cultured mammalian cells, employing a two-step strategy similar to an approach routinely used for genetic manipulation in yeast. Utilizing an APRT+ recombinant generated by targeted integration of plasmid sequences (including a functional copy of the gpt gene) at the CHO APRT locus, we have been able to select gpt- "pop-out" recombinants that have arisen by intrachromosomal recombination between APRT direct repeats at the targeted integration site. Reciprocal exchanges leading to "pop-out" of integrated plasmid/gpt gene sequences occur at a rate of approximately 6.3 x 10(-6) per cell generation. Depending on the site of crossover, such "pop-out" events result in either replacement or restoration of the original APRT target gene sequence.
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
Scheerer
J B
JB
Brotherman
K A
KA
D000225:Adenine / Q000031:analogs & derivatives; D000228:Adenine Phosphoribosyltransferase / Q000235:genetics*; D000818:Animals; D015139:Blotting, Southern; D002460:Cell Line; D002872:Chromosome Deletion; D006224:Cricetinae; D003412:Cricetulus; D004351:Drug Resistance / Q000235:genetics; D005260:Female; D005818:Genetic Engineering / Q000379:methods*; D016297:Mutagenesis, Site-Directed; D010053:Ovary / Q000166:cytology; D011995:Recombination, Genetic; D013866:Thioguanine
D000228:Adenine Phosphoribosyltransferase; D013866:Thioguanine; D000225:Adenine; C006654:8-azaadenine