Targeting vector configuration and method of gene transfer influence targeted correction of the APRT gene in Chinese hamster ovary cells.
A 21-bp deletion in the third exon of the APRT gene in Chinese hamster ovary (CHO) cells was corrected by transfection with a plasmid containing hamster APRT sequences. Targeted correction frequencies in the range of 0.3-3.0 x 10(-6) were obtained with a vector containing 3.2 kb of APRT sequence homology. To examine the influence of vector configuration on targeted gene correction, a double-strand break was introduced at one of two positions in the vector prior to transfection by calcium phosphate-DNA coprecipitation or electroporation. A double-strand break in the region of APRT homology contained in the vector produced an insertion-type vector, while placement of the break just outside the region of homology produced a replacement-type vector. Gene targeting with both linear vector configurations yielded equivalent ratios of targeted recombinants to nontargeted vector integrants; however, targeting with the two different vector configurations resulted in different distributions of targeted recombination products. Analysis of 66 independent APRT+ recombinant clones by Southern hybridization showed that targeting with the vector in a replacement-type configuration yielded fewer targeted integrants and more target gene convertants than did the integration vector configuration. Targeted recombination was about fivefold more efficient with electroporation than with calcium phosphate-DNA coprecipitation; however, both gene transfer methods produced similar distributions of targeted recombinants, which depended only on targeting vector configuration. Our results demonstrate that insertion-type and replacement-type gene targeting vectors produce similar overall targeting frequencies in gene correction experiments, but that vector configuration can significantly influence the yield of particular recombinant types.
Nairn
R S
RS
Department of Carcinogenesis, University of Texas M.D. Anderson Cancer Center, Smithville 78957.
Adair
G M
GM
Porter
T
T
Pennington
S L
SL
Smith
D G
DG
Wilson
J H
JH
Seidman
M M
MM
D000228:Adenine Phosphoribosyltransferase / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004247:DNA; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010957:Plasmids; D012150:Polymorphism, Restriction Fragment Length; D011995:Recombination, Genetic; D012689:Sequence Homology, Nucleic Acid
D004247:DNA; D000228:Adenine Phosphoribosyltransferase