Abstract
A 21-bp deletion in the third exon of theAPRT gene in Chinese hamster ovary (CHO) cells was corrected by transfection with a plasmid containing hamsterAPRT sequences. Targeted correction frequencies in the range of 0.3–3.0×10−6 were obtained with a vector containing 3.2 kb ofAPRT 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 ofAPRT 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.
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Nairn, R.S., Adair, G.M., Porter, T. et al. Targeting vector configuration and method of gene transfer influence targeted correction of theAPRT gene in Chinese hamster ovary cells. Somat Cell Mol Genet 19, 363–375 (1993). https://doi.org/10.1007/BF01232748
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DOI: https://doi.org/10.1007/BF01232748