Double-strand gap repair in a mammalian gene targeting reaction.
To better understand the mechanism of homologous recombination in mammalian cells that facilitates gene targeting, we have analyzed the recombination reaction that inserts a plasmid into a homologous chromosomal locus in mouse embryonic stem cells. A partially deleted HPRT gene was targeted with various plasmids capable of correcting the mutation at this locus, and HPRT+ recombinants were directly selected in HAT medium. The structures of the recombinant loci were then determined by genomic Southern blot hybridizations. We demonstrate that plasmid gaps of 200, 600, and 2,500 bp are efficiently repaired during the integrative recombination reaction. Targeting plasmids that carry a double-strand break or gap in the region of DNA homologous to the target locus produce 33- to 140-fold more hypoxanthine-aminopterin-thymidine-resistant recombinants than did these same plasmids introduced in their uncut (supercoiled) forms. Our data suggest that double-strand gaps and breaks may be enlarged prior to the repair reaction since sequence heterologies carried by the incoming plasmids located close to them are often lost. These results extend the known similarities between mammalian and yeast recombination mechanisms and suggest several features of the insertional (O-type) gene targeting reaction that should be considered when one is designing mammalian gene targeting experiments.
Valancius
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Department of Pathology, School of Medicine, University of North Carolina, Chapel Hill 27599-7525.
Smithies
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O
D000818:Animals; D015139:Blotting, Southern; D004247:DNA / Q000235:genetics*; D004260:DNA Repair; D004274:DNA, Recombinant; D005821:Genetic Techniques; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D051379:Mice; D014411:Neoplastic Stem Cells; D010957:Plasmids; D011995:Recombination, Genetic; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D004274:DNA, Recombinant; D004247:DNA; D007041:Hypoxanthine Phosphoribosyltransferase