title: Reexamination of gene targeting frequency as a function of the extent of homology between the targeting vector and the target locus.
abstract: Mutations were targeted to the Hprt locus of mouse embryo-derived stem cells by using 22 different sequence replacement and sequence insertion vectors. The targeting frequency was examined at two sites within the Hprt locus as a function of the extent of homology between the targeting vector and the target locus. The targeting frequency was also compared by using vectors prepared from isogenic and nonisogenic DNA sources. With one exception, all of the vectors showed the same exponential dependence of targeting efficiency on the extent of homology between the targeting vector and the target locus. This was true regardless of whether they were sequence replacement or sequence insertion vectors, whether they were directed toward either of the two different sites within the Hprt locus, or whether they were prepared from isogenic or nonisogenic DNA sources. Vectors prepared from isogenic DNA targeted four to five times more efficiently than did the corresponding vectors prepared from nonisogenic DNA. The single case of unexpectedly low targeting efficiency involved one of the vectors prepared from nonisogenic DNA and could be attributed to an unfavorable distribution of heterology between the Hprt sequences present in the targeting vector and the endogenous Hprt gene.
authors.affiliation: Howard Hughes Medical Institute, University of Utah School of Medicine, Salt Lake City 84112.
authors.forename: C
authors.initials: C
authors.lastname: Deng
authors.forename: M R
authors.initials: MR
authors.lastname: Capecchi
mesh_terms: D000818:Animals; D002460:Cell Line; D004247:DNA / Q000235:genetics*; D004251:DNA Transposable Elements; D004622:Embryo, Mammalian; D005091:Exons; D005822:Genetic Vectors; D015698:Genomic Library; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D051379:Mice; D010957:Plasmids; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013234:Stem Cells / Q000201:enzymology
chemical_list: D004251:DNA Transposable Elements; D004247:DNA; D007041:Hypoxanthine Phosphoribosyltransferase