Efficiency of insertion versus replacement vector targeting varies at different chromosomal loci.
We have analyzed the targeting frequencies and recombination products generated with isogenic vectors at the fah and fgr loci in embryonic stem cells. A single vector which could be linearized at different sites to generate either a replacement or an insertion vector was constructed for each locus. A replacement event predominated when the vectors were linearized at the edge of the homologous sequences, while an insertion event predominated when the vectors were linearized within the homologous sequences. However, the ratio of the targeting frequencies exhibited by the different vector configurations differed for the two loci. When the fgr vector was linearized as an insertion vector, the ratio of targeted to random integrations was four- to eightfold greater than when the vector was linearized as a replacement vector. By contrast, the ratio of targeted to random integrations at the fah locus did not vary with the linearization site of the vector. The different relationships between the targeting frequency and the vector configuration at the fgr and fah loci may indicate a DNA sequence or chromatin structure preference for different targeting pathways.
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030.
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Hasty
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Crist
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Grompe
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Bradley
D000818:Animals; D002478:Cells, Cultured; D002874:Chromosome Mapping; D002875:Chromosomes / Q000648:ultrastructure; D018390:Gene Targeting; D005796:Genes; D005822:Genetic Vectors; D006867:Hydrolases / Q000235:genetics; D066298:In Vitro Techniques; D051379:Mice; D011505:Protein-Tyrosine Kinases / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D011995:Recombination, Genetic; D015183:Restriction Mapping; D019061:src-Family Kinases
D011518:Proto-Oncogene Proteins; D011505:Protein-Tyrosine Kinases; C053102:proto-oncogene proteins c-fgr; D019061:src-Family Kinases; D006867:Hydrolases; C020156:fumarylacetoacetase