title: Targeting of the Gi2 alpha gene in ES cells with replacement and insertion vectors.
abstract: Five replacement vectors (RV) and one insertion vector (IV) were constructed in which ca. 10 kb of genomic Gi2 alpha sequence, flanked on one (IV) or both (RV) sides by a thymidine kinase (TK) marker, were disrupted by a Neo marker inserted into the NcoI site of exon 3. G418RFIAUR clones corresponding to ca. 4 x 10(8) ES cells electroporated with replacement vectors were analyzed and revealed no targeting event. The insertion vector, however, was integrated by a single reciprocal recombination resulting in a duplication of homology (Hit step; G418RFIAUS), which was lost--together with the plasmid and the TK sequences--by intrachromosomal recombination (Run step; G418RFIAUR). Thus, the Hit and Run strategy can be used with a selectable marker disrupting the targeted gene, giving rise to the same targeted product that would have been expected to arise from a double crossover with a replacement vector.
authors.affiliation: Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030.
authors.forename: U
authors.initials: U
authors.lastname: Rudolph
authors.forename: P
authors.initials: P
authors.lastname: Brabet
authors.forename: J
authors.initials: J
authors.lastname: Kaplan
authors.forename: P
authors.initials: P
authors.lastname: Hasty
authors.forename: A
authors.initials: A
authors.lastname: Bradley
authors.forename: L
authors.initials: L
authors.lastname: Birnbaumer
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D019204:GTP-Binding Proteins / Q000235:genetics*; D005819:Genetic Markers / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection / Q000379:methods
chemical_list: D005819:Genetic Markers; D011993:Recombinant Fusion Proteins; D019204:GTP-Binding Proteins