FIG. 1.

KO vector and strategies for characterizing homologous recombination by Southern blotting and genotyping by PCR after electroporation of the Gα15 replacement vector. (A) Wild-type (wt) allele and the targeting vector. The restriction sites are shown to scale. tk, thymidine kinase. (B) The KO allele. The pgk::neo insertion cassette is not drawn to scale. The SpeI, EcoRV, and HindIII sites used in characterizing the wild-type and KO alleles by Southern blot are shown on each allele. The inserted pgk::neo cassette is 2.1 kb in size and contains both EcoRV and SpeI restriction sites. The 5′ probe hybridizes to a 14-kb fragment in the wild-type allele and 9.5 kb in the KO allele when DNA is digested with EcoRV. The 3′ probe recognizes 9.2- and 7-kb fragments in wild-type and KO mice after digest with SpeI and HindIII. The Neo probe hybridizes to a 14-kb SpeI fragment in the KO allele. The relative positions of PCR products obtained from amplification of the wild-type allele with primers CT115 and CT133 (550 bp) and the KO allele with primers TW30 and TW144 (720 bp) are indicated. (C) Results obtained from genotyping Gα15^+/+, Gα15^+/−, and Gα15^−/− tail DNA by Southern blot using the 5′ probe after digest with EcoRV; ethidium bromide-stained 2.5% agarose gel of PCR products obtained from amplifying the same tail DNA with the primers described above.