Targeted disruption of the Gab2 locus.

(A) Restriction map of the Gab2 locus and targeting vector. The deleted region contains an exon encoding part of the PH domain. This region was replaced by the neomycin-resistance gene (neo). H, HindIII; B, BamHI; S, SpeI. A λFixII 129/Sv mouse strain genomic library (Stratagene) was screened by hybridization with the mouse Gab2 cDNA fragment containing an exon encoding the PH domain (amino acids 1-128). A targeting vector was designed to replace theBamHI-SalI fragment containing the PH domain with the neomycin-resistance gene (neo). To construct the targeting vector, the 1.5-kb HindIII-BamHI fragment of mouse Gab2 genomic DNA, the 2.0-kb BamHI-XbaI fragment of pgk-neo, the 9.0-kb SpeI-BamHI fragment of mouse Gab2 genomic DNA, and the 2.0-kb XhoI fragment of the thymidine kinase gene were subcloned into the HindIII andXhoI sites of pBluescript SK+. Further details are available upon request. (B) Southern blot analysis for genotyping.EcoRI and BglII-digested DNA from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice was hybridized with the 5′ probe shown in (A). (C) Immunoblotting analysis of Gab2. Gab2 immunoprecipitated from the testis of Gab2+/+, +/−, and −/−mice was analyzed by immunoblotting with anti–Gab2 antibody. Lysate from each of the same samples were immunoblotted with anti–Gab1 or anti–SHP-2 antibodies for loading control.