Generation of Insr knock-ins. (A) Targeting vector. Exons are represented by dark gray rectangles. The INSR cDNA was cloned downstream of the TAG stop codon, an internal ribosomal entry site (IRES) and a lox-flanked (“floxed”) neomycin-resistance (neo) cassette. (B) Southern blot analysis. DNA was digested with PstI. Blots were hybridized with an external probe 5′ of the targeted locus. The WT band is about 10 kbp and the recombinant band generated by homologous recombination (βac/INSR allele) is about 3 kbp. Mice carrying the bac/INSR allele were mated with transgenic mice expressing cre to remove the loxP-neo-loxP cassette. Excision of the neo cassette resulted in the generation of a βac/INSR δlox allele (right panel, right lane). The upper band corresponds to the intact βac/INSR allele and the lower band, to the cre-excised βac/INSR δlox allele. (C) Western blot analysis of Insr (left panel) and tubulin expression (right) in several brain sections, liver, muscle, and heart in Syn-Insr, Camk-Insr, Nes-Insr, and Hs6-Insr mice. (D) Generation of pancreatic β cell–specific Insr knock-in. We show PCR analysis of Rip/cre–mediated excision of the βac/INSR allele in the pancreas. We isolated DNA from pancreata dissected from 2-day-old βac/INSR δlox/Insr–/– mice and amplified it by PCR with three sets of primers specific for the Rip-cre transgene (upper panel), βac/INSR allele (middle panel), and βac/INSR δlox allele (bottom panel).