Figure 1.

Creation of fIrs2 mice and cr²-mediated recombination/deletion of fIrs2. (A) A schematic diagram of the 4 steps in the generation of the fIrs2 containing embryonic stem cells (see Methods for details). Homol recomb, homologous recombination. (B) Recombination/deletion of fIrs2 was revealed in various mouse tissues at 4 weeks of age by PCR analysis of genomic DNA. L, liver; K, kidney; H, heart; S, spleen; B, brain, F, fat; M, muscle. The intact fIrs2 allele (fIrs2⁺) yields a 750-bp fragment, whereas the recombined/deleted allele (fIrs2^_) yields a 250-bp fragment. (C) Real-time PCR analysis of Irs2 expression in isolated islets, normalized by cyclophilin expression. (D) PCR analysis of hypothalamic DNA to reveal recombination event. (E) Immunoprecipitation and Western blot analysis of hypothalamus with antibodies against Irs2. (F) Brain sections were immunostained with antibodies against Irs2 (red) and Cre (green), and the images were merged in Openlab Image software (version 3.14; Improvision). Similar results were obtained with multiple sections from 2 to 8 mice. Asterisks indicate third ventricle. Magnification, ×20.