Generation of Irs2flox mice and characteristics of RIPCre and POMCCre mice. (A) Schema of targeting construct design, simplified restriction map of the Irs2 locus, the locus after homologous recombination and the deletion of neomycin cassette (Neo), and Southern blotting and PCR genotyping strategies used to identify these events. External probe A was used to identify homologous recombination (HR), probe B to detect the selection cassette, and probe C to detect the coding region of Irs2. HSV-tk, herpes simplex virus thymidine kinase. (B) Southern blot analysis with probe A demonstrating homologous recombination after targeting. (C and D) Southern blots using probe B after Cre-mediated recombination demonstrating deletion (Del) of the neomycin cassette and using probe C to demonstrate retention of Irs2 coding region confirming type 2 recombination. (E) PCR analysis with primers P1 and P2 of HR clone that has lost the neomycin cassette but retained the loxP site downstream of the Irs2 coding region. (F–H) We examined Cre expression in RIPCre mice by analyzing GFP expression in pancreatic sections costained with insulin and hypothalamic sections from RIPCreZEG mice. (I) We examined Cre expression in POMCCre mice by analyzing hypothalamic sections from POMCCreZEG mice. (J) Combined ISH for POMC and ICC for GFP was performed in POMCCreZEG mice to confirm Cre expression in POMC neurons. (K) Immunohistochemistry for IRS2 (red) was performed in hypothalamic sections from POMCCreZEG mice. Scale bars: 50 μm (H, I, and K) and 10 μm (J). 3V, third ventricle; ME, median eminence.