Generation and Characterization of Ins1-Cre and Ins1-CreERT2 Mice

Mice Ins1^Cre (Ins1^tm1(cre)Thor) and Ins1^CreERT2 (Ins1^{tm1(CreERT2)Thor}) knock-in mice were generated by Genoway (Lyon, France) and kept on a C57Bl/6J genetic background. Briefly, a targeting vector was created by inserting the Cre or CreERT2 recombinase genes by homologous recombination in the second exon of the Ins1 gene so that the coding region of the recombinase starts at the initiation codon and replaces the Ins1 coding sequence (Fig. 1). Following transfection of the targeting vector into embryonic stem cells, negative and positive selections were performed by diphtheria toxin and neomycin (neo) selection, respectively. Cells with confirmed homologous recombination were used to generate chimeric mice. These were crossed with C57Bl/6J mice, and mice with germline transmission were crossed with C57Bl/6J flippase (Flp) deleter mice to eliminate the neo cassette. Mice with germline transmission of the recombined allele and deletion of the neo cassette were then crossed with Rosa26-eYFP mice [20 (link)] or Rosa26-tdTomato mice [21 (link)]. Animals were housed four to five per cage at 23°C on a 12 h light/dark cycle and were fed a standard rodent chow (Diet 3436; Provimi Kliba, Penthallaz, Switzerland). To induce recombination in Ins1^CreERT2 crossed with Rosa26 reporter mice, tamoxifen (T5648; Sigma-Aldrich, St Louis, MO, USA) was dissolved in corn oil at 20 mg/ml, and 2 mg was injected subcutaneously four times over a 2 week period.Fig. 1

Generation of Ins1^Cre and Ins1^CreERT2 mice. (a) Structure of the Ins1 locus, of the targeting vector, of the targeted allele, and of the Ins1^Cre locus following flipase (Flp)-dependent removal of the flipase recognition site (Frt)-flanked neo cassette. DTA, diphtheria toxin receptor gene; Ex, exon; ATG, translation initiation codon. (b) The Ins1^CreERT2 allele was obtained using the targeting strategy depicted in (a). (c) Structure of the Rosa26-eYFP/tdTomato transgene and localisation of the primers (arrows) used for PCR analysis of the recombined locus. CMV/A, cytomegalovirus/actin promoter; (d) PCR analysis shows recombination in the pancreas but not in the liver, cortex, hypothalamus or cerebellum of Ins1^Cre/+;Rosa26-eYFP. No recombination was observed in the pancreas of Rosa26-eYFP littermates; amplification of the Gapdh gene is used as a control