Mtmg reporter mice

C57BL6 mice were used at indicated age unless otherwise stated. For the lineage-tracing study, Ascl1^CreERT2 knock-in mice (Kim et al., 2011 (link)) and mTmG reporter mice (Jackson Laboratory, stock 007576) (Muzumdar et al., 2007 (link)) were used. All mice were housed at the University of Washington; protocols were approved by the University of Washington Institutional Animal Care and Use Committee.

To suppress Cnot3 expression in β cells, we crossed mice carrying Cnot3 floxed alleles (Cnot3^flox/flox)^{16 (link),26 (link)} with Ins1-Cre mice (Riken Bioresource Center, # RBRC09525), in which Cre recombinase is knocked in at the Ins1 locus^{68 (link)}. Recombination is expected to be restricted largely to β cells, and to be minimal at other sites, including the brain, where Ins1 is not expressed^{69 (link)}. mTmG reporter mice (Jackson Laboratory, # 007676) were used to trace β cells in which Ins1-Cre-mediated recombination was induced. Primers used for genotyping are listed in Supplementary Table 2. β-cell-specific Cnot3 KO mice (Cnot3βKO) were used for experiments, and their littermates (Cnot3^flox/flox) were used as controls, unless otherwise stated (Ins1-Cre mice were used as controls for Cnot3βKO expressing mTmG reporter gene). All experiments were performed on 8–10-week-old male mice, unless stated otherwise. db/db mice were used as a model of T2D^{22 (link)} and compared with +/db mice, purchased from CLEA Japan. We maintained mice on a 12-h light/dark cycle in a temperature-controlled (22 °C) barrier facility with free access to water and either a normal chow diet (NCD, CA-1, CLEA Japan) or a HFD (HFD32, CLEA Japan). All mouse experiments were approved by the Animal Care and Use Committee of Okinawa Institute of Science and Technology (OIST) Graduate University, Okinawa, Japan.

Mice expressing homozygously floxed Tsc2 alleles (Tsc2^flox/flox) and mice expressing Cre recombinase under the control of the CC10 promoter (CC10^Cre) were provided by Elizabeth Henske (Brigham and Women’s Hospital, Boston, MA). CC10^Cre mice were bred with Tsc2^flox/flox to generate mice with airway epithelial specific deletion of Tsc2. mT/mG reporter mice were obtained from Jackson Laboratory (007576). CC10^Cre mice were bred to mT/mG reporter mice (22 (link)) to confirm airway epithelium-specific expression of the Cre recombinase. Genotyping was performed by Transnetyx using real-time PCR for both Tsc2 and Cre alleles. All mice were backcrossed to a pure C57BL/6 background, which was confirmed by genome scanning performed by Jackson Laboratory. WT C57BL/6 mice (000664) were obtained from Jackson Laboratory. Deletion of Tsc2 was confirmed by immunostaining lung sections for phosphorylated ribosomal S6 protein and immunoblotting for tuberin and phosphorylated ribosomal S6 with protein lysate from isolated tracheobronchial epithelial cells. All mice were housed in pathogen free conditions in vivariums at Brigham and Women’s Hospital and The Davis Heart and Lung Research Institute. Mice were used for experiments at 6–12 weeks of age and provided food and water ad lib.

Mice were maintained in the Animal Research Center and experiments were performed under a protocol approved by the Institutional Animal Care and Use Committee of Yale University. For inducible Cre-mediated recombination, Nck1−/−;Nck2flox/flox mice^{29 (link)} (129/SVJ), Pdgf-bflox/flox mice^{32 (link)} (C57BL/6J) and mTmG reporter mice (The Jackson Laboratory, 129/SVJ) were bred with Cdh5-CreERT2 mice^{59 (link)} (C57BL/6J), Pdgfrβ-CreERT2 mice^{60 (link),61 (link)} (129/SV;C57BL/6J mixed background), SMACreERT2 mice^{62 (link)} (or ACTA2-CreERT2, FVB/NJ), and Myh11CreERT2 mice (The Jackson Laboratory, FVB/NJ). Gene deletion was induced by intraperitoneal injections with 50 μg tamoxifen (Sigma, T5648; 1 mg/ml) to pups at P0, P1, and P2, and mice were sacrificed at P5. The Cre-negative, tamoxifen-treated littermates are used as control mice. For genetic cell fate tracking, CRE activity was induced by two consecutive intraperitoneal injections of pups at P6 and P7 with 50 μg tamoxifen solution.

All mice were housed at the Montana State University, and protocols were approved by the Montana State University Institutional Animal Care and Use Committee. Both male and female mice were used for this study. Ikbkap CKO mice were generated by crossing Tα1-Cre, which targets postmitotic neurons (Gloster et al., 1994 (link); Coppola et al., 2004 (link); Coksaygan et al., 2006 (link)) and Ikbkap-floxed mice (International Knockout Mouse Consortium; Chaverra et al., unpublished observations). Mice were used at ages detailed below, and littermate Cre^-;Ikbkap^f/f mice were used as controls. To determine Cre expression in the retina, Tα1-Cre mice were crossed to mTmG reporter mice (stock #007576; Jackson Laboratory, Bar Harbor, ME; Muzumdar et al., 2007 (link)). To analyze endogenous expression of Ikbkap in the retina, LacZ reporter mice (Ikbkap:β-gal) were used (International Knockout Mouse Consortium; George et al., 2013 (link)).