Genetic Manipulation of β-Cell Cnot3 Expression

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.