Generation of ΔRING-Trim41 Mutant Mice

ΔRING-Trim41 mice were generated by microinjection described previously [49 (link)]. First, a gRNA solution was prepared by annealing two tracrRNAs (Sigma-Aldrich, St. Louis, MO, USA) and crRNA (Sigma-Aldrich). The target genomic sequences are listed in S1 Table Then, the gRNA solution and Cas9 nuclease solution (Thermo Fisher Scientific, Waltham, MA, USA) were mixed: 40 ng/μL gRNA each and 108 ng/μL Cas9 nucleases. The obtained complex was then microinjected into fertilized eggs (B6D2F1) using a programmable microinjector (FemtoJet 4i, Eppendorf, Hamburg, Germany). The microinjected eggs were then transplanted into the oviduct ampulla of pseudopregnant mice (ICR) on the following day. After 19 days, pups were delivered through Caesarean section and placed with foster mothers (ICR). To generate heterozygous mutant mice, F0 mice were mated with WT B6D2F1. Mouse colonies with the desired mutation were maintained by sibling matings. The genotyping primers (GeneDesign) are listed in S1 Table The mutant mouse line is available from Riken BRC (#11041) and CARD (#2948).