Generation of Tmem2-FLAG Knock-in Mice

Pronuclear stage embryos from C57BL6/J mice were purchased from ARK Resource (Kumamoto, Japan). Recombinant Cas9 protein, crRNA and tracrRNA were obtained from Integrated DNA technology. Single-stranded oligodeoxynucleotides for insertion of a FLAG epitope were designed with 30 bp sequence homologies on each side of the Cas9-mediated double strand break (see S2 Table for sequence information). For generation of Tmem2-FLAG knock-in (Tmem2-FLAG^KI) mice, we used the Technique for Animal Knockout System by Electroporation (TAKE) [68 (link)]. Briefly, embryos were washed twice with Opti-MEM solution and aligned in the electrode gap filled with 50 μl of Cas9/gRNA(crRNA-tracrRNA complex)/ssODN (200/100/100 ng/μl) mixture. The intact embryos were subjected to electroporation using poring (225V) and transfer pulses (20V). After electroporation, embryos were returned to KSOM Mouse Embryo Media (Millipore Sigma) at 37°C. Genome edited 2-cell embryos were transferred to oviducts of pseudopregnant ICR female mice, and genomic DNA from newborn mice was analyzed by PCR (see S1 Table for primer sequences).