Mice were genotyped by polymerase chain reaction (PCR) on tail biopsy samples using the following primers: forward, 5′-AGGCAGCGCCTTTGCTGCGTC-3′; reverse, 5′-TCCTGGTCACAGAGGTCCTTA-3′. PCR mix contained 1.0 μl of DNA, 0.2 μl of each primer, 0.4 μl of DreamTaq DNA Polymerase (Thermo Fisher Scientific, EP0703), and PCR buffer [containing tris-HCl (pH 8.8), ammonium sulfate (Sigma-Aldrich), MgCl2 (Sigma-Aldrich), 2-mercaptoethanol (Merck), EDTA (pH 8.0) (Sigma-Aldrich), nucleoside triphosphates (2′-deoxyadenosine 5′-triphosphate, 2′-deoxycytidine 5′-triphosphate, 2′-deoxyguanosine 5′-triphosphate, and 3′-deoxythymidine 5′-triphosphate) (Promega), bovine serum albumin (BSA; Ambion—Life Technologies), and H2O to a final volume of 20 μl]. PCR conditions were as follows: 3 min of initial denaturation at 94°C, followed by 36 cycles of 30-s denaturation at 94°C, 30-s annealing at 60°C, and 60-s elongation at 72°C. Final elongation was performed for 7 min at 72°C. Enzymatic digestion was performed with StyI (10 U/μl) (New England Biolabs, R0500S). The PCR product was separated by 3% gel electrophoresis (+/+ 600 bp, +/− 600 bp + 350 bp + 250 bp, −/− 350 bp + 250 bp).

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