Fig. 1.

Generation of mice with RyR2-L3591D mutation. A: schematic representation of the mouse Ryr2 gene and targeting construct. S represents SphI restriction site. Neomycin-resistant gene (neo), Cre recombinase gene driven by testis-specific angiotensin-converting enzyme promoter (tACE-cre), and thymidine kinase gene (TK) are indicated. Arrows and x indicate the position of primers for screening and a mutation site, respectively. B: Southern blot of genomic DNA. After restriction enzyme digestion of genomic DNA with SphI, 5′ and 3′ probes identify 13.5-kb and 11-kb fragments in the targeted allele, respectively. Both probes hybridize with 20.5-kb fragments in the wild-type (WT) allele. C: DNA sequence analysis of RyR2-L3591D. cDNA encoding the CaM binding site of RyR2 was amplified from total RNA from homozygous (hybrid genetic background) mouse cardiac muscle and sequenced. The RyR2-L3591D mutation was confirmed. A HinfI site created by the L3591D mutation (GACTC) was used for screening the mutant allele.