(A) Targeted engineering of the S-to-A mutation of S2814 in the mouse RyR2 locus. A genomic clone containing exons 56 and 57 of the mouse Ryr2 gene was isolated from a 129/SvJ λKO-1 library and cloned into a pDTA4B vector using homologous recombination. (B) The S2814A mutation was introduced into exon 56 of RyR2 along with a new ClaI site. A cassette containing a loxP-flanked NeoR gene expressed from the phosphoglycerate kinase promoter (PGK-NeoR) was cloned into intron 56 to obtain the final targeting vector. (C) Targeting vector was linearized with Pme1 and electroporated into AB2.2 129Sv/J ES cells. Homologous-targeted integrands were identified using Southern blot analysis. (D) Following germline transmission and crossing with Meox2-Cre mice, final allele without Neo cassette was obtained. (E) Southern blot analysis reveals homologous-targeted mutant allele (*). (F) Heterozygous mice (HET) are bred to obtain homozygous knockin mice (HOM), identified by PCR genotyping. (G) Autoradiograph showing that CaMKII phosphorylation in the presence of γ-ATP³² was greatly inhibited in RyR2 from S2814A mouse heart. Immunoprecipitated RyR2 (input) is shown (top). Half the sample was CaMKII phosphorylated in the presence or absence of KN-93 (bottom). Noncontiguous lanes are separated by white lines. (H) Electrophysiology studies revealed that AF could be induced by cardiac pacing in 44% of WT mice after carbachol injection (50 ng/g body weight i.p.) compared with 7.7% of Ryr2^S2814A mice (*P < 0.05).