Figure 4. Generation of the RyR2-V2475F mouse.

A, Strategy for generation of the transgenic mice by homologous recombination. A, (first line) The region of the WT RYR2 gene containing exons 47, 48, 49 and 51 (numbered boxes). (Second line) the RyR2-V2475F targeting vector containing the V2475F substitution (*), a translationally silent Mlu I restriction site (M), the loxP flanked pGK promoter/EM7 promoter-NEO-pGHpA cassette (NEO), the location of the EcoRI restriction sites (R1) used in genotyping and the MC1-HSV-Tk cassette (TK). (Third line) Homologous recombination between the endogenous RyR2 locus and the V2475F targeting vector resulted in a chromosome carrying the V2475F substitution and the loxP flanked Neo cassette. (Last line): the V2475F allele after Cre excision of the Neo cassette. B, The 5′ and 3′ Southern blot probes used in genotyping the NeoR ES cells. C, PCR confirmation of WT and heterozygous V2475F mice. The expected 1Kb band is seen in the heterozygotes. The targeting vector was used as positive control. D, H&E-stained hearts indicate no structural alterations in V2475F heterozygous mice compared to WT. E, heart rate, and F, fractional shortening, were not significantly different between WT and V2475F heterozygous mice. G, Non-Mendelian propagation of V2475F heterozygous mice yields ~20% WT, ~80% heterozygotes, and 0% homozygotes.