Fig. 1. Generation of R163C mice. ( A ) Construct design and recombination schematic. ( 1 ) Targeting construct to introduce R163C mutation into ryanodine receptor type 1 (RyR1) exon 6. ( 2 ) Wild-type (WT) RyR1 locus. ( 3 ) The same locus after recombination. ( 4 ) The same locus after breeding R163C mice to Tnap-Cre (tissue-nonspecific alkaline phosphatase promoter driven bacterial Cre recombinase gene) mice to remove neomycin/cytosine deaminase ( neo/CD ) cassettes. ( B ) Primer designation for reverse-transcriptase polymerase chain reaction to confirm transcription of WT and the R163C knock-in (KI) allele. ( C ) Result of reverse-transcriptase polymerase chain reaction for WT ( lanes 1 and 2 ), R163C heterozygous ( lanes 3 and 4 ), and R163C homozygous ( lanes 5 and 6 ) mice. Primer pair WT-s/WT-as was used for lanes 1 , 3 and 5 . Primer pair KI-s/KI-as was used for lanes 2 , 4 , and 6 . ( D ) Partial sequencing result of all four bands from reverse-transcriptase polymerase chain reaction showing the transcribed WT and KI allele. Bands in lanes 1 and 3 (in B ) are identical and partially shown in the upper panel . Bands in lanes 4 and 6 (in B ) are identical and partially shown in the lower panel . The exact locations of primer WT-s and KI-s are labeled. The mutated four nucleotides in the 160–base pair (bp) product are underlined . ( E ) Western blot analysis of RyR1 expression in age-matched R163C homozygous ( lane 1 ), R163C heterozygous ( lane 2 ), and WT ( lane 3 ) mouse embryos. Polyacrylamide gel, 7%, was used. Protein, 25 μg, was loaded in each lane. cDNA = complementary DNA; loxP = locus of crossover in P1.