Generation of SMSrD348E mice. A: Scheme of homologous recombination for generation of SMSrD348E mice. B: Genotyping PCR for wild-type (+/+), heterozygous (+/SMSrD348E), and homozygous (SMSrD348E) mice; characteristic DNA fragments 393 bp (wild-type), 493 bp (D348E), 580 bp (Neo; mice with neomycin selection cassette). C: The sequence exchange leading to the SMSrD348E point mutation additionally produced an MfeI restriction site. This was validated by MfeI digestion of a 433 bp PCR amplicon generated from the D348E allele. The fragments of 320 bp and 113 bp after MfeI treatment confirm the identity of the point mutation. D: Southern blot analysis of SMSrD348E mice. Expected fragment sizes: BclI restriction, wild-type 12.2 kbp; D348E 13.8 kbp.