Generation of a T cell–specific Crry mutant mouse. (A) Schematic diagram of the wild-type Crry gene locus, targeting vector, and recombinant Crry gene locus (from top to bottom). The targeting vector contained 2 loxP sites (arrowheads) flanking exon 5. The neomycin (neo) gene cassette was flanked by 2 FRT sites (diamonds). HindIII cut produces a 6.3-kb fragment in the wild-type allele (top) and a 8.3-kb fragment in the recombinant allele (bottom). DT indicates diphtheria toxin. (B) Southern blot data showing that the positive ES cell clone (flox-neo/+) produced 2 HindIII restriction fragments, whereas a representative nontargeted ES cell clone (+/+) produced only the 6.3-kb band. (C) PCR genotyping of WT and floxed Crry gene alleles (after neo excision) with the use of a pair of primers flanking exon 5. WT alleles produced a 970-bp product, whereas the floxed allele produced a 1100-bp product. Horizontal arrows denote the approximate locations of primers (Tx1, E5S2) used in PCR, and solid arrowheads indicate the loxP sites. (D) PCR analysis showing thymus-specific deletion of exon 5 of the Crry gene in CD4-Cre⁺ and Lck-Cre⁺ Crry^flox/flox mice. A 350-bp fragment indicative of mutated Crry gene allele was detected in Cre-positive mouse thymus (Thy) only. It was not present in the kidney (Kid) or in Cre-negative mouse thymus.