Generation of p38α^Y323F mice. (A) A 9.3-kb genomic fragment of p38α locus (top) was cloned with the use of AvaI- and EcoRI-specific restriction sites and used to create the targeting vector (middle), into which a Y323F mutation and LoxP-flanked neomycin resistance gene were inserted. Insertion of the Y323F mutation created a new BclI restriction site in the knockin allele. Positions of the forward (Fwd) and reverse (Rev) primers used in long template PCR are shown (bottom). (B) Genotyping of p38α^Y323F mice by long template PCR, followed by digestion with BclI restriction enzyme generated a nondigested 4172-bp wild-type (WT/WT) band and 3546-bp knockin (Y323F/Y323F) band together with a small 626-bp band (not shown).