Generation of PAR-2–deficient mice. (a) Structure of the PAR-2 targeting vector (top), the wild-type PAR-2 allele (middle), and targeted allele (bottom) resulting from replacement recombination at the dashed crosses. The null allele was created by substitution of a reporter/selection cassette (dark and light blue boxes) for most of the exon 2 sequence (filled gray box). Non-exon–containing chromosomal and cloned genomic DNA sequence is shown by a thick black line and pBluescript plasmid sequence by a thin black line. Restriction enzyme sites ClaI (C), HindIII (H), KpnI (K), NotI (N), and SalI (S) are indicated by small arrows, and the sizes of relevant restriction fragments are shown by dotted lines. The targeted allele was identified by KpnI digestion and hybridization with the 5′ and 3′ flanking probe fragments (gray rectangles). (b) Southern blot analysis of KpnI-digested genomic DNA prepared from pups from an intercross mating of PAR-2 mice heterozygous (+/–) for the null allele, demonstrating the presence of viable mice homozygous for the null allele (–/–). (c) RT-PCR for β-actin (548 bp) and PAR-2 (380 bp) mRNA expression in articular (lane 1 and lane 3, respectively) and intestinal (positive control; lane 2 and lane 4, respectively) tissue from PAR-2^+/+ mice. (d) High-power view (oil immersion, differential interference contrast) of a synovial arteriole from a PAR-2^–/– mouse. (e) Synovial tissue from a PAR-2^+/+ mouse. β-galactosidase activity absent in endothelial cells (arrow 1) from PAR-2^+/+ mice compared with PAR-2^–/– tissue. Erythrocytes (arrowheads) are clearly visible in the lumen as are surrounding smooth muscle cells (arrow 2). Scale bar: 10 μm.