Gene targeting strategy for B-hVPAC1 mice. The exons 2~13 of mouse Vpac1 gene that encode the full-length protein exclude the signal peptide region were replaced by human VPAC1 exons 2~13 in B-hVPAC1 mice.

Strain specific analysis of VPAC1 mRNA expression in wild-type C57BL/6 mice and B-hVPAC1 mice by RT-PCR. Small intestine RNA were isolated from wild-type C57BL/6 mice (+/+) and homozygous B-hVPAC1 mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human VPAC1 primers. Mouse Vpac1 mRNA was detectable only in wild-type C57BL/6 mice. Human VPAC1 mRNA was detectable only in homozygous B-hVPAC1 mice.

Western blot analysis of VPAC1 protein expression in homozygous B-hVPAC1 mice. Various tissue lysates were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hVPAC1 mice (H/H), and then analyzed by western blot with anti-VPAC1 antibody. 30 μg total proteins were loaded for western blotting analysis. VPAC1 was detected in lung and small intestine of wild-type and homozygous mice, as anti-VPAC1 antibody was cross-reactive between human and mouse.