Gene targeting strategy for B-hPROK2 mice. The exons 1~4 of mouse Prok2 gene that encode the full-length protein were replaced by human PROK2 exons 1~4 in B-hPROK2 mice.

Strain specific analysis of PROK2 mRNA expression in wild-type C57BL/6 mice and B-hPROK2 mice by RT-PCR. Testis RNA were isolated from wildtype C57BL/6 mice (+/+) and homozygous B-hPROK2 mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human PROK2 primers. Mouse PROK2 mRNA was only detectable in wild-type C57BL/6 mice. Human PROK2 mRNA was only detectable in homozygous B-hPROK2 mice but not in wild-type mice.

Western blot analysis of PROK2 protein expression in homozygous B-hPROK2 mice. Various tissue lysates were collected from wild-type C57BL/6N (+/+) mice and homozygous B-hPROK2 mice (H/H), and then analyzed by western blot with anti-PROK2 antibody. 40 μg total proteins were loaded for western blotting analysis. PROK2 was detected in hippocampus, cerebellum, bladder and spleen.