Gene targeting strategy for B-hMC4R mice. The exon 1 including 3’UTR of mouse Mc4r gene that encode the full-length protein were replaced by human MC4R exon 1 and  3’UTR in B-hMC4R mice.

Strain specific analysis of MC4R mRNA expression in wild-type C57BL/6N mice and B-hMC4R mice by RT-PCR. Brain RNA were isolated from wild-type C57BL/6N mice (+/+) and homozygous B-hMC4R mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human MC4R primers. Mouse Mc4r mRNA was detectable only in wild-type C57BL/6N mice. Human MC4R mRNA was detectable only in homozygous B-hMC4R mice but not in wild-type mice.

Western blot analysis of MC4R protein expression in homozygous B-hMC4R mice. Various tissue lysates were collected from wild-type C57BL/6N mice (+/+) and homozygous B-hMC4R mice (H/H), and then analyzed by protein simple-western blot with anti-MC4R antibody. 3 μg total proteins were loaded for western blotting analysis. MC4R protein was detected in the brain，kidney，spleen，heart and liver of wild-type C57BL/6N mice and homozygous B-MC4R mice, as the antibody is cross-recognize both human and mouse MC4R.