Figure 1. Validation of mouse MC3R replacement by human MC3R.

Quantitative real-time PCR for relative hypothalamic mRNA expression normalized by β-actin expression by the 2–ΔΔCt method (n=3/group) in 4-month-old female C57BL/6 mice (MC3R^+/+) or knock-in mice that were homozygous (MC3R^hWT/hWT) or heterozygous (MC3R^hWT/+) for the common alleles for the human MC3R or homozygous (MC3R^hDM/hDM) or heterozygous (MC3R^hDM/+) for the human MC3R sequence variants C17A+G241A. Expression was determined using human-specific MC3R primers (a,b) and mouse-specific MC3R primers (c,d). MC3R protein expression (e) was measured by western blotting (for MC3R^hWT/hWT n=5; for MC3R^hDM/hDM n=4) in homozygous mice and in the hypothalamic N8 murine cell line, which does not express MC3R mRNA. MC3R protein expression adjusted for GAPDH was quantified using Image J (f). Data are represented as mean±s.e.m. A different letter represents significant differences at P<0.05 compared with the other groups. Similar results were found for male mice (data not shown). Groups were compared by one-way analysis of variance followed by Bonferroni post-tests (a–d) and Student's t-test (two-tailed) (f).