Breeding and identification of Metrnlloxp/loxp Villin-cre mice (Metrnl−/−). (A) Breeding strategy for Metrnl conditional knockout mice. (B) Genotyping of Metrnl-floxed mice. The PCR product of 243 bp is amplified from the conditional floxed (loxp-flanked) Metrnl allele, and the PCR product of 131 bp is amplified from the wild-type Metrnl allele. (C) Genotyping of Cre-positive mice, including Metrnlloxp/wt Villin-cre and Metrnlloxp/loxp Villin-cre mice. (D) Metrnl mRNA expression in the tissues of Metrnl−/− and wild-type mice. WAT, white adipose tissue. BAT, brown adipose tissue. n=6. The data are expressed as the mean±SEM. **P<0.01. (E) Immunohistochemical staining for Metrnl in the colons of Metrnl−/− and wild-type mice. Bar, 100 μm.