The Mutation Effect of PRDM16
To determine the biochemical function of the substitution inPRDM16 , 3T3-L1 cells (preadipocyte cell line) that ectopically expressed cattle PRDM16 and PRDM16 MU (L779P mutation ofPRDM16 ) coding sequences were generated and induced to differentiate towards beige adipocytes (Additional file 1: Fig. S8 ). The overexpression efficiency was kept at equivalent levels (Fig. 3a ). After full differentiation, no differences in morphological characteristics between the PRDM16 and PRDM16 MU groups was observed (Additional file 1: Fig. S9 ). In addition, no significant differences in the mRNA and protein expression levels of PPARγ, a key adipogenesis-regulating gene (Fig. 3b and 3c ). Notably, the differentiation efficiency was lower for the control group (cells infected with an empty vector) than the two other groups, supporting the idea that a lack of PRDM16 significantly impeded the differentiation of brown adipocytes, and the overexpression of PRDM16 would significantly increase the number of brown adipocytes [17]. Despite the similar efficiency in differentiation between the two ectopic PRDM16 overexpressing groups, the mRNA expression levels of four BAT-selective genes (UCP1 , C/EBPβ , PGC1-α and CIDEΑ ) were significantly lower in the PRDM16 MU group than in the PRDM16 group (Fig. 3d ). Moreover, overexpression of PRDM16 MU largely increased UCP1 expression, which was far inferior to PRDM16 overexpression (Fig. 3d , 3e ). The mRNA levels of other BAT-selective genes (C/EBPβ ,PGC1-α and CIDEΑ ) were significantly lower in PRDM16 MU-overexpressing adipocytes than in PRDM16 -overexpressing adipocytes (Fig. 3d ). The above results indicate that L779P mutation significantly impaired the normal function of PRDM16 in the formation of brown adipocytes in southern cattle, which live in a warmer area relative to northern cattle.