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. 2008 Dec 17;105(52):20970–20975. doi: 10.1073/pnas.0806962106

Fig. 3.

Fig. 3.

Generation of MC2R-AS-BMAL1 TG mice. (A) Schematic diagrams of the MC2R-AS-BMAL1 and MC2R-S-BMAL1 constructs (Upper). pA indicates the poly(A) signal from the bovine growth hormone gene. Effect of pMC2R-AS-BMAL1 or pMC2R-S-BMAL1 on endogenous BMAL1 protein levels in Y1 cells (Lower). Cotransfected GFP was used as an internal control (CTL). (B and C) Y1 cells were transfected with MC2R-S-BMAL1 or MC2R-AS-BMAL1. After serum shock, StAR (B) and Per2 (C) mRNA levels were examined. Data were normalized with TATA-binding protein (TBP) and are expressed as mean ± SE arbitrary units (A.U.), where the mean value of the MC2R-S-BMAL1 group at 0 h was set at 1 (n = 3). (D) Schematic diagrams of the MC2R-AS-BMAL1 transgene and the endogenous MC2R gene (Upper). Primer binding sites for genotyping are indicated by arrowheads. The copy number of MC2R-AS-BMAL1 TG was determined by competitive PCR genotyping (Lower). The arrowhead indicates PCR products derived from the MC2R-AS-BMAL1, and arrows indicate those from the endogenous MC2R gene. (E) Adrenal gland-specific mRNA expression of AS-BMAL1 TG as revealed by competitive RT-PCR. Arrowhead indicates PCR products derived from MC2R-AS-BMAL1, and arrows from endogenous Bmal1 mRNA. (F) Adrenal gland-specific knockdown of BMAL1 expression. BMAL1 protein levels at circadian time (CT)00 and CT12 were examined in the indicated tissues by immunoblotting.