Construction of CHOP-10 promoter–reporter and transient transfection. Genomic DNA was prepared from the 3T3-L1 preadipocytes by using Wizard Genomic DNA Purification Kit (Promega). CHOP-10 RO5185426 (starting ?977 upstream to +19 downstream) was amplified by PCR method and cloned into pGL-3 luciferase reporter construct. Two oligonucleotides used for PCR are: Forward primer: 5′-CGAACGCGTCA GTCCAGGGCAATCATGCGT-3′; Reverse primer: 5′-CGATCTAGATGTTAGGCTCAAGATAACTGACCT-3′.
In this PCR amplification, we used the primers tailed at the 5′ end with a MluI and BgIII restriction endonuclease recognition sequence. Transient transfection experiments in preadipocytes were performed by jetPEI (Polyplus Transfection) based on the manufacturer’s instructions. Equal amount p-RL plasmid was co-transfected as internal transfection efficiency control. Forty-eight hours later, both Firefly and Renilla luciferase activities were quantified using dual-luciferase reporter assay system (Promega) with Luminoskan TL plus Luminometer (Labsystem, Germany). YY1 expression plasmids were generously provided by Dr. Y. Shi (Department of Pathology, Harvard Medical School).
View User's Journal
The tooth sections were examined
 |
