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Plasmid pR1C-4.1CAT that contains 4.1 kb 5′-flanking sequence of mouse Ren-1c gene was provided by K.W. Gross (Roswell Park Cancer Institute, Buffalo, New York, USA). To generate pGL-117bp reporter plasmid, the 123-bp renin minimal promoter fragment (+6 to –117) was released from pR1C-4.1CAT with XbaI and BamHI and inserted into the HindIII site of pGL3-basic vector (Promega Corp.). To generate pGL-4.1kb reporter plasmid, the BamHI fragment (–4.1 kb to –118 bp) from pR1C-4.1CAT was inserted into the BglII site of pGL-117bp. To analyze the activity of renin gene promoter, As4.1-hVDR cells were transfected with the reporter plasmids by electroporation according to the method of Shi et al using a Gene Pulser (Bio-Rad Laboratories Inc.). pCMV–β-galactosidase (β-gal) plasmid was cotransfected as an internal control. pGL3-control plasmid (Promega Corp.) was used as the positive control. The transfected cells were treated with ethanol or 10–8 M 1,25(OH)2D3 in Opti-MEM medium (Invitrogen Life Technologies) containing 2% charcoal-treated FBS 4 hours after electroporation, and luciferase activity was determined at 48 hours after initial transfection using the Luciferase Assay System (Promega Corp.). Luciferase activity was normalized to β-gal activity obtained from the same electroporation, and presented as fold induction based on the basal activity of pGL3-basic empty vector determined in the same experiment. |
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