Pgl 3 basic luciferase report vector

The 5'-regulatory region of Hsd3b was cloned from chicken genomic DNA by long PCR (S1 Table). The amplified fragment spans the region between −3544 to +7 bp of the chicken Hsd3b gene, where +1 is the transcription initiation site. PCR products were cloned into the pGL-3 basic luciferase report vector (Promega) using the NheI and XhoI restriction sites. This construct was named −3544 HSD3Bpr-luc. Deletion constructs −2220 HSD3Bpr-luc, −1457 HSD3Bpr-luc, −665 HSD3Bpr-luc, and −215 HSD3Bpr-luc were generated by PCR, with −3544 HSD3Bpr-luc as the template, and confirmed by bidirectional sequencing. Granulosa cells were plated on 24-well plates for transient transfection experiments. The cells were transfected with the luciferase reporter plasmids (800 ng/well) using Lipofectamine 2000 (Invitrogen). Transfection efficiency was normalized by cotransfection of 30 ng of the Renilla luciferase reporter plasmid (pRL-CMV vector; Promega). At 24 h after transfection, recombinant TGF-β1 was added. At 48h after transfection, the cells were lysed and assayed for promoter activity using the dual-luciferase reporter assay system. The enzymatic activity of luciferase was measured with a luminometer (Modulus TM, Turner Biosystems).