Renilla luciferase plasmid dna

The reporter CD44P pGL3, a gift from Dr. Robert Weinberg (Addgene plasmid # 19122), contains 2021 bp of the human CD44 promoter/enhancer upstream of the translation initiation site. CD44P pGL3 reporter plasmid was co-transfected with 0.2 μg Renilla Luciferase plasmid DNA (Promega, Mannheim, Germany) as a control for transfection efficiency. After 48 hr incubation, transfected cells were lysed using the Dual-Luciferase Reporter Assay System (Promega). Promoter-driven luciferase activity was measured on the Llumat LB 9507 machine (EG&G Berthold Co.) and normalized to the Renilla Luciferase activity. Each experiment was carried out in triplicates and repeated three times.

1 × 10⁵ A549 or E2-A549 cells were seeded in each well of 6-well plates for 16 h, and adenoviruses, adeno-GFP and adeno-GFP-Sp1, were added as described. Two days after adenovirus infection, CD44P/pGL3 promoter-reporter construct (Addgene, Plasmid #19,122) or reporter plasmids containing aldehyde dehydrogenase 1 (ALDH1) and (sex determining region Y)-box 2 (Sox2) promoter regions were transfected into cells. Reporter plasmid DNA (1 μg) was co-transfected with Renilla Luciferase plasmid DNA (1 μg) (Promega, Mannheim, Germany) as a control for transfection efficiency. After 24 h incubation, transfected cells were lysed using the Dual-Luciferase Reporter Assay System (Promega). Promoter-driven luciferase activity was measured by luminometer (LB9506; Berthold Technologies, Bad Wildbad, Germany) and normalized to the Renilla Luciferase activity. Each experiment was carried out in triplicates and repeated three times. For construction, genomic DNA of A549 cells were prepared. The promoters of Sox2 and ALDH1 were produced by PCR using primers (Additional file 7: Table S1). After amplification and purification, the DNA fragments were ligated to pGL2 vector using restriction enzymes, KpnI/ NheI for pGL2-ALDH1 and pGL2-Sox2 (New England Biolabs, Ipswich, MA).