Stayner, C.K., Cunliffe, H.E., Ward, T.A. and Eccles, M.R.
Notes: MDCK and 293 cells were previously shown to express PAX2 endogenously. Transfections were carried out using FuGENE® 6 Transfection Reagent with 10% serum without antibiotics as follows. Cells (COS-7, NIH-3T3, 293 and MDCK cell lines; 5 × 105) were seeded into 60mm dishes and transfected at approximately 50–60% confluency, 16–18 hours later. Cells were harvested 24 hours after transfection. A total of 2.05μg of DNA was used in transfections to analyze the PAX2 promoter deletion series of constructs. These reactions contained 2μg of PAX2 promoter-reporter firefly luciferase plasmid (pGL2-Basic Vector) plus 0.05μg of internal control Renilla luciferase plasmid (pRL-TK Vector). For cotransfection experiments, a total of 4.05μg of DNA was used. The reactions contained 1μg of PAX2 promoter-reporter firefly luciferase plasmid and either 3μg of pBluescript DNA or 3μg of WT1 expression plasmid, plus 0.05μg of internal control Renilla luciferase plasmid (pRL Control Vector).
The DNA solutions were combined at a 1:3 ratio with FuGENE® 6 reagent (e.g. 4.05 μg of DNA to 12 μl of FuGENE® 6 reagent for transfection of each cell line. The cells were overlaid with a final 2ml of medium, for 24 hours prior to lysis. Cell lysates were assayed for luciferase production using a Dual-Luciferase® Assay System. Luminescence was measured with a luminometer. (4302)