Brad Swanson, Aileen Paguio, Denise Garvin, Pete Stecha, Frank Fan and Keith Wood
Promega Corporation, 2800 Woods Hollow Rd., Madison, WI 53711
Luciferase reporter assays are critical tools for the cancer researcher because they are well suited for quantifying important cellular processes including proliferation, differentiation, and death. This is due to their wide dynamic range, low endogenous activity, and ease of use. In particular, luciferase reporter assays have been crucial tools in elucidating transcriptional regulatory mechanisms that control many aspects of cell physiology and function. Recent improvements within the pGL4 Family of Luciferase Reporter Vectors have further improved the utility of these vectors to meet the demands researchers in both academic and pharmaceutical settings. Our efforts to improve the luciferase reporter gene have enhanced both the dynamic range and the temporal response of luciferase reporter assays. We codon optimized the luc2 and hRluc reporter gene open reading frames to facilitate more efficient expression leading to higher levels of luciferase protein.
Comparison of several different cell lines transfected with luc2 versus luc+ reporter vectors validates that luc2 transfected cells have significantly higher light output than luc+ transfected cells. To enable more rapid assay kinetics and reducing assay time, we incorporated protein destabilization domains into the luciferase protein sequence. The combination of these improvements results in a reporter gene that allows high luciferase protein expression that closely mirrors the transcriptional status of the luciferase gene. In addition the pGL4 Vector backbone has been optimized to significantly reduce the number of potential consensus transcription factor binding sites and lower uninduced levels of the luciferase reporter. These improvements have been incorporated into our GloResponse™ Reporter Cell Lines to generate high quality stable reporter cell lines that facilitate study of the cAMP and NFAT signaling pathways in HEK293 cells.