Brad Swanson, Denise Garvin, Aileen Paguio, Tracy Worzella, Frank Fan and Keith Wood
Promega Corporation, 2800 Woods Hollow Road, Madison WI 53711
Bioluminescent reporter technologies are uniquely suited for high-throughput screening due to their inherent high sensitivity, wide dynamic range and low susceptibility to compound interference. Improvement of data quality and reduction of false positives caused by cytotoxic compounds can be achieved by incorporating a control reporter (e.g. a second luciferase) and ratiometric measurement. Introducing both reporters into the cell on the same plasmid backbone can result in aberrant expression from cross-interference between promoters and response elements. Therefore we have developed a strategy of generating dual-luciferase stable cell lines for GPCR assays using a two-plasmid system. Plasmid one features both a firefly luciferase gene regulated by the response element of interest (e.g., CRE or NFAT-RE) and a hygromycin-selectable marker. The second plasmid expresses the target GPCR (e.g., muscarinic receptor 3) and a Renilla luciferase-neomycin selectable marker fusion. Destabilized luciferase reporters provide significant benefit by reducing assay time, which limits the duration of cytotoxic compound interactions. This dual-luciferase assay system enables rapid screening and improves data quality.