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Abstract for Investigation of Muscarinic 3 Receptor (M3R)
Activation of the NFAT and cAMP Signaling Pathways Using Dual-Luciferase
Stable Cell Lines
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. |
