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Abstract for Multiplexing Using Dual-Luciferase Reporters for
GPCR Assays
Frank Fan, Denise Garvin, Aileen Paguio, Brad Swanson, Tracy
Worzella 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 inherently high sensitivity,
wide dynamic range and low susceptibility to compound interference.
Improvement of data quality and reduction of false hits can be
achieved by incorporating a control reporter (e.g., a second
luciferase). 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) 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.
Multiplexing using this dual-luciferase assay system enables rapid
screening and improves data quality. |
