Download
poster
228kb
pdf? |
|
Abstract for G-Protein Coupled Receptor Assay Using Dual-Luciferase
Stable Cell Lines
Aileen Paguio, Frank Fan and Keith Wood
Promega Corporation, 2800 Woods Hollow Road, Madison WI 53711
Bioluminescence-based technologies are great tools for
high-throughput drug screening because of their high sensitivity,
wide dynamic range and low interference by the compounds. To reduce
false positives generated by cytotoxicity of the screening
compounds, it is desirable to include a control reporter (e.g.,
another luciferase) in the same assay system. However, such a dual-
luciferase assay could not be configured simply by placing two
luciferase genes on one vector due to cross-interference between
promoters and response elements (unpublished results). Therefore, we
have developed a strategy of generating dual-luciferase stable cell
lines for a GPCR assay. It involves two plasmids: one expressing
firefly luciferase genes under the control of a response element
(e.g., CRE) and a hygromycin-selectable marker, and the other
expressing target GPCR (e.g., dopamine receptor D1) and a Renilla
luciferase-neomycin-selectable marker fusion. In addition,
destabilized luciferases were used to achieve more rapid signal
response and shorter assay time. This helps to further the reduction
of cytotoxicity caused by extensive contact time between the cells
and the screening compounds. This dual-luciferase assay allows more
rapid screening and improves data quality. |
