G-Protein Coupled Receptor Assay Using Dual-Luciferase Stable Cell Lines Scientific Poster
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simply by placing two luciferase genes on one vector due to cross-interference between promoters and response elements (unpublished results).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.
- Part# PS035
- Printed in USA.