Optimized reporter gene assays based on a synthetic multifunctional promoter and a secreted luciferase.
Efficient screening for ligands of seven-transmembrane, G-protein-coupled receptors, whether transfected or endogenously expressed, often involves cell-based reporter assays. Here we describe the development of reporter gene assays in HeLa cells. The reporter construct includes a synthetic multifunctional promoter with several different response motifs (NF-kappaB, STAT, and AP-1) and hence efficiently funnels several signaling pathways. The assay, performed with the resulting reporter cell line HFF11, has an exceptional high Z-factor and a large signal-to-background ratio. To facilitate cell handling during screening, we introduced a secreted Renilla luciferase as a reporter enzyme. HR36 reporter cells, equipped with the construct, were added to ligands present in a multiwell plate and after addition of coelenterazine they produced a luminescence readout. This procedure economizes cell handling and at the same time increases assay quality and sensitivity
Kotarsky
Knut
K
Division of Molecular Neurobiology, Wallenberg Neuroscience Center, Lund University, Sweden. Knut.Kotarsky@mphy.lu.se
Antonsson
Liselotte
L
Owman
Christer
C
Olde
Björn
B
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D005434:Flow Cytometry; D017930:Genes, Reporter; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2 / Q000378:metabolism; D013997:Time Factors; D014162:Transfection / Q000379:methods
D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D008156:Luciferases