Optimized reporter gene assays based on a synthetic multifunctional promoter and a secreted luciferase.
Forskningsoutput: Tidskriftsbidrag › Artikel i vetenskaplig tidskrift
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
|Enheter & grupper|
Ämnesklassifikation (UKÄ) – OBLIGATORISK
|Status||Published - 2003|
|Peer review utförd||Ja|