Optimized reporter gene assays based on a synthetic multifunctional promoter and a secreted luciferase

Anal Biochem. 2003 May 15;316(2):208-15. doi: 10.1016/s0003-2697(03)00082-4.


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

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Cell Line
  • Flow Cytometry
  • Genes, Reporter*
  • HeLa Cells / metabolism
  • Humans
  • Luciferases / analysis
  • Luciferases / biosynthesis
  • Luciferases / genetics*
  • Microscopy, Fluorescence
  • Molecular Sequence Data
  • Plasmids / genetics
  • Promoter Regions, Genetic*
  • Purinergic P2 Receptor Antagonists
  • Receptors, Purinergic P2 / metabolism
  • Time Factors
  • Transfection / methods


  • Purinergic P2 Receptor Antagonists
  • Receptors, Purinergic P2
  • Luciferases