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

Knut Kotarsky, Liselotte Antonsson, Christer Owman, Björn Olde

Research output: Contribution to journalArticlepeer-review

7 Citations (SciVal)

Abstract

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
Original languageEnglish
Pages (from-to)208-215
JournalAnalytical Biochemistry
Volume316
Issue number2
DOIs
Publication statusPublished - 2003

Bibliographical note

The information about affiliations in this record was updated in December 2015.
The record was previously connected to the following departments: Molecular Neurobiology (0131000140), Drug Target Discovery (013212045), Cardiology (013230026), Mucosal Immunology (013212072)

Subject classification (UKÄ)

  • Neurosciences

Keywords

  • Luciferase
  • Cell surface receptors
  • Reporter genes
  • Assay system
  • Preclinical drug evaluation
  • Biological assay

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