Optimized reporter gene assays based on a synthetic multifunctional promoter and a secreted luciferase.
Author
Summary, in English
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
Department/s
- Mucosal Immunology
- Drug Target Discovery
- Cardiology
Publishing year
2003
Language
English
Pages
208-215
Publication/Series
Analytical Biochemistry
Volume
316
Issue
2
Links
Document type
Journal article
Publisher
Elsevier
Topic
- Neurosciences
Keywords
- Luciferase
- Cell surface receptors
- Reporter genes
- Assay system
- Preclinical drug evaluation
- Biological assay
Status
Published
Research group
- Mucosal Immunology
- Drug Target Discovery
ISBN/ISSN/Other
- ISSN: 1096-0309