Optimized reporter gene assays based on a synthetic multifunctional promoter and a secreted luciferase.
Författare
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
Avdelning/ar
- Slemhinnans immunologi
- Drug Target Discovery
- Kardiologi
Publiceringsår
2003
Språk
Engelska
Sidor
208-215
Publikation/Tidskrift/Serie
Analytical Biochemistry
Volym
316
Issue
2
Länkar
Dokumenttyp
Artikel i tidskrift
Förlag
Elsevier
Ämne
- Neurosciences
Nyckelord
- Luciferase
- Cell surface receptors
- Reporter genes
- Assay system
- Preclinical drug evaluation
- Biological assay
Status
Published
Forskningsgrupp
- Mucosal Immunology
- Drug Target Discovery
ISBN/ISSN/Övrigt
- ISSN: 1096-0309