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

Knut Kotarsky, Liselotte Antonsson, Christer Owman, Björn Olde

Forskningsoutput: TidskriftsbidragArtikel i vetenskaplig tidskriftPeer review

7 Citeringar (SciVal)

Sammanfattning

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
Originalspråkengelska
Sidor (från-till)208-215
TidskriftAnalytical Biochemistry
Volym316
Utgåva2
DOI
StatusPublished - 2003

Bibliografisk information

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)

Ämnesklassifikation (UKÄ)

  • Neurovetenskaper

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