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

In English

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

Publiceringsår

2003

Språk

Engelska

Sidor

208-215

Publikation/Tidskrift/Serie

Analytical Biochemistry

Volym

316

Issue

2

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