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

Kotarsky, Knut LU ; Antonsson, Liselotte LU ; Owman, Christer LU and Olde, Björn LU (2003) In Analytical Biochemistry 316(2). p.208-215
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... (More)
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 (Less)
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author
; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Luciferase, Cell surface receptors, Reporter genes, Assay system, Preclinical drug evaluation, Biological assay
in
Analytical Biochemistry
volume
316
issue
2
pages
208 - 215
publisher
Elsevier
external identifiers
  • pmid:12711342
  • wos:000182606500009
  • scopus:0037447842
ISSN
1096-0309
DOI
10.1016/S0003-2697(03)00082-4
language
English
LU publication?
yes
additional info
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)
id
09259c9b-0101-4fd6-9b75-dacb07d3ce01 (old id 113181)
alternative location
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12711342&dopt=Abstract
date added to LUP
2016-04-01 11:42:49
date last changed
2021-10-03 05:20:34
@article{09259c9b-0101-4fd6-9b75-dacb07d3ce01,
  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},
  author       = {Kotarsky, Knut and Antonsson, Liselotte and Owman, Christer and Olde, Björn},
  issn         = {1096-0309},
  language     = {eng},
  number       = {2},
  pages        = {208--215},
  publisher    = {Elsevier},
  series       = {Analytical Biochemistry},
  title        = {Optimized reporter gene assays based on a synthetic multifunctional promoter and a secreted luciferase.},
  url          = {http://dx.doi.org/10.1016/S0003-2697(03)00082-4},
  doi          = {10.1016/S0003-2697(03)00082-4},
  volume       = {316},
  year         = {2003},
}