Optimized reporter gene assays based on a synthetic multifunctional promoter and a secreted luciferase.
(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)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/113181
- author
- Kotarsky, Knut LU ; Antonsson, Liselotte LU ; Owman, Christer LU and Olde, Björn LU
- organization
- publishing date
- 2003
- 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
- 2022-01-26 17:06:32
@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}}, keywords = {{Luciferase; Cell surface receptors; Reporter genes; Assay system; Preclinical drug evaluation; Biological assay}}, 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}}, }