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A chimeric reporter gene allowing for clone selection and high-throughput screening of reporter cell lines expressing G-protein-coupled receptors

Kotarsky, Knut LU ; Owman, Christer LU and Olde, Björn (2001) In Analytical Biochemistry 288(2). p.209-215
Abstract
Efficient screening of ligands interacting with G-protein-coupled receptors is central for modern drug development. Here, we describe an optimized reporter vector primarily intended for use in reporter cell lines expressing such receptors. The construct consists of a synthetic enhancer containing 9x TRE (12-O-tetradecanoylphorbol-13-acetate-responsive elements) fused to a minimal CMV (cytomegalovirus) promoter. Activation of the promoter construct leads to the expression of a chimeric reporter protein based on the genes for enhanced green fluorescent protein and Photinus luciferase. The chimeric protein allows for both clonal selection by fluorescence, which facilitates the selection of optimal reporter cell lines and high-throughput... (More)
Efficient screening of ligands interacting with G-protein-coupled receptors is central for modern drug development. Here, we describe an optimized reporter vector primarily intended for use in reporter cell lines expressing such receptors. The construct consists of a synthetic enhancer containing 9x TRE (12-O-tetradecanoylphorbol-13-acetate-responsive elements) fused to a minimal CMV (cytomegalovirus) promoter. Activation of the promoter construct leads to the expression of a chimeric reporter protein based on the genes for enhanced green fluorescent protein and Photinus luciferase. The chimeric protein allows for both clonal selection by fluorescence, which facilitates the selection of optimal reporter cell lines and high-throughput screening by luminescens. In designing the vector, increasing numbers of TRE motifs were tested in front of two different minimal promoters. The reporter gene was more strongly inducible with increasing numbers of TRE motifs. The constructs were tested in two cell lines, CHO and HeLa. The latter regulated reporter gene activity stronger in response to PMA (phorbol 12-myristate 13-acetate) stimulation and were used to construct HF1 reporter cell lines. Model experiments were carried out on these reporter cells transfected with the human BLTR, human CCR5, or the rat alpha(1b) receptor. After maximal agonist stimulation reporter gene activity was increased 200-, 15-, and 50-fold, respectively. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
GPCR, GFP, luciferase, reporter gene assay, activating protein 1
in
Analytical Biochemistry
volume
288
issue
2
pages
209 - 215
publisher
Elsevier
external identifiers
  • pmid:11152592
  • scopus:0035863914
ISSN
1096-0309
DOI
10.1006/abio.2000.4898
language
English
LU publication?
yes
id
96b299bf-b07f-4750-9f69-a409402b255a (old id 1119923)
date added to LUP
2008-07-01 14:53:10
date last changed
2018-05-29 11:56:00
@article{96b299bf-b07f-4750-9f69-a409402b255a,
  abstract     = {Efficient screening of ligands interacting with G-protein-coupled receptors is central for modern drug development. Here, we describe an optimized reporter vector primarily intended for use in reporter cell lines expressing such receptors. The construct consists of a synthetic enhancer containing 9x TRE (12-O-tetradecanoylphorbol-13-acetate-responsive elements) fused to a minimal CMV (cytomegalovirus) promoter. Activation of the promoter construct leads to the expression of a chimeric reporter protein based on the genes for enhanced green fluorescent protein and Photinus luciferase. The chimeric protein allows for both clonal selection by fluorescence, which facilitates the selection of optimal reporter cell lines and high-throughput screening by luminescens. In designing the vector, increasing numbers of TRE motifs were tested in front of two different minimal promoters. The reporter gene was more strongly inducible with increasing numbers of TRE motifs. The constructs were tested in two cell lines, CHO and HeLa. The latter regulated reporter gene activity stronger in response to PMA (phorbol 12-myristate 13-acetate) stimulation and were used to construct HF1 reporter cell lines. Model experiments were carried out on these reporter cells transfected with the human BLTR, human CCR5, or the rat alpha(1b) receptor. After maximal agonist stimulation reporter gene activity was increased 200-, 15-, and 50-fold, respectively.},
  author       = {Kotarsky, Knut and Owman, Christer and Olde, Björn},
  issn         = {1096-0309},
  keyword      = {GPCR,GFP,luciferase,reporter gene assay,activating protein 1},
  language     = {eng},
  number       = {2},
  pages        = {209--215},
  publisher    = {Elsevier},
  series       = {Analytical Biochemistry},
  title        = {A chimeric reporter gene allowing for clone selection and high-throughput screening of reporter cell lines expressing G-protein-coupled receptors},
  url          = {http://dx.doi.org/10.1006/abio.2000.4898},
  volume       = {288},
  year         = {2001},
}