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Evaluation of a CRE-directed luciferase reporter gene assay as an alternative to measuring cAMP accumulation

George, Samantha E., Bungay, Peter J., Naylor, Louise H. (1997) Evaluation of a CRE-directed luciferase reporter gene assay as an alternative to measuring cAMP accumulation. Journal of Biomolecular Screening, 2 (4). pp. 235-240. ISSN 1087-0571. (doi:10.1177/108705719700200408) (The full text of this publication is not currently available from this repository. You may be able to access a copy if URLs are provided) (KAR id:18068)

The full text of this publication is not currently available from this repository. You may be able to access a copy if URLs are provided.
Official URL:
http://dx.doi.org/10.1177/108705719700200408

Abstract

A CHO reporter cell line expressing the firefly luciferase gene under the control of six cAMP response elements (CREs) was used to investigate the relationship between cAMP accumulation and cAMP dependent reporter gene expression and therefore, to assess the reporter gene system as an alternative functional assay. Timecourse experiments showed that cAMP accumulation preceded luciferase expression and that longer incubations (>2 h) were required to gain results with the reporter gene system. However, forskolin concentration dose-response studies revealed a 100-fold amplification of the signal measured by luciferase expression compared with direct cAMP accumulation, indicating that the reporter gene system afforded greater sensitivity. EC50 values determined for agonist activation of an inhibitory (5-HT1B-like) G-protein-coupled receptor (GPCR) were the same, and for a stimulatory GPCR (calcitonin Cia-like) were 10-fold lower, using the reporter gene system compared to cAMP accumulation assays, indicating the suitability of the reporter system for measuring the activity of receptors differentially coupled to the cAMP pathway. The phorbol ester, PMA, and the Ca2+ ionophore, A23187, were able to potentiate forskolin-stimulated luciferase expression but not cAMP accumulation, suggesting that the former could also be used to monitor cross-talk between different signal transduction pathways at the level of gene transcription.

Item Type: Article
DOI/Identification number: 10.1177/108705719700200408
Subjects: Q Science
Q Science > QR Microbiology
Divisions: Divisions > Division of Natural Sciences > Biosciences
Depositing User: M.A. Ziai
Date Deposited: 06 Mar 1914 09:32 UTC
Last Modified: 16 Nov 2021 09:56 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/18068 (The current URI for this page, for reference purposes)

University of Kent Author Information

Naylor, Louise H..

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