Current Chemical Genomics and Translational Medicine


ISSN: 2213-0886 ― Volume 8, 2014

Improved Dual-Luciferase Reporter Assays for Nuclear Receptors



Aileen Paguio, Pete Stecha, Keith V Wood , Frank Fan*
Promega Corporation, Madison, Wisconsin, USA

Abstract

Nuclear receptors play important roles in many cellular functions through control of gene transcription. It is also a large target class for drug discovery. Luciferase reporter assays are frequently used to study nuclear receptor function because of their wide dynamic range, low endogenous activity, and ease of use. Recent improvements of luciferase genes and vectors have further enhanced their utilities. Here we applied these improvements to two reporter formats for studying nuclear receptors. The first assay contains a Murine Mammary Tumor Virus promoter upstream of a destabilized luciferase. The presence of response elements for nuclear hormone receptor in this promoter allows the studies of endogenous and/or exogenous full length receptors. The second assay contains a ligand binding domain (LBD) of a nuclear receptor fused to the GAL4 DNA binding domain (DBD) on one vector and multiple Gal4 Upstream Activator Sequences (UAS) upstream of luciferase reporter on another vector. We showed that codon optimization of luciferase reporter genes increased expression levels in conjunction with the incorporation of protein destabilizing sequences into luciferase led to a larger assay dynamic range in both formats. The optimum number of UAS to generate the best response was determined. The expression vector for nuclear receptor LBD/GAL4 DBD fusion also constitutively expresses a Renilla luciferase-neoR fusion protein, which provides selection capability (G418 resistance, neoR) as well as an internal control (Renilla luciferase). This dual-luciferase format allowed detecting compound cytotoxicity or off-target change in expression during drug screening, therefore improved data quality. These luciferase reporter assays provided better research and drug discovery tools for studying the functions of full length nuclear receptors and ligand binding domains.

Keywords: Nuclear receptor, luciferase, reporter assays, HTS, drug discovery, cytotoxicity.


Identifiers and Pagination:

Year: 2010
Volume: 4
First Page: 43
Last Page: 49
Publisher Id: CCGTM-4-43
DOI: 10.2174/1875397301004010043

Article History:

Received Date: 19/2/2010
Revision Received Date: 24/3/2010
Acceptance Date: 25/3/2010
Electronic publication date: 26/05/2010
Collection year: 2010

Article Information:

© Paguio et al.; Licensee Bentham Open.

open-access license: This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.


* Address correspondence to this author at the Promega Corporation, Madison, Wisconsin, USA; Tel: 608-277-2531; Fax: 608-298-4818 E-mail: frank.fan@promega.com



Browse Contents


Webmaster Contact: urooj@benthamopen.com
Copyright © 2015 Bentham Open