Current Chemical Genomics and Translational Medicine




(Discontinued)

ISSN: 2213-9885 ― Volume 12, 2018

HTS-Compatible β-Lactamase Transcriptional Reporter Gene Assay for Interrogating the Heat Shock Response Pathway



Michael K Hancock1, Menghang Xia2, Elizabeth S Frey1, Srilatha Sakamuru2, Kun Bi*, 1
1 Invitrogen Corporation, Discovery Assays and Services, 501 Charmany Drive, Madison, WI 53719, USA
2 NIH Chemical Genomics Center, National Institutes of Health, 9800 Medical Center Drive, Bethesda, MD 20893, USA

Abstract

Moderate environmental and physiological stressors are known to initiate protective heat shock response (HSR) leading to cell survival. HSR is largely mediated by the activation of heat shock factor (HSF), resulting in increased heat shock protein expression. Dysregulation of the HSR signaling has been associated with various diseases including cancer, inflammation and neurodegenerative disorders. Compounds that can modulate HSR have been pursued for the treatment of these diseases. To facilitate the discovery of HSR modulators, we developed a high-throughput amenable betalactamase transcriptional reporter gene assay for monitoring the function of HSF. HeLa cells were engineered to express the beta-lactamase reporter under the control of HSF response elements (HSE) present in the HSP70 gene promoter. The HSE-beta lactamase (HSE-bla) reporter gene assay was validated by using HSF-specific siRNAs and known small molecule modulators. Taking the advantage of fluorescence resonance energy transfer (FRET)-based cell permeable betalactamase substrate, this assay can be miniaturized into 1536-well format. Our results demonstrate that the assay is robust and can be applied to high-throughput screening (HTS) for modulators of HSR.



Article Information


Identifiers and Pagination:

Year: 2009
Volume: 3
First Page: 1
Last Page: 6
Publisher Id: CCGTM-3-1
DOI: 10.2174/1875397300903010001

Article History:

Received Date: 09/11/2008
Revision Received Date: 10/12/2008
Acceptance Date: 14/12/2008
Electronic publication date: 05/2/2009
Collection year: 2009

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© Hancock et al.; Licensee Bentham Open.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.5/), which permits unrestrictive use, distribution, and reproduction in any medium, provided the original work is properly cited.


* Address correspondence to this author at the Invitrogen Corporation, Discovery Assays and Services, 501 Charmany Drive, Madison, WI 53719, USA; E-mail: kun.bi@invitrogen.com


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