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Nucleic Acids Research 2005 33(3):e27; doi:10.1093/nar/gni030
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Published online 16 February 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oupjournals.org

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Use of mRNA- and protein-destabilizing elements to develop a highly responsive reporter system

Dominic C. Voon1,2, Lily S. Subrata1,2, Svetlana Baltic1,2, Marco P. Leu1,2, Joanna M. Whiteway1,2,3, Agnes Wong1,2,3, Samuel A. Knight1,2, Frank T. Christiansen2,3 and John M. Daly1,2,*

1 GeneStream Pty Ltd 96 Chipping Road, City Beach, WA 6015, Australia 2 Department of Clinical Immunology and Biochemical Genetics, Royal Perth Hospital Wellington Street, Perth WA 6000, Australia 3 School of Surgery and Pathology, The University of Western Australia Crawley WA 6009, Australia

*To whom correspondence should be addressed. Tel/Fax: +61 8 92051149; Email: johndaly{at}genestream.com.au

Received October 26, 2004. Revised December 24, 2004. Accepted January 23, 2005.

Reporter assays are widely used in applications that require measurement of changes in gene expression over time (e.g. drug screening). With standard reporter vectors, the measurable effect of a treatment or compound (altered reporter activity) is substantially diluted and delayed, compared with its true effect (altered transcriptional activity). This problem is caused by the relatively long half-lives of both the reporter protein and its mRNA. As a result, the activities of compounds, ligands or treatments that have a relatively minor effect, or a substantial but transient effect, often remain undetected. To circumvent this problem, we introduced modular protein- and mRNA-destabilizing elements into a range of commonly used reporters. Our data show that both elements are required for maximal responses to both increases and decreases in transcriptional activity. The double-destabilized reporter vectors showed markedly improved performance in drug screening, kinetic assays and dose–response titrations.

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors


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