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Nucleic Acids Research Advance Access published online on April 10, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm103
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© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

RNA

Optimization and characterization of tRNA-shRNA expression constructs

Lisa J. Scherer1, Richard Frank2,3 and John J. Rossi1,3,*

1Department of Molecular Biology and 2Division of Hematology & Hematopoietic Cell Transplantation and 3Graduate School of Biological Sciences, Beckman Research Institute of the City of Hope, 1450 E. Duarte Road, Duarte, California 91010, USA

*To whom correspondence should be addressed. Tel: +1-626-301-8360; Fax: +1-626-301-8271; Email: jrossi{at}coh.org, jrossi{at}bricoh.edu

Received November 6, 2006. Revised January 12, 2007. Accepted February 6, 2007.

Expression of short hairpin RNAs via the use of PolIII-based transcription systems has proven to be an effective mechanism for triggering RNAi in mammalian cells. The most popular promoters for this purpose are the U6 and H1 promoters since they are easily manipulated for expression of shRNAs with defined start and stop signals. Multiplexing (the use of siRNAs against multiple targets) is one strategy that is being developed by a number of laboratories for the treatment of HIV infection since it increases the likelihood of suppressing the emergence of resistant virus in applications. In this context, the development of alternative small PolIII promoters other than U6 and H1 would be useful. We describe tRNALys3-shRNA chimeric expression cassettes which produce siRNAs with comparable efficacy and strand selectivity to U6-expressed shRNAs, and show that their activity is consistent with processing by endogenous 3' tRNAse. In addition, our observations suggest general guidelines for expressing effective tRNA-shRNAs with the potential for graded response, to minimize toxicities associated with competition for components of the endogenous RNAi pathway in cells.


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