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Biotechniques. 2012 Aug;0(0):1-5. doi: 10.2144/000113909.

Attenuated protein expression vectors for use in siRNA rescue experiments.

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  • 1Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT, USA.

Abstract

Transient transfection of small interfering RNA (siRNA) provides a powerful approach for studying cellular protein functions, particularly when the target protein can be re-expressed from an exogenous siRNA-resistant construct in order to rescue the knockdown phenotype, confirm siRNA target specificity, and support mutational analyses. Rescue experiments often fail, however, when siRNA-resistant constructs are expressed at suboptimal levels. Here, we describe an ensemble of mammalian protein expression vectors with CMV promoters of differing strengths. Using CHMP2A rescue of HIV-1 budding, we show that these vectors can combine high-transfection efficiencies with tunable protein expression levels to optimize the rescue of cellular phenotypes induced by siRNA transfection.

PMID:
22877307
[PubMed - in process]
PMCID:
PMC3759224
Free PMC Article

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