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Attenuated protein expression vectors for use in siRNA rescue experiments.

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.

SUBMITTER: Morita E

PROVIDER: S-EPMC3759224 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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Attenuated protein expression vectors for use in siRNA rescue experiments.

Morita Eiji E Arii Jun J Christensen Devin D Votteler Jörg J Sundquist Wesley I WI

BioTechniques 20120801 0

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 exp ...[more]

PMID: 22877307

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