title: 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.
authors.affiliation: Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT, USA.
authors.forename: Eiji
authors.initials: E
authors.lastname: Morita
authors.forename: Jun
authors.initials: J
authors.lastname: Arii
authors.forename: Devin
authors.initials: D
authors.lastname: Christensen
authors.forename: Jörg
authors.initials: J
authors.lastname: Votteler
authors.forename: Wesley I
authors.initials: WI
authors.lastname: Sundquist