Transposon-based RNAi delivery system for generating knockdown cell lines

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Transposon-based RNAi delivery system for generating knockdown cell lines
April 9, 2004
Arnold D. Heggestad, Lucia Notterpek and Bradley S. Fletcher
Biochemical and Biophysical Research Communications
Abstract
RNA interference is rapidly becoming a powerful tool for genetic analyses in mammalian systems. A potential drawback to transient small inhibitory RNA silencing is the short duration of downregulation it confers, usually only 24?72 h. Viral-based vector systems for the long-term delivery of RNA hairpins have been developed, yet they require expertise in viral production and transduction. Here we describe a simple plasmid-based system for the generation of long-term gene knockdown utilizing RNA interference combined with the gene delivery capabilities of the mammalian Tc1-like transposon Sleeping Beauty. Designated Maleficent, this system is shown to downregulate exogenous expression of GFP in a constitutively positive cell line. In addition, targeting of the endogenously expressed lamin A gene results in long-term silencing with significant reduction in protein levels (>95%). Maleficent therefore provides a relatively easy, efficient, and stable means of delivering RNAi hairpins to generate long-term gene-specific knockdown cell lines.
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