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PubMed · 15033448

Transposon-based RNAi delivery system for generating knockdown cell lines.

Arnold D Heggestad·Lucia Notterpek·Bradley S Fletcher
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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 24-72h. 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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AnimalsAnimals, Genetically ModifiedBase SequenceBlotting, WesternCell LineCell SeparationChickensDNA Transposable ElementsDown-RegulationFlow CytometryGene SilencingGenetic TechniquesGenetic VectorsGreen Fluorescent ProteinsHeLa CellsHumansLamin Type ALuminescent ProteinsMicroscopy, FluorescenceMolecular Sequence DataPlasmidsRNA InterferenceRNA, Small InterferingTime FactorsTransfectionJournal ArticleResearch Support, Non-U.S. Gov't

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Arnold D Heggestad, Lucia Notterpek, Bradley S Fletcher. 2004-04-09. Transposon-based RNAi delivery system for generating knockdown cell lines.. https://doi.org/10.1016/j.bbrc.2004.02.090

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