title: Transposon-based RNAi delivery system for generating knockdown cell lines.
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.
authors.affiliation: Department of Pharmacology and Therapeutics, University of Florida, College of Medicine, Gainesville, FL 32610, USA.
authors.forename: Arnold D
authors.initials: AD
authors.lastname: Heggestad
authors.forename: Lucia
authors.initials: L
authors.lastname: Notterpek
authors.forename: Bradley S
authors.initials: BS
authors.lastname: Fletcher
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002469:Cell Separation; D002645:Chickens; D004251:DNA Transposable Elements; D015536:Down-Regulation; D005434:Flow Cytometry; D020868:Gene Silencing; D005821:Genetic Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D034904:Lamin Type A / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
chemical_list: D004251:DNA Transposable Elements; D034904:Lamin Type A; D008164:Luminescent Proteins; D034741:RNA, Small Interfering; C440308:lamin C; D049452:Green Fluorescent Proteins