Heggestad Arnold D, Notterpek Lucia, Fletcher Bradley S
Department of Pharmacology and Therapeutics, University of Florida, College of Medicine, Gainesville, FL 32610, USA.
Biochem Biophys Res Commun. 2004 Apr 9;316(3):643-50. doi: 10.1016/j.bbrc.2004.02.090.
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.
RNA干扰正迅速成为哺乳动物系统中进行遗传分析的强大工具。瞬时小干扰RNA沉默的一个潜在缺点是其下调作用持续时间较短，通常仅为24 - 72小时。已开发出用于长期递送RNA发夹的基于病毒的载体系统，但它们需要病毒生产和转导方面的专业知识。在此，我们描述了一种简单的基于质粒的系统，该系统利用RNA干扰结合哺乳动物类Tc1转座子睡美人的基因递送能力来实现长期基因敲低。该系统命名为“Maleficent”，在组成型阳性细胞系中可下调绿色荧光蛋白（GFP）的外源表达。此外，靶向内源性表达的核纤层蛋白A基因可导致长期沉默，蛋白水平显著降低（> 95%）。因此，“Maleficent”提供了一种相对简便、高效且稳定的方法来递送RNAi发夹，以产生长期基因特异性敲低的细胞系。
