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通过重组杆状病毒载体介导的人原代细胞中的RNA干扰。

RNA interference mediated in human primary cells via recombinant baculoviral vectors.

作者信息

Nicholson Linda J, Philippe Marie, Paine Alan J, Mann Derek A, Dolphin Colin T

机构信息

Department of Oncology, Guy's, King's and St Thomas' School of Medicine, The Rayne Institute, St Thomas' Hospital, London SE1 7EH, UK.

出版信息

Mol Ther. 2005 Apr;11(4):638-44. doi: 10.1016/j.ymthe.2004.12.010.

Abstract

The success of RNA interference (RNAi) in mammalian cells, mediated by siRNAs or shRNA-generating plasmids, is dependent, to an extent, upon transfection efficiency. This is a particular problem with primary cells, which are often difficult to transfect using cationic lipid vehicles. Effective RNAi in primary cells is thus best achieved with viral vectors, and retro-, adeno-, and lentivirus RNAi systems have been described. However, the use of such human viral vectors is inherently problematic, e.g., Class 2 status and requirement of secondary helper functions. Although insect cells are their natural host, baculoviruses also transduce a range of vertebrate cell lines and primary cells with high efficiency. The inability of baculoviral vectors to replicate in mammalian cells, their Class 1 status, and the simplicity of their construction make baculovirus an attractive alternative gene delivery vector. We have developed a baculoviral-based RNAi system designed to express shRNAs and GFP from U6 and CMV promoters, respectively. Transduction of Saos2, HepG2, Huh7, and primary human hepatic stellate cells with a baculoviral construct expressing shRNAs targeting lamin A/C resulted in effective knockdown of the corresponding mRNA and protein. Development of this baculoviral-based system provides an additional shRNA delivery option for RNAi-based investigations in mammalian cells.

摘要

由小干扰RNA(siRNA)或产生短发夹RNA(shRNA)的质粒介导的RNA干扰(RNAi)在哺乳动物细胞中的成功,在一定程度上取决于转染效率。对于原代细胞来说,这是一个特别的问题,因为使用阳离子脂质载体转染原代细胞通常很困难。因此,在原代细胞中实现有效的RNAi最好使用病毒载体,并且已经描述了逆转录病毒、腺病毒和慢病毒RNAi系统。然而,使用这类人类病毒载体本身存在问题,例如2类病毒的性质以及对辅助功能的需求。尽管昆虫细胞是杆状病毒的天然宿主,但杆状病毒也能高效转导一系列脊椎动物细胞系和原代细胞。杆状病毒载体无法在哺乳动物细胞中复制,它们属于1类病毒,并且构建简单,这使得杆状病毒成为一种有吸引力的替代基因递送载体。我们开发了一种基于杆状病毒的RNAi系统,该系统设计为分别从U6和巨细胞病毒(CMV)启动子表达shRNA和绿色荧光蛋白(GFP)。用表达靶向核纤层蛋白A/C的shRNA的杆状病毒构建体转导骨肉瘤细胞系(Saos2)、肝癌细胞系(HepG2)、人肝癌细胞系(Huh7)和原代人肝星状细胞,导致相应的mRNA和蛋白被有效敲低。这种基于杆状病毒的系统的开发为在哺乳动物细胞中基于RNAi的研究提供了另一种shRNA递送选择。

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