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RNA interference mediated in human primary cells via recombinant baculoviral vectors

Lookup NU author(s): Professor Derek Mann

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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.


Publication metadata

Author(s): Nicholson LJ, Philippe M, Paine AJ, Mann DA, Dolphin CT

Publication type: Article

Publication status: Published

Journal: Molecular Therapy

Year: 2005

Volume: 11

Issue: 4

Pages: 638-644

ISSN (print): 1525-0016

ISSN (electronic): 1525-0024

Publisher: Nature Publishing Group

URL: http://dx.doi.org/10.1016/j.ymthe.2004.12.010

DOI: 10.1016/j.ymthe.2004.12.010

Notes: 1525-0016 (Print) Journal Article


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