RNA interference mediated in human primary cells via recombinant baculoviral vectors.
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.
Nicholson
Linda J
LJ
Department of Oncology, Guy's, King's and St Thomas' School of Medicine, The Rayne Institute, St Thomas' Hospital, London SE1 7EH, UK.
Philippe
Marie
M
Paine
Alan J
AJ
Mann
Derek A
DA
Dolphin
Colin T
CT
D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D014161:Transduction, Genetic / Q000379:methods*
D034741:RNA, Small Interfering; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins