Baculovirus-mediated gene silencing in insect cells using intracellularly produced long double-stranded RNA.
Double-stranded RNA-mediated interference (RNAi) has recently emerged as a powerful reverse genetics tool to silence gene expression in multiple organisms, including plants, nematodes and insects. In this study, DNA vectors capable of promoting the synthesis of long hairpin dsRNAs in vivo from a DNA template to suppress gene expression in insect cells have been successfully constructed. The inhibition of the expression of a gene encoding enhanced green fluorescent protein (eGFP) in insect cells was demonstrated by using plasmid or baculovirus vectors. Both plasmid and baculovirus vectors were able to inhibit eGFP expression in a dose dependent manner. Complete inhibition was obtained when co-transfection ratios of target plasmid to inhibition plasmid were 1:1 and 1:0.1. Eighty percent suppression was still maintained even when the ratio of eGFP plasmid to 'hairpin' plasmid was as high as 1:0.01. When the hairpin dsRNAs were encoded in a baculovirus, the suppression was about 50% when the ratio of 'target' baculovirus to 'inhibition' baculovirus reached 1:10. Therefore, the designed plasmid and baculovirus vectors are useful to induce RNAi in insect cell systems.
Huang
Yi
Y
State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, People's Republic of China.
Deng
Fei
F
Hu
Zhihong
Z
Vlak
Just M
JM
Wang
Hanzhong
H
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000096:biosynthesis / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000096:biosynthesis*; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D018411:Spodoptera; D014162:Transfection / Q000379:methods
D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins