Abstract
BackgroundRNA interference (RNAi) is a robust tool for inhibiting specific gene expression, but it is limited by the uncertain efficiency of siRNA or shRNA constructs. It has been shown that the overexpression of ARGONAUTE 2 (AGO2) protein increases silencing efficiency. However, the key elements required for AGO2-mediated enhancement of gene silencing in lentiviral vector has not been well studied.ResultsTo explore the application of AGO2-based shRNA system in mammalian cells, we designed shRNA vectors targeting the EGFP reporter gene and evaluated the effects of various factors on silencing efficiency including stem length, loop sequence, antisense location as well as the ratio between AGO2 and shRNA. We found that 19 ~ 21-bp stem and 6- or 9-nt loop structure in the sense-loop-antisense (S-L-AS) orientation was an optimal design in the AGO2-shRNA system. Then, we constructed a single lentiviral vector co-expressing shRNA and AGO2 and demonstrated that the simultaneous expression of shRNA and AGO2 can achieve robust silencing of exogenous DsRed2 and endogenous ID1 and P65 genes. However, the titers of packaged lentivirus from constitutive expression of AGO2 vector were extremely low, severely limiting its broad application. For the first time, we demonstrated that the problem can be significantly improved by using the inducible expression of AGO2 lentiviral system.ConclusionsWe reported a novel lentiviral vector with an optimal design of shRNA and inducible AGO2 overexpression which provides a new tool for RNAi research.
Highlights
RNA interference (RNAi) is a robust tool for inhibiting specific gene expression, but it is limited by the uncertain efficiency of siRNA or shRNA constructs
Co‐expression of ARGONAUTE 2 (AGO2) protein strongly enhances shRNA silencing activities We first examined the efficiency of two shRNA sequences in silencing EGPF
U6 promoter-driven shEGFP417, shEGFP450 or an unrelated control vector was co-transfected with pENTR/CMV-enhanced green florescence protein (EGFP) and pDsRed2c1 plasmids into HEK293 cells
Summary
RNA interference (RNAi) is a robust tool for inhibiting specific gene expression, but it is limited by the uncertain efficiency of siRNA or shRNA constructs. RNAi can be used to repress the expression of any genes. It is still much unknown about the siRNA biogenesis and action of mechanism. The application of RNAi technique is limited by the inconsistent efficiency of targeting sequences [6]. The existing computational approaches for siRNA selection allow the identification of potential sequences, but they do not ensure that each selected siRNA has sufficient silencing efficiency. About 25 % of the selected siRNA/shRNA sequences are functional with a knockdown efficiency
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