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Plasmid-based shRNA lentiviral particle production for RNAi applications.

ABSTRACT: Lentiviral vectors have become mainstream gene transfer vehicles for their ability to deliver and integrate into host cells. In RNA interference (RNAi) applications, lentiviral constructs constitutively express double-stranded RNA molecules, usually as short hairpin RNA (shRNA), enabling long-term gene silencing, and, when pseudotyped with a broad host glycoprotein envelope, allow a multitude of cell types to be transduced. Their successful use ultimately relies on the production of lentiviral particles in high titer and uniformity. Typical methods require the transfection of three or more plasmids in which essential viral elements have been encoded separated so as to remain replication deficient. These transfection procedures are of critical importance; however, methods often vary among laboratories, making it difficult to assess the overall efficiency of lentiviral particle production. In this report, we focus exclusively on this step and compare the overall impact of the commercial transfection reagent FuGENE 6 with FuGENE HD. We found that FuGENE HD resulted in at least 5-fold improvement in viral particle titer as assessed by the p24 standard enzyme-linked immunosorbent assay. We present the complete optimized workflow and demonstrate this utility in which a single modification of this transfection step improved the lentiviral particle production.

SUBMITTER: Shum D

PROVIDER: S-EPMC4170048 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Plasmid-based shRNA lentiviral particle production for RNAi applications.

Shum David D Djaballah Hakim H

Journal of biomolecular screening 20140617 9

Lentiviral vectors have become mainstream gene transfer vehicles for their ability to deliver and integrate into host cells. In RNA interference (RNAi) applications, lentiviral constructs constitutively express double-stranded RNA molecules, usually as short hairpin RNA (shRNA), enabling long-term gene silencing, and, when pseudotyped with a broad host glycoprotein envelope, allow a multitude of cell types to be transduced. Their successful use ultimately relies on the production of lentiviral p ...[more]

PMID: 24939963

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Project description:BackgroundChikungunya virus (CHIKV) is a re-emerging alphavirus that causes chikungunya fever and persistent arthralgia in humans. Currently, there is no effective vaccine or antiviral against CHIKV infection. Therefore, this study evaluates whether RNA interference which targets at viral genomic level may be a novel antiviral strategy to inhibit the medically important CHIKV infection.MethodsPlasmid-based small hairpin RNA (shRNA) was investigated for its efficacy in inhibiting CHIKV replication. Three shRNAs designed against CHIKV Capsid, E1 and nsP1 genes were transfected to establish stable shRNA-expressing cell clones. Following infection of stable shRNA cells clones with CHIKV at M.O.I. 1, viral plaque assay, Western blotting and transmission electron microscopy were performed. The in vivo efficacy of shRNA against CHIKV replication was also evaluated in a suckling murine model of CHIKV infection.ResultsCell clones expressing shRNAs against CHIKV E1 and nsP1 genes displayed significant inhibition of infectious CHIKV production, while shRNA Capsid demonstrated a modest inhibitory effect as compared to scrambled shRNA cell clones and non-transfected cell controls. Western blot analysis of CHIKV E2 protein expression and transmission electron microscopy of shRNA E1 and nsP1 cell clones collectively demonstrated similar inhibitory trends against CHIKV replication. shRNA E1 showed non cell-type specific anti-CHIKV effects and broad-spectrum silencing against different geographical strains of CHIKV. Furthermore, shRNA E1 clones did not exert any inhibition against Dengue virus and Sindbis virus replication, thus indicating the high specificity of shRNA against CHIKV replication. Moreover, no shRNA-resistant CHIKV mutant was generated after 50 passages of CHIKV in the stable cell clones. More importantly, strong and sustained anti-CHIKV protection was conferred in suckling mice pre-treated with shRNA E1.ConclusionTaken together, these data suggest the promising efficacy of anti-CHIKV shRNAs, in particular, plasmid-shRNA E1, as a novel antiviral strategy against CHIKV infection.

Dataset Information

Plasmid-based shRNA lentiviral particle production for RNAi applications.

Publications

Plasmid-based shRNA lentiviral particle production for RNAi applications.

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