title: 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.
authors.affiliation: HTS Core Facility, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
authors.forename: David
authors.initials: D
authors.lastname: Shum
authors.affiliation: HTS Core Facility, Memorial Sloan Kettering Cancer Center, New York, NY, USA djaballh@mskcc.org.
authors.forename: Hakim
authors.initials: H
authors.lastname: Djaballah
mesh_terms: D018929:Cell Culture Techniques; D002460:Cell Line; D005822:Genetic Vectors / Q000096:biosynthesis* / Q000235:genetics*; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D010957:Plasmids / Q000235:genetics* / Q000302:isolation & purification; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*
chemical_list: D034741:RNA, Small Interfering
keywords: DNA; FuGENE 6; FuGENE HD; RNAi; lentiviral particles; plasmid; shRNA; viral titer