New protocol for lentiviral vector mass production

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TypeBook Chapter
Book titleLentivirus Gene Engineering Protocols: Second Edition
Pages3952; # of pages: 14
SubjectAnimals; bio; Bioreactor; Bioreactors; Biotechnology; Calcium; Cell Line; Cells; Chromatography; HEK-293; Heparin; Lentiviral vectors; Purification; serum-free medium; Transfection; Ultracentrifugation
AbstractMultiplasmid transient transfection is the most widely used technique for the generation of lentiviral vectors. However, traditional transient transfection protocols using 293 T adherent cells and calcium phosphate/DNA co-precipitation followed by ultracentrifugation are tedious, time-consuming, and difficult to scale up. This chapter describes a streamlined protocol for the fast mass production of lentiviral vectors and their purification by affinity chromatography. Lentiviral particles are generated by transient transfection of suspension growing HEK 293 cells in serum-free medium using polyethylenimine (PEI) as transfection reagent. Lentiviral vector production is carried out in Erlenmeyer flasks agitated on orbital shakers requiring minimum supplementary laboratory equipment. Alternatively, the method can be easily scaled up to generate larger volumes of vector stocks in bioreactors. Heparin affinity chromatography allows for selective concentration and purification of lentiviral particles in a singlestep directly from vector supernatants. The method is suitable for the production and purification of different vector pseudotypes
Publication date
PublisherHumana Press
AffiliationNational Research Council Canada (NRC-CNRC); NRC Biotechnology Research Institute
Peer reviewedYes
NRC numberBCBIO15
NPARC number16225325
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Record identifierb697a5ce-3795-4339-baf9-20b19f28799b
Record created2010-11-05
Record modified2016-05-09
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