title: Optimization of lentiviral vector production using polyethylenimine-mediated transfection.
abstract: The aim of the present study was to optimize the polyethylenimine (PEI)-mediated transfection method in order to simplify the efficient production of lentiviral vectors (LvVs), and to compare the CaPO4- and PEI-mediated transfection methods for producing LvVs. Different titration methods of LvV stocks, as well as different culture media, culture durations, cell densities and DNA quantities were compared to obtain an optimized procedure for the production of LvVs. Optimization of the production method for LvVs was achieved using PEI-mediated transient transfections. Serum-free Opti-MEM®was used to directly produce LvVs that could be harvested 48 h after transfection. Furthermore, a cell density of 15×106cells/10-cm plate and a DNA concentration of 1X were selected for the optimum production of LvVs. The optimized LvV titration method was simple and direct; it involved LvVs carrying fluorescent reporters, which proved to be faster than the standard methods but equally as sensitive. In conclusion, a scalable process for production of LvVs by PEI-mediated transfection was established and optimized. The optimized PEI-mediated transfection method was easy to use, as well as providing greater reliability with a higher degree of reproducibility and consistency. Despite using less DNA, the PEI-mediated transfection method resulted in viral titers that were the same as those achieved using the CaPO4-mediated method.
authors.affiliation: Department of Urology, Affiliated Cancer Hospital of Guangxi Medical University, Nanning, Guangxi 530021, P.R. China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Tang
authors.affiliation: Centre for Cancer Therapeutics, Ottawa Health Research Institute, University of Ottawa, Ottawa, Ontario K1H 8L6, Canada.
authors.forename: Kenneth
authors.initials: K
authors.lastname: Garson
authors.affiliation: Department of Gynecologic Oncology, Affiliated Cancer Hospital of Guangxi Medical University, Nanning, Guangxi 530021, P.R. China.
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.affiliation: Department of Cellular and Molecular Medicine, Centre for Cancer Therapeutics, Ottawa Health Research Institute, University of Ottawa, Ottawa, ON K1H 8L6, Canada.
authors.forename: Barbara C
authors.initials: BC
authors.lastname: Vanderhyden
keywords: calcium phosphate; lentivirus; polyethylenimine; vector production