Optimization of lentiviral vector production using polyethylenimine-mediated transfection

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 stoc...

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Main Authors:	TANG, YONG, GARSON, KENNETH, LI, LI, VANDERHYDEN, BARBARA C.
Format:	Online
Language:	English
Published:	D.A. Spandidos 2015
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4246624/

id	pubmed-4246624
recordtype	oai_dc
spelling	pubmed-42466242014-11-28 Optimization of lentiviral vector production using polyethylenimine-mediated transfection TANG, YONG GARSON, KENNETH LI, LI VANDERHYDEN, BARBARA C. Articles 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 15×106 cells/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. D.A. Spandidos 2015-01 2014-11-07 /pmc/articles/PMC4246624/ /pubmed/25435933 http://dx.doi.org/10.3892/ol.2014.2684 Text en Copyright © 2015, Spandidos Publications http://creativecommons.org/licenses/by/3.0 This is an open-access article licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License. The article may be redistributed, reproduced, and reused for non-commercial purposes, provided the original source is properly cited.
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	TANG, YONG GARSON, KENNETH LI, LI VANDERHYDEN, BARBARA C.
spellingShingle	TANG, YONG GARSON, KENNETH LI, LI VANDERHYDEN, BARBARA C. Optimization of lentiviral vector production using polyethylenimine-mediated transfection
author_facet	TANG, YONG GARSON, KENNETH LI, LI VANDERHYDEN, BARBARA C.
author_sort	TANG, YONG
title	Optimization of lentiviral vector production using polyethylenimine-mediated transfection
title_short	Optimization of lentiviral vector production using polyethylenimine-mediated transfection
title_full	Optimization of lentiviral vector production using polyethylenimine-mediated transfection
title_fullStr	Optimization of lentiviral vector production using polyethylenimine-mediated transfection
title_full_unstemmed	Optimization of lentiviral vector production using polyethylenimine-mediated transfection
title_sort	optimization of lentiviral vector production using polyethylenimine-mediated transfection
description	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 15×106 cells/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.
publisher	D.A. Spandidos
publishDate	2015
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4246624/
_version_	1613161682874400768

Optimization of lentiviral vector production using polyethylenimine-mediated transfection

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