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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 CaPO(4)- and PEI-mediated transfection methods for producing LvVs. Different titration methods of LvV st...

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Autores principales: TANG, YONG, GARSON, KENNETH, LI, LI, VANDERHYDEN, BARBARA C.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: D.A. Spandidos 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4246624/
https://www.ncbi.nlm.nih.gov/pubmed/25435933
http://dx.doi.org/10.3892/ol.2014.2684
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author TANG, YONG
GARSON, KENNETH
LI, LI
VANDERHYDEN, BARBARA C.
author_facet TANG, YONG
GARSON, KENNETH
LI, LI
VANDERHYDEN, BARBARA C.
author_sort TANG, YONG
collection PubMed
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 CaPO(4)- 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 15×10(6) 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 CaPO(4)-mediated method.
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spelling pubmed-42466242014-11-28 Optimization of lentiviral vector production using polyethylenimine-mediated transfection TANG, YONG GARSON, KENNETH LI, LI VANDERHYDEN, BARBARA C. Oncol Lett 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 CaPO(4)- 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 15×10(6) 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 CaPO(4)-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.
spellingShingle Articles
TANG, YONG
GARSON, KENNETH
LI, LI
VANDERHYDEN, BARBARA C.
Optimization of lentiviral vector production using polyethylenimine-mediated transfection
title 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_short Optimization of lentiviral vector production using polyethylenimine-mediated transfection
title_sort optimization of lentiviral vector production using polyethylenimine-mediated transfection
topic Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4246624/
https://www.ncbi.nlm.nih.gov/pubmed/25435933
http://dx.doi.org/10.3892/ol.2014.2684
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