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Simplified production and concentration of lentiviral vectors to achieve high transduction in primary human T cells
BACKGROUND: Lentiviral vectors have emerged as efficient vehicles for transgene delivery in both dividing and non-dividing cells. A number of different modifications in vector design have increased biosafety and transgene expression. However, despite these advances, the transduction of primary human...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2013
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3830501/ https://www.ncbi.nlm.nih.gov/pubmed/24215295 http://dx.doi.org/10.1186/1472-6750-13-98 |
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author | Cribbs, Adam P Kennedy, Alan Gregory, Bernard Brennan, Fionula M |
author_facet | Cribbs, Adam P Kennedy, Alan Gregory, Bernard Brennan, Fionula M |
author_sort | Cribbs, Adam P |
collection | PubMed |
description | BACKGROUND: Lentiviral vectors have emerged as efficient vehicles for transgene delivery in both dividing and non-dividing cells. A number of different modifications in vector design have increased biosafety and transgene expression. However, despite these advances, the transduction of primary human T cells is still challenging and methods to achieve efficient gene transfer are often expensive and time-consuming. RESULTS: Here we present a simple optimised protocol for the generation and transduction of lentivirus in primary human CD45RA(+) T cells. We show that generation of high-titre lentivirus with improved primary T cell transduction is dependent upon optimised ultracentrifuge speed during viral concentration. Moreover, we demonstrate that transduction efficiency can be increased with simple modifications to the culturing conditions. Overall, a transduction efficiency of up to 89% in primary human CD45RA(+) cells is achievable when these modifications are used in conjunction. CONCLUSION: The optimised protocol described here is easy to implement and should facilitate the production of high-titre lentivirus with superior transduction efficiency in primary human T cells without the need for further purification methods. |
format | Online Article Text |
id | pubmed-3830501 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-38305012013-11-17 Simplified production and concentration of lentiviral vectors to achieve high transduction in primary human T cells Cribbs, Adam P Kennedy, Alan Gregory, Bernard Brennan, Fionula M BMC Biotechnol Methodology Article BACKGROUND: Lentiviral vectors have emerged as efficient vehicles for transgene delivery in both dividing and non-dividing cells. A number of different modifications in vector design have increased biosafety and transgene expression. However, despite these advances, the transduction of primary human T cells is still challenging and methods to achieve efficient gene transfer are often expensive and time-consuming. RESULTS: Here we present a simple optimised protocol for the generation and transduction of lentivirus in primary human CD45RA(+) T cells. We show that generation of high-titre lentivirus with improved primary T cell transduction is dependent upon optimised ultracentrifuge speed during viral concentration. Moreover, we demonstrate that transduction efficiency can be increased with simple modifications to the culturing conditions. Overall, a transduction efficiency of up to 89% in primary human CD45RA(+) cells is achievable when these modifications are used in conjunction. CONCLUSION: The optimised protocol described here is easy to implement and should facilitate the production of high-titre lentivirus with superior transduction efficiency in primary human T cells without the need for further purification methods. BioMed Central 2013-11-12 /pmc/articles/PMC3830501/ /pubmed/24215295 http://dx.doi.org/10.1186/1472-6750-13-98 Text en Copyright © 2013 Cribbs et al.; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Methodology Article Cribbs, Adam P Kennedy, Alan Gregory, Bernard Brennan, Fionula M Simplified production and concentration of lentiviral vectors to achieve high transduction in primary human T cells |
title | Simplified production and concentration of lentiviral vectors to achieve high transduction in primary human T cells |
title_full | Simplified production and concentration of lentiviral vectors to achieve high transduction in primary human T cells |
title_fullStr | Simplified production and concentration of lentiviral vectors to achieve high transduction in primary human T cells |
title_full_unstemmed | Simplified production and concentration of lentiviral vectors to achieve high transduction in primary human T cells |
title_short | Simplified production and concentration of lentiviral vectors to achieve high transduction in primary human T cells |
title_sort | simplified production and concentration of lentiviral vectors to achieve high transduction in primary human t cells |
topic | Methodology Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3830501/ https://www.ncbi.nlm.nih.gov/pubmed/24215295 http://dx.doi.org/10.1186/1472-6750-13-98 |
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