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Overcoming the Specific Toxicity of Large Plasmids Electrotransfer in Primary Cells In Vitro

Gene electrotransfer is a safe and efficient nonviral technique for the transfer of nucleic acids of all sizes. Using a small reporter plasmid (3.5 kbp), electrotransfer of more than 90% of the cells, with ~70% viability, can be routinely achieved even in primary cells like mesenchymal stem cells. H...

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Autores principales: Lesueur, Léa L, Mir, Lluis M, André, Franck M
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5014460/
https://www.ncbi.nlm.nih.gov/pubmed/27111417
http://dx.doi.org/10.1038/mtna.2016.4
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author Lesueur, Léa L
Mir, Lluis M
André, Franck M
author_facet Lesueur, Léa L
Mir, Lluis M
André, Franck M
author_sort Lesueur, Léa L
collection PubMed
description Gene electrotransfer is a safe and efficient nonviral technique for the transfer of nucleic acids of all sizes. Using a small reporter plasmid (3.5 kbp), electrotransfer of more than 90% of the cells, with ~70% viability, can be routinely achieved even in primary cells like mesenchymal stem cells. However, under the same experimental conditions, electrotransfer of larger plasmids (from 6 to 16 kbp) results in very low viability and transfection efficacy. Here, we show that these strong decreases are directly linked to the physical size of the plasmid molecule. Moreover, large plasmids are toxic only when the cells are exposed to electrotransfer pulses. This specific toxicity of large plasmids during electrotransfer is not due to transgene expression and occurs within less than 45 minutes. Indeed, postpulses recovery times of up to 45 minutes are able to entirely abolish the specific toxicity of large plasmid electrotransfer, resulting in a survival and transfection efficacy identical to that of small plasmids. Finally, electrotransfer of small and large plasmids can reach 90–99% of transfection with 60–90% survival considering the findings here reported.
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spelling pubmed-50144602016-09-19 Overcoming the Specific Toxicity of Large Plasmids Electrotransfer in Primary Cells In Vitro Lesueur, Léa L Mir, Lluis M André, Franck M Mol Ther Nucleic Acids Original Article Gene electrotransfer is a safe and efficient nonviral technique for the transfer of nucleic acids of all sizes. Using a small reporter plasmid (3.5 kbp), electrotransfer of more than 90% of the cells, with ~70% viability, can be routinely achieved even in primary cells like mesenchymal stem cells. However, under the same experimental conditions, electrotransfer of larger plasmids (from 6 to 16 kbp) results in very low viability and transfection efficacy. Here, we show that these strong decreases are directly linked to the physical size of the plasmid molecule. Moreover, large plasmids are toxic only when the cells are exposed to electrotransfer pulses. This specific toxicity of large plasmids during electrotransfer is not due to transgene expression and occurs within less than 45 minutes. Indeed, postpulses recovery times of up to 45 minutes are able to entirely abolish the specific toxicity of large plasmid electrotransfer, resulting in a survival and transfection efficacy identical to that of small plasmids. Finally, electrotransfer of small and large plasmids can reach 90–99% of transfection with 60–90% survival considering the findings here reported. Nature Publishing Group 2016-03 2016-03-08 /pmc/articles/PMC5014460/ /pubmed/27111417 http://dx.doi.org/10.1038/mtna.2016.4 Text en Copyright © 2016 Official journal of the American Society of Gene & Cell Therapy http://creativecommons.org/licenses/by-nc-sa/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/4.0/
spellingShingle Original Article
Lesueur, Léa L
Mir, Lluis M
André, Franck M
Overcoming the Specific Toxicity of Large Plasmids Electrotransfer in Primary Cells In Vitro
title Overcoming the Specific Toxicity of Large Plasmids Electrotransfer in Primary Cells In Vitro
title_full Overcoming the Specific Toxicity of Large Plasmids Electrotransfer in Primary Cells In Vitro
title_fullStr Overcoming the Specific Toxicity of Large Plasmids Electrotransfer in Primary Cells In Vitro
title_full_unstemmed Overcoming the Specific Toxicity of Large Plasmids Electrotransfer in Primary Cells In Vitro
title_short Overcoming the Specific Toxicity of Large Plasmids Electrotransfer in Primary Cells In Vitro
title_sort overcoming the specific toxicity of large plasmids electrotransfer in primary cells in vitro
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5014460/
https://www.ncbi.nlm.nih.gov/pubmed/27111417
http://dx.doi.org/10.1038/mtna.2016.4
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