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Effects of Circular DNA Length on Transfection Efficiency by Electroporation into HeLa Cells

The ability to produce extremely small and circular supercoiled vectors has opened new territory for improving non-viral gene therapy vectors. In this work, we compared transfection of supercoiled DNA vectors ranging from 383 to 4,548 bp, each encoding shRNA against GFP under control of the H1 promo...

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Autores principales: Hornstein, Benjamin D., Roman, Dany, Arévalo-Soliz, Lirio M., Engevik, Melinda A., Zechiedrich, Lynn
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5137892/
https://www.ncbi.nlm.nih.gov/pubmed/27918590
http://dx.doi.org/10.1371/journal.pone.0167537
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author Hornstein, Benjamin D.
Roman, Dany
Arévalo-Soliz, Lirio M.
Engevik, Melinda A.
Zechiedrich, Lynn
author_facet Hornstein, Benjamin D.
Roman, Dany
Arévalo-Soliz, Lirio M.
Engevik, Melinda A.
Zechiedrich, Lynn
author_sort Hornstein, Benjamin D.
collection PubMed
description The ability to produce extremely small and circular supercoiled vectors has opened new territory for improving non-viral gene therapy vectors. In this work, we compared transfection of supercoiled DNA vectors ranging from 383 to 4,548 bp, each encoding shRNA against GFP under control of the H1 promoter. We assessed knockdown of GFP by electroporation into HeLa cells. All of our vectors entered cells in comparable numbers when electroporated with equal moles of DNA. Despite similar cell entry, we found length-dependent differences in how efficiently the vectors knocked down GFP. As vector length increased up to 1,869 bp, GFP knockdown efficiency per mole of transfected DNA increased. From 1,869 to 4,257 bp, GFP knockdown efficiency per mole was steady, then decreased with increasing vector length. In comparing GFP knockdown with equal masses of vectors, we found that the shorter vectors transfect more efficiently per nanogram of DNA transfected. Our results rule out cell entry and DNA mass as determining factors for gene knockdown efficiency via electroporation. The length-dependent effects we have uncovered are likely explained by differences in nuclear translocation or transcription. These data add an important step towards clinical applications of non-viral vector delivery.
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spelling pubmed-51378922016-12-21 Effects of Circular DNA Length on Transfection Efficiency by Electroporation into HeLa Cells Hornstein, Benjamin D. Roman, Dany Arévalo-Soliz, Lirio M. Engevik, Melinda A. Zechiedrich, Lynn PLoS One Research Article The ability to produce extremely small and circular supercoiled vectors has opened new territory for improving non-viral gene therapy vectors. In this work, we compared transfection of supercoiled DNA vectors ranging from 383 to 4,548 bp, each encoding shRNA against GFP under control of the H1 promoter. We assessed knockdown of GFP by electroporation into HeLa cells. All of our vectors entered cells in comparable numbers when electroporated with equal moles of DNA. Despite similar cell entry, we found length-dependent differences in how efficiently the vectors knocked down GFP. As vector length increased up to 1,869 bp, GFP knockdown efficiency per mole of transfected DNA increased. From 1,869 to 4,257 bp, GFP knockdown efficiency per mole was steady, then decreased with increasing vector length. In comparing GFP knockdown with equal masses of vectors, we found that the shorter vectors transfect more efficiently per nanogram of DNA transfected. Our results rule out cell entry and DNA mass as determining factors for gene knockdown efficiency via electroporation. The length-dependent effects we have uncovered are likely explained by differences in nuclear translocation or transcription. These data add an important step towards clinical applications of non-viral vector delivery. Public Library of Science 2016-12-05 /pmc/articles/PMC5137892/ /pubmed/27918590 http://dx.doi.org/10.1371/journal.pone.0167537 Text en © 2016 Hornstein et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Hornstein, Benjamin D.
Roman, Dany
Arévalo-Soliz, Lirio M.
Engevik, Melinda A.
Zechiedrich, Lynn
Effects of Circular DNA Length on Transfection Efficiency by Electroporation into HeLa Cells
title Effects of Circular DNA Length on Transfection Efficiency by Electroporation into HeLa Cells
title_full Effects of Circular DNA Length on Transfection Efficiency by Electroporation into HeLa Cells
title_fullStr Effects of Circular DNA Length on Transfection Efficiency by Electroporation into HeLa Cells
title_full_unstemmed Effects of Circular DNA Length on Transfection Efficiency by Electroporation into HeLa Cells
title_short Effects of Circular DNA Length on Transfection Efficiency by Electroporation into HeLa Cells
title_sort effects of circular dna length on transfection efficiency by electroporation into hela cells
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5137892/
https://www.ncbi.nlm.nih.gov/pubmed/27918590
http://dx.doi.org/10.1371/journal.pone.0167537
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