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In Vitro Validation of Phosphorodiamidate Morpholino Oligomers

One of the crucial aspects of screening antisense oligonucleotides destined for therapeutic application is confidence that the antisense oligomer is delivered efficiently into cultured cells. Efficient delivery is particularly vital for antisense phosphorodiamidate morpholino oligomers, which have a...

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Autores principales: Aung-Htut, May T., McIntosh, Craig S., West, Kristin A., Fletcher, Sue, Wilton, Steve D.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6719133/
https://www.ncbi.nlm.nih.gov/pubmed/31408997
http://dx.doi.org/10.3390/molecules24162922
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author Aung-Htut, May T.
McIntosh, Craig S.
West, Kristin A.
Fletcher, Sue
Wilton, Steve D.
author_facet Aung-Htut, May T.
McIntosh, Craig S.
West, Kristin A.
Fletcher, Sue
Wilton, Steve D.
author_sort Aung-Htut, May T.
collection PubMed
description One of the crucial aspects of screening antisense oligonucleotides destined for therapeutic application is confidence that the antisense oligomer is delivered efficiently into cultured cells. Efficient delivery is particularly vital for antisense phosphorodiamidate morpholino oligomers, which have a neutral backbone, and are known to show poor gymnotic uptake. Here, we report several methods to deliver these oligomers into cultured cells. Although 4D-Nucleofector™ or Neon™ electroporation systems provide efficient delivery and use lower amounts of phosphorodiamidate morpholino oligomer, both systems are costly. We show that some readily available transfection reagents can be used to deliver phosphorodiamidate morpholino oligomers as efficiently as the electroporation systems. Among the transfection reagents tested, we recommend Lipofectamine 3000™ for delivering phosphorodiamidate morpholino oligomers into fibroblasts and Lipofectamine 3000™ or Lipofectamine 2000™ for myoblasts/myotubes. We also provide optimal programs for nucleofection into various cell lines using the P3 Primary Cell 4D-Nucleofector™ X Kit (Lonza), as well as antisense oligomers that redirect expression of ubiquitously expressed genes that may be used as positive treatments for human and murine cell transfections.
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spelling pubmed-67191332019-09-10 In Vitro Validation of Phosphorodiamidate Morpholino Oligomers Aung-Htut, May T. McIntosh, Craig S. West, Kristin A. Fletcher, Sue Wilton, Steve D. Molecules Article One of the crucial aspects of screening antisense oligonucleotides destined for therapeutic application is confidence that the antisense oligomer is delivered efficiently into cultured cells. Efficient delivery is particularly vital for antisense phosphorodiamidate morpholino oligomers, which have a neutral backbone, and are known to show poor gymnotic uptake. Here, we report several methods to deliver these oligomers into cultured cells. Although 4D-Nucleofector™ or Neon™ electroporation systems provide efficient delivery and use lower amounts of phosphorodiamidate morpholino oligomer, both systems are costly. We show that some readily available transfection reagents can be used to deliver phosphorodiamidate morpholino oligomers as efficiently as the electroporation systems. Among the transfection reagents tested, we recommend Lipofectamine 3000™ for delivering phosphorodiamidate morpholino oligomers into fibroblasts and Lipofectamine 3000™ or Lipofectamine 2000™ for myoblasts/myotubes. We also provide optimal programs for nucleofection into various cell lines using the P3 Primary Cell 4D-Nucleofector™ X Kit (Lonza), as well as antisense oligomers that redirect expression of ubiquitously expressed genes that may be used as positive treatments for human and murine cell transfections. MDPI 2019-08-12 /pmc/articles/PMC6719133/ /pubmed/31408997 http://dx.doi.org/10.3390/molecules24162922 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Aung-Htut, May T.
McIntosh, Craig S.
West, Kristin A.
Fletcher, Sue
Wilton, Steve D.
In Vitro Validation of Phosphorodiamidate Morpholino Oligomers
title In Vitro Validation of Phosphorodiamidate Morpholino Oligomers
title_full In Vitro Validation of Phosphorodiamidate Morpholino Oligomers
title_fullStr In Vitro Validation of Phosphorodiamidate Morpholino Oligomers
title_full_unstemmed In Vitro Validation of Phosphorodiamidate Morpholino Oligomers
title_short In Vitro Validation of Phosphorodiamidate Morpholino Oligomers
title_sort in vitro validation of phosphorodiamidate morpholino oligomers
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6719133/
https://www.ncbi.nlm.nih.gov/pubmed/31408997
http://dx.doi.org/10.3390/molecules24162922
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