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...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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MDPI AG
2019-08-01
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| Series: | Molecules |
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| Online Access: | https://www.mdpi.com/1420-3049/24/16/2922 |
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| author | May T. Aung-Htut Craig S. McIntosh Kristin A. West Sue Fletcher Steve D. Wilton |
| author_facet | May T. Aung-Htut Craig S. McIntosh Kristin A. West Sue Fletcher Steve D. Wilton |
| author_sort | May T. Aung-Htut |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-23T13:06:27Z |
| format | Article |
| id | doaj.art-f8ef51a203554f95ae6f3c21d41e2ba5 |
| institution | Directory Open Access Journal |
| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-12-23T13:06:27Z |
| publishDate | 2019-08-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Molecules |
| spelling | doaj.art-f8ef51a203554f95ae6f3c21d41e2ba52022-12-21T17:45:53ZengMDPI AGMolecules1420-30492019-08-012416292210.3390/molecules24162922molecules24162922In Vitro Validation of Phosphorodiamidate Morpholino OligomersMay T. Aung-Htut0Craig S. McIntosh1Kristin A. West2Sue Fletcher3Steve D. Wilton4Centre for Molecular Medicine and Innovative Therapeutics, Murdoch University, Perth, WA 6150, AustraliaCentre for Molecular Medicine and Innovative Therapeutics, Murdoch University, Perth, WA 6150, AustraliaCentre for Molecular Medicine and Innovative Therapeutics, Murdoch University, Perth, WA 6150, AustraliaCentre for Molecular Medicine and Innovative Therapeutics, Murdoch University, Perth, WA 6150, AustraliaCentre for Molecular Medicine and Innovative Therapeutics, Murdoch University, Perth, WA 6150, AustraliaOne 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.https://www.mdpi.com/1420-3049/24/16/2922antisense oligonucleotidemorpholinoPMOtransfectionelectroporationexon skipping |
| spellingShingle | May T. Aung-Htut Craig S. McIntosh Kristin A. West Sue Fletcher Steve D. Wilton In Vitro Validation of Phosphorodiamidate Morpholino Oligomers Molecules antisense oligonucleotide morpholino PMO transfection electroporation exon skipping |
| 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 | antisense oligonucleotide morpholino PMO transfection electroporation exon skipping |
| url | https://www.mdpi.com/1420-3049/24/16/2922 |
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