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In Vitro Validation of Phosphorodiamidate Morpholino Oligomers

1
Centre for Molecular Medicine and Innovative Therapeutics, Murdoch University, Perth, WA 6150, Australia
2
Perron Institute for Neurological and Translational Science, the University of Western Australia, Perth, WA 6009, Australia
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Academic Editor: Rakesh N. Veedu
Molecules 2019, 24(16), 2922; https://doi.org/10.3390/molecules24162922
Received: 19 June 2019 / Revised: 8 August 2019 / Accepted: 8 August 2019 / Published: 12 August 2019
(This article belongs to the Special Issue Antisense Oligonucleotide Chemistry and Applications)
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Abstract

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. View Full-Text
Keywords: antisense oligonucleotide; morpholino; PMO; transfection; electroporation; exon skipping antisense oligonucleotide; morpholino; PMO; transfection; electroporation; exon skipping
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Aung-Htut, M.T.; McIntosh, C.S.; West, K.A.; Fletcher, S.; Wilton, S.D. In Vitro Validation of Phosphorodiamidate Morpholino Oligomers. Molecules 2019, 24, 2922.

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