In Vitro Validation of Phosphorodiamidate Morpholino Oligomers

被引:14
|
作者
Aung-Htut, May T. [1 ,2 ]
McIntosh, Craig S. [1 ,2 ]
West, Kristin A. [1 ]
Fletcher, Sue [1 ,2 ]
Wilton, Steve D. [1 ,2 ]
机构
[1] Murdoch Univ, Ctr Mol Med & Innovat Therapeut, Perth, WA 6150, Australia
[2] Univ Western Australia, Perron Inst Neurol & Translat Sci, Perth, WA 6009, Australia
来源
MOLECULES | 2019年 / 24卷 / 16期
基金
澳大利亚国家健康与医学研究理事会;
关键词
antisense oligonucleotide; morpholino; PMO; transfection; electroporation; exon skipping; ANTISENSE OLIGONUCLEOTIDE; PHOSPHOROTHIOATE; INHIBITION; ETEPLIRSEN; DELIVERY; MODEL;
D O I
10.3390/molecules24162922
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 (TM) or Neon (TM) 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 (TM) for delivering phosphorodiamidate morpholino oligomers into fibroblasts and Lipofectamine 3000 (TM) or Lipofectamine 2000 (TM) for myoblasts/myotubes. We also provide optimal programs for nucleofection into various cell lines using the P3 Primary Cell 4D-Nucleofector (TM) 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.
引用
收藏
页数:11
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