Centre for Molecular Medicine and Innovative Therapeutics, Murdoch University, Perth, WA 6150, Australia.
Perron Institute for Neurological and Translational Science, the University of Western Australia, Perth, WA 6009, Australia.
Molecules. 2019 Aug 12;24(16):2922. doi: 10.3390/molecules24162922.
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.
筛选用于治疗应用的反义寡核苷酸的关键方面之一是确信反义寡聚物能够有效地递送到培养的细胞中。对于具有中性骨架的反义磷酰胺吗啉寡聚物，有效的递送尤其重要，因为已知其具有较差的电穿孔摄取。在这里，我们报告了几种将这些寡聚物递送到培养细胞中的方法。虽然 4D-Nucleofector™或 Neon™电穿孔系统提供了有效的递送，并且使用较少的磷酰胺吗啉寡聚物，但这两个系统都很昂贵。我们表明，一些现成的转染试剂可用于有效地递送电穿孔磷酰胺吗啉寡聚物，与电穿孔系统一样有效。在测试的转染试剂中，我们推荐 Lipofectamine 3000™用于将磷酰胺吗啉寡聚物递送到成纤维细胞中，以及 Lipofectamine 3000™或 Lipofectamine 2000™用于肌母细胞/肌管。我们还提供了使用 P3 Primary Cell 4D-Nucleofector™ X Kit（Lonza）进行各种细胞系核转染的最佳方案，以及重定向表达普遍表达基因的反义寡聚物，这些基因可作为人类和鼠细胞转染的阳性治疗方法。
