Bergan R, Connell Y, Fahmy B, Neckers L
Clinical Pharmacology Branch, NCI, NIH, Bethesda, MD 20892.
Nucleic Acids Res. 1993 Jul 25;21(15):3567-73. doi: 10.1093/nar/21.15.3567.
Obtaining high transfection efficiencies and achieving appropriate intracellular concentrations and localization are two of the most important barriers to the implementation of gene targeted therapy. The efficiency of endogenous uptake of oligodeoxynucleotides (ODNs) varies from cell type to cell type and may be a limiting factor of antisense efficacy. The use of electroporation to obtain high intracellular concentrations of a synthetic ODN in essentially 100% of viable cells is described. It is also shown that the transfected ODNs initially localize to the nucleus and remain there for at least 48 hours. The cellular trafficking of electroporated ODNs is shown to be an energy dependent process. Targeting of the c-myc proto-oncogene of U937 cells by electroporation of phosphorothioate-modified ODNs results in rapid and specific suppression of this gene at ODN concentrations much lower than would otherwise be required. This technique appears to be applicable to a variety of cell types and may represent a powerful new investigate tool as well as a promising approach to the ex vivo treatment of hematologic disorders.
获得高转染效率以及实现适当的细胞内浓度和定位是基因靶向治疗实施过程中两个最重要的障碍。寡脱氧核苷酸（ODN）的内源性摄取效率因细胞类型而异，可能是反义疗效的限制因素。本文描述了使用电穿孔在基本上100%的活细胞中获得高细胞内浓度的合成ODN的方法。研究还表明，转染的ODN最初定位于细胞核，并在那里停留至少48小时。电穿孔ODN的细胞运输过程是一个能量依赖过程。通过电穿孔硫代磷酸酯修饰的ODN靶向U937细胞的c-myc原癌基因，在ODN浓度远低于其他所需浓度时，可快速、特异性地抑制该基因。该技术似乎适用于多种细胞类型，可能代表一种强大的新研究工具，也是血液系统疾病体外治疗的一种有前景的方法。
