PDF(Pubmed)
Abstract：
Gene electrotransfer (GET) is non-viral gene delivery technique, also known as electroporation-mediated gene delivery or electrotransfection. GET is a method used to introduce foreign genetic material (such as DNA or RNA) into cells by applying external pulsed electric fields (PEFs) to create temporary pores in the cell membrane. This study was undertaken to examine the impact of buffer composition on the efficiency of GET in mammalian cells Also, we specifically compared the effectiveness of high-frequency nanosecond (ns) pulses with standard microsecond (µs) pulses. For the assessment of cell transfection efficiency and viability, flow cytometric analysis, luminescent assays, and measurements of metabolic activity were conducted. The efficiency of electrotransfection was evaluated using two different proteins encoding plasmids (pEGFP-N1 and Luciferase-pcDNA3). The investigation revealed that the composition of the electroporation buffer significantly influences the efficacy of GET in CHO-K1 cell line. The different susceptibility of cell lines to the electric field and the plasmid cytotoxicity were reported. It was also shown that electroporation with nanosecond duration PEF protocols ensured equivalent or even better transfection efficiency than standard µsPEF. Additionally, we successfully performed long-term transfection of the murine 4T1 cell line using high-frequency nanosecond PEFs and confirmed its\' applicability in an in vivo model. The findings from the study can be applied to optimize electrotransfection conditions.
摘要：
基因电转移（GET）是非病毒基因传递技术，也称为电穿孔介导的基因递送或电转染。GET是一种通过施加外部脉冲电场（PEF）以在细胞膜上产生临时孔来将外源遗传物质（例如DNA或RNA）引入细胞的方法。本研究旨在研究缓冲液组成对哺乳动物细胞中GET效率的影响。我们特别比较了高频纳秒（ns）脉冲与标准微秒（µs）脉冲的有效性。为了评估细胞转染效率和活力，流式细胞仪分析,发光测定，并进行代谢活性的测量。使用两种不同的蛋白质编码质粒(pEGFP-N1和荧光素酶-pcDNA3)评估电转染的效率。研究表明，电穿孔缓冲液的组成显着影响GET在CHO-K1细胞系中的功效。报道了细胞系对电场和质粒细胞毒性的不同敏感性。还显示,具有纳秒持续时间PEF方案的电穿孔确保了与标准μsPEF相当或甚至更好的转染效率。此外,我们成功地使用高频纳秒PEFs对小鼠4T1细胞系进行了长期转染,并证实了其在体内模型中的适用性.该研究的发现可用于优化电转染条件。
