Molecular and Cellular Cardiology Laboratory, IC/FUC, Av. Princesa Isabel 370-3 degrees andar, CEP: 90620-00, Porto Alegre, RS, Brazil,
Cytotechnology. 2006 Jul;51(3):141-8. doi: 10.1007/s10616-006-9028-1. Epub 2006 Nov 14.
The improvement of gene therapy protocols is intimately related to the establishment of efficient gene transfer methods. Electroporation has been increasingly employed in in vitro and in vivo protocols, and much attention has been given to increasing its transfection potential. The method is based on the application of an electric field of short duration and high voltage to the cells, forming reversible pores through which molecules can enter the cell. In this work, we describe the optimization of a protocol for the electroporation of K562 cells involving the combination of electric field, resistance and capacitance values. Using RPMI 1640 as pulsing buffer and 30 mug of pEGFP-N1 plasmid, 875 V cm(-1), 500 muF and infinite resistance, we achieved transfection rates of 82.41 +/- 3.03%, with 62.89 +/- 2.93% cell viability, values higher than those reported in the literature. Analyzing cell cycle after electroporation, with three different electric field conditions, we observed that in a heterogeneous population of cells, viability of G(1) cells is less affected by electroporation than that of cells in late S and G(2)/M phases. We also observed that efficiency of electroporation can be improved using the DNAse inhibitor Zn, immediately after the pulse. These results can represent a significant improvement of current methods of electroporation of animal and plant cells.
基因治疗方案的改进与高效基因转移方法的建立密切相关。电穿孔技术已越来越多地应用于体外和体内方案中，并且人们越来越关注提高其转染潜力。该方法基于对细胞施加短时间和高电压的电场，形成可逆的孔，使分子可以进入细胞。在这项工作中，我们描述了优化 K562 细胞电穿孔方案的过程，涉及电场、电阻和电容值的组合。使用 RPMI 1640 作为脉冲缓冲液和 30 微克 pEGFP-N1 质粒，875 V cm(-1)、500 微法和无限电阻，我们实现了 82.41% +/- 3.03%的转染率，细胞活力为 62.89% +/- 2.93%，高于文献报道的值。在电穿孔后分析细胞周期，使用三种不同的电场条件，我们观察到在异质细胞群体中，G(1)期细胞的活力比晚期 S 和 G(2)/M 期细胞的活力受电穿孔的影响更小。我们还观察到，在脉冲后立即使用 DNAse 抑制剂 Zn 可以提高电穿孔的效率。这些结果可以代表动物和植物细胞电穿孔当前方法的显著改进。
