Pavlin Mojca, Haberl Sasa A, Rebersek Matej, Miklavcic Damijan, Kanduser Masa
Department of Fundamentals of Electrical Engineering, Mathematics and Physics, University of Ljubljana.
J Vis Exp. 2011 Sep 12(55):3309. doi: 10.3791/3309.
Gene electrotransfer is a physical method used to deliver genes into the cells by application of short and intense electric pulses, which cause destabilization of cell membrane, making it permeable to small molecules and allows transfer of large molecules such as DNA. It represents an alternative to viral vectors, due to its safety, efficacy and ease of application. For gene electrotransfer different electric pulse protocols are used in order to achieve maximum gene transfection, one of them is changing the electric field direction and orientation during the pulse delivery. Changing electric field direction and orientation increase the membrane area competent for DNA entry into the cell. In this video, we demonstrate the difference in gene electrotransfer efficacy when all pulses are delivered in the same direction and when pulses are delivered by changing alternatively the electric field direction and orientation. For this purpose tip with integrated electrodes and high-voltage prototype generator, which allows changing of electric field in different directions during electric pulse application, were used. Gene electrotransfer efficacy is determined 24h after pulse application as the number of cells expressing green fluorescent protein divided with the number of all cells. The results show that gene transfection is increased when the electric field orientation during electric pulse delivery is changed.
基因电穿孔是一种物理方法，通过施加短暂而强烈的电脉冲将基因导入细胞，这种电脉冲会导致细胞膜不稳定，使其对小分子具有通透性，并允许诸如DNA等大分子的转移。由于其安全性、有效性和易于应用，它代表了病毒载体的一种替代方法。对于基因电穿孔，使用不同的电脉冲方案以实现最大的基因转染，其中之一是在脉冲传递过程中改变电场方向和取向。改变电场方向和取向会增加DNA进入细胞的有效膜面积。在本视频中，我们展示了所有脉冲沿同一方向传递以及通过交替改变电场方向和取向来传递脉冲时基因电穿孔效率的差异。为此，使用了带有集成电极的尖端和高压原型发生器，该发生器允许在施加电脉冲期间在不同方向上改变电场。在施加脉冲24小时后，将表达绿色荧光蛋白的细胞数量除以所有细胞的数量来确定基因电穿孔效率。结果表明，当在电脉冲传递过程中改变电场取向时，基因转染会增加。
