Birck Nanotechnology Center, Purdue University, West Lafayette, Indiana, USA.
Nat Protoc. 2011 Jul 21;6(8):1192-208. doi: 10.1038/nprot.2011.360.
Electroporation is a high-efficiency and low-toxicity physical gene transfer method. Classical electroporation protocols are limited by the small volume of cell samples processed (less than 10(7) cells per reaction) and low DNA uptake due to partial permeabilization of the cell membrane. Here we describe a flow-through electroporation protocol for continuous transfection of cells, using disposable devices, a syringe pump and a low-cost power supply that provides a constant voltage. We show transfection of cell samples with rates ranging from 40 μl min(-1) to 20 ml min(-1) with high efficiency. By inducing complex migrations of cells during the flow, we also show permeabilization of the entire cell membrane and markedly increased DNA uptake. The fabrication of the devices takes 1 d and the flow-through electroporation typically takes 1-2 h.
电穿孔是一种高效低毒的物理基因转移方法。经典的电穿孔方案受到处理的细胞样品体积小（每个反应小于 10(7)个细胞）和细胞膜部分通透导致 DNA 摄取率低的限制。在这里，我们描述了一种使用一次性装置、注射器泵和低成本电源（提供恒压）的连续转染的流电穿孔方案。我们展示了以 40 μl min(-1)到 20 ml min(-1)的速率进行的细胞转染，效率很高。通过在流动过程中诱导细胞的复杂迁移，我们还显示了整个细胞膜的通透性和 DNA 摄取量的显著增加。器件的制造需要 1 天，而流电穿孔通常需要 1-2 小时。
