Bestman Jennifer E, Ewald Rebecca C, Chiu Shu-Ling, Cline Hollis T
Cold Spring Harbor Laboratory, Watson School of Biological Sciences, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA.
Nat Protoc. 2006;1(3):1267-72. doi: 10.1038/nprot.2006.186.
Single-cell electroporation allows transfection of plasmid DNA or macromolecules into individual living cells using modified patch electrodes and common electrophysiological equipment. This protocol is optimized for rapid in vivo electroporation of Xenopus laevis tadpole brains with DNA, dextrans, morpholinos and combinations thereof. Experienced users can electroporate roughly 40 tadpoles per hour. The technique can be adapted for use with other charged transfer materials and in other systems and tissues where cells can be targeted with a micropipette. Under visual guidance, an electrode filled with transfer material is placed in a cell body-rich area of the tadpole brain and a train of voltage pulses applied, which electroporates a nearby cell. We show examples of successfully electroporated single cells, instances of common problems and troubleshooting suggestions. Single-cell electroporation is an affordable method to fluorescently label and genetically manipulate individual cells. This powerful technique enables observation of single cells in an otherwise normal environment.
单细胞电穿孔技术可利用改良的膜片电极和普通的电生理设备，将质粒DNA或大分子转染到单个活细胞中。本实验方案针对非洲爪蟾蝌蚪脑的DNA、葡聚糖、吗啉代化合物及其组合的快速体内电穿孔进行了优化。经验丰富的用户每小时大约可对40只蝌蚪进行电穿孔操作。该技术可适用于其他带电转移材料，以及可通过微量移液器靶向细胞的其他系统和组织。在视觉引导下，将充满转移材料的电极置于蝌蚪脑富含细胞体的区域，并施加一串电压脉冲，从而对附近的细胞进行电穿孔。我们展示了成功电穿孔的单细胞示例、常见问题实例及故障排除建议。单细胞电穿孔是一种经济实惠的方法，可对单个细胞进行荧光标记和基因操作。这项强大的技术能够在其他方面正常的环境中观察单个细胞。
