In vivo single-cell electroporation for transfer of DNA and macromolecules
In vivo single-cell electroporation for transfer of DNA and macromolecules
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DOI：
10.1038/nprot.2006.186
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Cline, Hollis T.
中科院分区：
文献类型：
--
作者：
Bestman, Jennifer E.;Ewald, Rebecca C.;Cline, Hollis T.
单细胞电穿孔允许使用改良的贴片电极和普通电生理设备将质粒DNA或大分子转染到单个活细胞中。本方案针对非洲爪蟾蝌蚪脑的DNA、右旋糖酐、morpholinos及其组合在体内快速电穿孔进行了优化。有经验的用户每小时可以电穿孔大约40只蝌蚪。该技术可以适用于其他带电转移材料和其他系统和组织，其中细胞可以用微移液管靶向。在视觉引导下，一个充满转移材料的电极被放置在蝌蚪大脑的细胞体丰富的区域，并施加一系列电压脉冲，使附近的细胞电穿孔。我们展示了成功电穿孔单细胞的例子，常见问题的实例和故障排除建议。单细胞电穿孔是一种负担得起的荧光标记和基因操作单个细胞的方法。这种强大的技术可以在正常环境中观察单个细胞。
Single-cell electroporation allows transfection of plasmid DNA or macrocmolecules 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.