Gärtner Annette, Collin Ludovic, Lalli Giovanna
MRC Laboratory for Molecular Cell Biology, University College London, UK.
Methods Enzymol. 2006;406:374-88. doi: 10.1016/S0076-6879(06)06027-7.
Efficient gene transfer is an important tool for the study of neuronal function and biology. This has proved difficult and inefficient with traditional transfection strategies, which can also be fairly toxic, whereas viral-mediated gene transfer, although highly efficient, is often time-consuming. The recently developed Amaxa Nucleofector technology, based on electroporation with preset parameters in a cell-type-specific solution, enables direct delivery of DNA, small interfering (si)RNA oligonucleotides and siRNA vectors into the cell nucleus. This strategy results in reproducible, rapid, and efficient transfection of a broad range of cells, including primary neurons. Nucleofected neurons survive for up to 3 weeks and remain functional. We are currently using this transfection method to examine the contribution of Rho GTPase signaling pathways in the establishment of neuronal polarity, neuronal migration, and neurite outgrowth. Here, we describe three protocols to efficiently nucleofect rat cerebellar granule, cortical, and hippocampal neurons.
高效的基因转移是研究神经元功能和生物学的重要工具。传统的转染策略已证明难以实现且效率低下，而且可能具有相当大的毒性，而病毒介导的基因转移虽然效率很高，但往往耗时。最近开发的Amaxa核转染技术基于在细胞类型特异性溶液中使用预设参数进行电穿孔，能够将DNA、小干扰（si）RNA寡核苷酸和siRNA载体直接递送至细胞核。这种策略可对包括原代神经元在内的多种细胞进行可重复、快速且高效的转染。经核转染后的神经元可存活长达3周并保持功能。我们目前正在使用这种转染方法来研究Rho GTPase信号通路在神经元极性建立、神经元迁移和神经突生长中的作用。在此，我们描述三种可有效对大鼠小脑颗粒神经元、皮质神经元和海马神经元进行核转染的方案。
