Single cell electroporation in vivo within the intact developing brain.
Single cell electroporation in vivo within the intact developing brain.
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DOI：
10.3791/705
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发表时间：
2008-07-11
期刊：
影响因子：
--
通讯作者：
Haas, Kurt
中科院分区：
文献类型：
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作者：
Hewapathirane, D Sesath;Haas, Kurt
单细胞电穿孔 (SCE) 是一种专门技术，可以将 DNA 或其他大分子递送到完整组织内的单个细胞中，包括体内制剂。该技术的独特优势在于，可以对单个细胞进行实验操作，同时保持周围组织不变，从而将细胞自主效应与整体治疗产生的效应区分开来。当与先进的体内成像技术相结合时，荧光标记物的 SCE 可以在几秒到几天的时间尺度内直接可视化细胞形态、细胞生长和细胞内事件。虽然该技术用于各种体内和离体制剂，但我们已经优化了该技术以用于非洲爪蟾蝌蚪。在这篇视频文章中，我们详细介绍了将荧光染料或质粒 DNA 注入白化非洲爪蟾蝌蚪完整大脑内神经元的程序。我们还讨论了优化产量的方法，并展示了 SCE 荧光标记的神经元的实时双光子荧光成像示例。
Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. The distinct advantage of this technique is that experimental manipulations may be performed on individual cells while leaving the surrounding tissue unaltered, thereby distinguishing cell-autonomous effects from those resulting from global treatments. When combined with advanced in vivo imaging techniques, SCE of fluorescent markers permits direct visualization of cellular morphology, cell growth, and intracellular events over timescales ranging from seconds to days. While this technique is used in a variety of in vivo and ex vivo preparations, we have optimized this technique for use in Xenopus laevis tadpoles. In this video article, we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the albino Xenopus tadpole. We also discuss methods to optimize yield, and show examples of live two-photon fluorescence imaging of neurons fluorescently labeled by SCE.