In vivo gene delivery to the postnatal ferret cerebral cortex by DNA electroporation
In vivo gene delivery to the postnatal ferret cerebral cortex by DNA electroporation
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DOI：
10.1016/j.jneumeth.2009.11.016
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发表时间：
2010-02-15
影响因子：
3
通讯作者：
Borrell, Victor
中科院分区：
文献类型：
--
作者：
Borrell, Victor
雪貂已被广泛用于揭示编码和处理视觉信息的神经机制，并确定这种复杂和微调的神经系统出现的潜在发育机制。近年来，已经产生了许多工具，可以以前所未有的力量研究神经系统。不幸的是，由于这些工具中有许多是基因编码的，它们对涉及“非基因”物种（如雪貂、猫和猴子）的研究影响有限。在这里，我展示了如何在体内电穿孔可以在出生后的雪貂套件进行传递遗传结构的大脑皮层的锥体神经元。GFP和DsRed编码质粒的电穿孔导致首先标记皮质祖细胞，然后迁移神经元，最后分化神经元及其过程。这种技术还允许在雪貂的皮质发育的遗传操作，如所示的显性负形式的Cdk 5的电穿孔。在电穿孔动物的成熟脑中，报告基因的表达揭示了皮质锥体的详细形态特征，包括它们的轴突和树突分支以及树突棘。我还表明，出生后的电穿孔可用于转染一个巨大的皮质领土，或者它可以专门针对一个子集的皮质区，甚至只有几个分散的金字塔沿着皮质地幔。因此，出生后雪貂的体内电穿孔是一种有效、快速、简单和高度通用的方法，用于将遗传构建体递送到这种动物，对于发育研究和成年解剖/功能研究都是最佳的。(C)2009 Elsevier B. V.保留所有权利。
Ferrets have been extensively used to unravel the neural mechanisms of coding and processing of visual information, and also to identify the developmental mechanisms underlying the emergence of such a complex and fine-tuned neural system. In recent years numerous tools have been generated that allow studying neural systems with unprecedented power. Unfortunately, because many of these tools are genetically encoded, they are having a limited impact on research involving "non-genetic" species, like ferret, cat and monkey. Here I show how in vivo electroporation can be performed in postnatal ferret kits to deliver genetic constructs to pyramidal neurons of the cerebral cortex. Electroporation of GFP- and DsRed-encoding plasmids results in labeling of cortical progenitors first, then migrating neurons, and finally differentiating neurons and their processes. This technique also allows for the genetic manipulation of cortical development in the ferret, as illustrated by electroporation of a dominant-negative form of Cdk5. In the mature brain of electroporated animals, expression of reporter genes reveals the detailed morphological traits of cortical pyramids, including their axonal and dendritic arborization, and dendritic spines. I also show that postnatal electroporation can be used for the transfection of a massive cortical territory, or it can be specifically directed to a subset of cortical areas, and even only to a few scattered pyramids along the cortical mantle. In vivo electroporation of postnatal ferrets is therefore an effective, rapid, simple and highly versatile method for delivering genetic constructs to this animal, optimal for both developmental studies and adult anatomical/functional studies. (C) 2009 Elsevier B.V. All rights reserved.