In utero electroporation followed by primary neuronal culture for studying gene function in subset of cortical neurons.
In utero electroporation followed by primary neuronal culture for studying gene function in subset of cortical neurons.
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DOI：
10.3791/2103
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发表时间：
2010-10-08
期刊：
影响因子：
--
通讯作者：
Young-Pearse, Tracy L
中科院分区：
文献类型：
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作者：
Rice, Heather;Suth, Seiyam;Young-Pearse, Tracy L
原代神经元培养物的体外研究允许对神经突生长进行定量分析。为了研究遗传改变如何影响神经元过程的生长，可以通过化学转染或病毒转导将 shRNA 或 cDNA 构建体引入原代神经元。然而，对于原代皮质细胞，使用这些方法转染不同类型的细胞类型（来自不同层的谷氨酸能神经元、抑制性神经元、神经胶质细胞）。使用子宫内电穿孔在胚胎啮齿动物皮层中引入 DNA 构建体可以靶向某些细胞亚群：早期胚胎皮层的电穿孔靶向皮层的深层，而胚胎晚期时间点的电穿孔则靶向更浅层的皮层。此外，电极在各个胚胎头部的不同放置导致针对皮层的背内侧和腹侧外侧区域。电穿孔后，可以将转染的细胞解剖、解离并在体外铺板，以定量分析神经突生长。在这里，我们提供了一种分步方法来定量测量皮质细胞子集中的神经元过程的生长。 Kriegstein 实验室的另外两篇 JoVE 文章详细描述了子宫内电穿孔的基本方案。我们将概述我们的子宫内电穿孔协议，重点关注最重要的细节，然后描述我们的协议，该协议适用于子宫内电穿孔研究神经元过程生长中的基因功能。
In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells. The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.