Name: methods for study of neuronal morphogenesis: ex vivo rnai electroporation in embryonic murine cerebral cortex
Uploaded: 2012-05-18T12:00:00
Description: Watch this Scientific Journal Video about Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex at JoVE.com

我们感谢提供的pSil-GFP的结构图1的插图，博士阿尔珀乌尊，共聚焦显微镜勒杜克生物成像设备，博士希林Bonni。电解金属锰支持从巴勒斯惠康基金，NARSAD奖的年轻研究者，美国国立卫生研究院NCRR COBRE P20的RR018728-01职业医学科学奖。借券支持由美国国立卫生研究院NCRR COBRE P20的RR018728-01，并已收到小灵通NRSA 5T32MH019118-20的支持。

1。准备培养液和媒体（不是视频） <ol><li>准备完整汉克平衡盐溶液（HBSS中）含有1X HBSS中，2.5毫米HEPES液（pH7.4），30毫米D-葡萄糖，1毫米氯化钙 ，硫酸镁4 1毫米和4毫米碳酸氢钠3 1升。加入双蒸水（DDH 2 O）的。过滤消毒用0.2微米的过滤器，并储存于4°C。 </li><li>准备片用35毫升基础培养基鹰媒体的培养基，完整的HBSS 12.9毫升，20毫米D-葡萄糖（1.35毫升的1 M的解决?...

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这些方法包括体外电击质粒编码双链RNA发夹和器官切片4提供了几个明显的优势文化。首先，这些方法可以快速评估为RNAi派生的表型的。 pSilencer载体包含U6启动子驱动的双链RNA发夹编码绿色荧光蛋白表达盒中列入允许细胞电穿孔与RNAi载体的快速识别和表征。除了时间效率，这些方法具有高度成本效益的几个淘汰赛行产生相比。第二，与生存电技术12，需要较长的培训和技能?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>汉密尔顿注射器</td><td>哈密​​尔顿</td><td> 80008 </td><td> 31计，0.5英寸长，PT-4（点斜边的水平），体积10μL </td></tr><tr><td>白金tweezertrodes </td><td> A型肉毒毒素</td><td> 45-0489 </td><td> 5mm大小</td></tr><tr><td> ECM830 electroporator </td><td> A型肉毒毒素</td><td> 45-0002 </td><td></td></tr><tr><td> BTX的脚踏</td><td> A型肉毒毒素</td><td> 45-0208 </td><td> ECM830 electroporator </td></tr><tr><td>振动刀片切片机</td><td>徕卡</td><td> VT1000小号</td><td></td></tr><tr><td> 6 - 以及菜用插入</td><td>鹰</td><td> 353502 </td><td>包含适合插入的缺口</td></tr><tr><td>组织培养插入</td><td>鹰</td><td> 353090 </td><td> 0.4微米</TD&gt; </tr><tr><td>快绿</td><td>西格玛</td><td> F7252 </td><td></td></tr><tr><td>低融点琼脂糖</td><td>稳</td><td> BP165-25 </td><td> DNA的等级</td></tr><tr><td>层粘连蛋白</td><td> Sigma-Aldrich公司</td><td> L2020 </td><td></td></tr><tr><td>聚-L-赖氨酸</td><td> Sigma-Aldrich公司</td><td> P5899 </td><td></td></tr><tr><td>培养基鹰</td><td> Sigma Aldrich公司</td><td> B - 1522 </td><td></td></tr><tr><td>无钙，镁10倍的HBSS </td><td> GIBCO公司</td><td> 14180-046 </td><td></td></tr><tr><td>肝素钠的游离酸</td><td> Sigma-Aldrich公司</td><td> H4034 </td><td></td></tr></tbody></table>

methods for study of neuronal morphogenesis: ex vivo rnai electroporation in embryonic murine cerebral cortex

大脑皮质的指示较高的认知功能。这6个层状结构会产生一个内部第一，外部最后的方式，其中第一个出生的神经元保持密切的心室，而去年出生的神经元迁移过去的第一个出生的神经细胞向大脑表面1。除了 ​​神经细胞迁移2，皮质功能正常的一个关键过程是调节神经元的形态3。虽然神经元形态，可在原代培养体外研究，有多少要了解到，从这些过程是如何在组织环境监管。 我们描述的技术来分析神经细胞迁移和/或在大脑皮层的器官切片4,6形态。用于修改一个pSilencer载体，其中包含同时U6启动子驱动的双链发夹RNA和一个单独的编码绿色荧光蛋白表达盒驱动b雅CMV启动子7-9。我们的方法允许候选基因的具体击倒后突起生长的缺陷快速评估，并已成功用于在屏幕上一个突起生长8监管。因为只有一部分细胞会表达的RNAi构造，器官切片允许镶嵌一个潜在的表型分析。此外，因为这种分析是在附近近似体内环境，它提供了一个低成本为皮质功能未知的基因，并迅速替代代转基因或基因敲除动物。最后，在体内电穿孔技术相比， 体外电击实验的成功是不依赖于熟练的手术技巧的发展，可以用更短的训练时间和技能进行。

Watch this Scientific Journal Video about Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex at JoVE.com

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

我们描述涉及的方法进行快速评估的功能基因在大脑皮层的发展，<em&gt;体外</em&gt;电质粒共表达抑制核糖核酸（RNA干扰）和绿色荧光蛋白在小鼠胚胎皮层。该协议是适合研究神经发育的各个方面，如神经，神经细胞迁移和神经元的形态，包括树突和轴突生长。

神经元形态的研究方法：<em&gt;体外</em&gt; RNAi技术在胚胎小鼠大脑皮质的电穿孔

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

Research

JoVE Journal

Neuroscience

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

科研

神经科学

In vivo Electroporation of Developing Mouse Retina

In vivo Electroporation of Developing Mouse Retina

在发展中国家小鼠视网膜体内电穿孔

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

检测内源NADH的荧光双光子成像的小鼠大脑皮层Microregional缺氧

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘<em&gt;在子宫内</em&gt;电穿孔

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

同时脑电图，实时测量的啮齿动物的大脑皮质中的神经元活动的乳酸浓度与Optogenetic的操作

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

听觉皮层在成年猫用高场磁共振成像功能成像

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

在子宫内电途径研究神经元亚群的兴奋性和单细胞连接

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

宫内电穿孔诱导小鼠大脑皮质中氧合酶的重组

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To ...