Name: transfection of mouse retinal ganglion cells by in vivo electroporation
Uploaded: 2011-04-17T14:00:00
Description: Watch this Scientific Journal Video about Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation at JoVE.com

pCAG gapEGFP质粒是博士S.麦康奈尔（斯坦福大学，加利福尼亚州）的礼物。 pCAG - tdTomato质粒博士M.费勒（伯克利分校，加利福尼亚州）的礼物。我们感谢爱德华Ruthazer博士建议使用一个单细胞标记和安妮Schohl验证两个质粒的Cre / loxP位技术支持战略的试点研究和Crair实验室成员（蒙特利尔，QC）的两个质粒战略。 R01 MH62639（MC），美国国立卫生研究院R01 EY015788（MC）和美国国立卫生研究院P30 EY000785（MC）的支持。

1。成套设备，电穿孔<ol><li> 电极 ：我们修改杜蒙＃5镊子作为电极使用。 <ol><li>分开，掰开镊子。 </li><li>焊接线，在每个双管齐下。包装线的连接和绝缘胶布离开暴露插脚的一角约25-30毫米的插头。 </li><li>任何合适的塑料垫片之间的两个插脚（如按钮）提供春风行动，把修改的镊子重新走到一起。 </li></ol></li><li> 电气设备：我们使用的电刺激电和示波器和音频监视器提供的电流脉...

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	<li>Huberman, A. D., Feller, M. B., Chapman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+underlying+development+of+visual+maps+and+receptive+fields.">Mechanisms underlying development of visual maps and receptive fields.</a> Annu Rev Neurosci. 31, 479-509 (2008).</li><li>McLaughlin, T., Torborg, C. L., Feller, M. B., O'Leary, D. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+utero+and+ex+vivo+electroporation+for+gene+expression+in+mouse+retinal+ganglion+cells.">In utero and ex vivo electroporation for gene expression in mouse retinal ganglion cells.</a> J Vis Exp. , (2009).</li><li>Ishikawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+GDNF+gene+transfer+into+axotomized+retinal+ganglion+cells+using+in+vivo+electroporation+with+a+contact+lens-type+electrode.">Effect of GDNF gene transfer into axotomized retinal ganglion cells using in vivo electroporation with a contact lens-type electrode.</a> Gene Ther. 12, 289-298 (2005).</li><li>Hewapathirane, D. 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在这个视频中， 我们证明在体内电协议，结果在与编码荧光蛋白质的DNA的结构，产后的小鼠视网膜神经细胞的单个或小群的标签。小群dLGN和SC荧光标记的研资局预测再现类似的投影模式，以前的研究使用亲油性染料研资局的标签，这表明电不干扰正常的研资局轴突乔木细化。我们利用这个协议在单一和不同的小鼠模型的视网膜神经节细胞组与正常，扰乱视觉地图水平的分析视网膜的地图。...

<table border="1"><tbody><tr><th>材料</th><th>公司</th><th>目录编号</th></tr><tr><td>杜蒙＃5镊子</td><td>精细科学的工具</td><td> 11252-20 </td></tr><tr><td>电刺激</td><td>草仪器</td><td> Model S4的</td></tr><tr><td>示波器</td><td>安捷伦</td><td>型号54621A </td></tr><tr><td>音频监听</td><td>草仪器</td><td>型号AM8B </td></tr><tr><td>拉紧器</td><td>萨特仪器</td><td> P - 97型</td></tr><tr><td> Vannas 剪刀 </td><td>世界精密仪器</td><td> 14003 </td></tr><tr><td>微型剪刀b </td><td>特德佩拉</td><td> 1347 </td></tr><tr><td>杜蒙机管局钳彗星 </td><td>精细科学的工具</td><td> 11210-20 </td></tr><tr><td> Nanoinject二系统</td><td>德拉蒙德科学</td><td> 3-000-204 </td></tr><tr><td>玻璃滴管</td><td>德拉蒙德科学</td><td> 3 - 000 - 203 - G / X </td></tr><tr><td>脚踏</td><td>德拉蒙德科学</td><td> 3-000-026 </td></tr><tr><td>矿物油</td><td> Sigma - Aldrich公司</td><td> M3516 </td></tr><tr><td> DII </td><td> Invitrogen公司</td><td> D - 383 </td></tr><tr><td> N，N -二甲基甲酰胺</td><td>西格玛</td><td> D4551 </td></tr></tbody></table>

transfection of mouse retinal ganglion cells by in vivo electroporation

研资局的预测中脑的针对性和细化，是一种流行和​​强大的模型系统，研究开发过程中神经连接的形式如何精确的模式。在小鼠实验中，retinofugal预测被安排在一个地形的方式和形式眼特定层在外侧膝状体，丘脑和上丘（SC）的（dLGN）。 retinofugal预测这些精确的模式的发展通常被标记RGC的人口与荧光染料和示踪剂，如辣根过氧化物酶 1-4研究。但是，这些方法都太粗到个别研资局轴突乔木形态的发展变化，是视网膜地图形成的基础上提供的洞察力。他们也不会允许的视网膜神经节细胞的遗传操作。 近日，电已成为一个有效的方法提供精确的时间和空间控制带电分子传递到视网膜5-11。当前视网膜电协议不允许基因操纵和跟踪retinofugal预测产后小鼠的视网膜神经节细胞的单件或小集群。有人认为，产后体内电穿孔是不是一个可行的方法转染视网膜神经节细胞，因为标签的效率极其低下，因而需要针对胚胎研资局祖细胞发生分化和增殖 6时的年龄。 在这个视频中，我们描述了在体内的基因靶向给药，均有产品，以及出生后小鼠视网膜神经节细胞的荧光右旋糖酐电协议。这种技术提供了一种经济有效，快速参与神经系统发育的几个方面，包括轴突回缩的候选基因的高效筛选和比较容易的平台，分支，层压，再生和突触形成电路开发的各个阶段。总之，我们在这里描述的一种有价值的工具将提供潜在的感官地图发展的分子机制的进一步了解。

Watch this Scientific Journal Video about Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation at JoVE.com

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

我们展示一个<em&gt;在体内</em&gt;电转染视网膜神经节细胞（RGC的）和其他类型的细胞在出生后的小鼠的年龄广泛视网膜的单件或小簇协议。标签和基因操纵产后视网膜神经节细胞的能力<em&gt;在体内</em&gt;是一个发展研究的有力工具。

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 ...

Research

JoVE Journal

Neuroscience

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

一个孤立的视网膜制备，记录从基因标记的视网膜神经节细胞的光反应

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

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 ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

在子宫内电穿孔：鼠标控制时空基因Transefection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

转基因小鼠内耳的发展<em&gt;在体内</em&gt;电穿孔

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and ...

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

在小鼠视网膜神经细胞，实现动态夹具与突触和人工电导

Ganglion cells are the output neurons of the  retina and their activity reflects the integration of multiple synaptic inputs  arising from specific neural ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

视网膜神经节细胞逆行标记的成年斑马鱼与荧光染料

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

体内基因转移到雪旺细胞在啮齿动物坐骨神经电穿孔

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

视网膜神经节细胞定义子集的单细胞RNA-Seq

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

光学相干断层扫描: 成像小鼠视网膜神经节细胞在体内

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

哺乳动物轴突再生: 成年小鼠背根神经节体内电穿孔的研究

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...