Name: stereotaxic injection of a viral vector for conditional gene manipulation in the mouse spinal cord
Uploaded: 2013-03-18T13:00:00
Description: Watch this Scientific Journal Video about Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord at JoVE.com

我们感谢Bakhos A. Tannous，博士为我们提供的rAAV-EGFP载体，载体的开发和生产在马萨诸塞州的查尔斯顿，马萨诸塞州总医院，神经科学中心的主任，约翰·王海提供技术援助。这项工作是支持的补助R01 NS050408（JS）由美国国家神经疾病与中风研究所。

描述的外科手术已经批准了哥伦比亚大学实验动物护理和使用委员会（IACUC）。 1。设备和病毒颗粒悬浮液的制备<ol><li>清洁和消毒的设备，消毒的外科手术器械和V形缺口，将用于修复椎骨L1尖峰。 </li><li>拉和锥玻璃吸管。我们使用的移液管的前端的直径为40μm，并在以20°的角度倾斜的。消毒的玻璃吸管。 </li><li>设置的立体定位框架，安装到操纵器上的微量喷射器，喷射器?...

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立体定向的载体注射可针对脊髓神经元的应用程序，如神经网络映射的基础上跨突触传播的病毒6,7或optogenetic解剖，轴突引导再生过程中从伤病中9,10，或基因治疗用于预防或治疗神经退行性疾病11， 12。病毒载体用于基因操作的脊髓研究躯体感觉，运动和植物神经途径9,10,13-15。鼠标是最广泛使用的模式生物的研究涉及立体病毒载体注射到大脑或脊髓，但该技术已?...

有条件的基因操作在小鼠的先进技术，使多方面的探索中枢神经系统的的突触途径和功能连接方法。转基因可能是受小分子效应如强力霉素，四环素控制的反式激活，它可以被设计为充当一个阻遏或激活基因转录，或他莫昔芬承认的雌激素受体​​1的配体结合域的突变作用于。通常是不可逆的转基因修饰的实现由脱氧核糖核酸（DNA）的重组酶。华创（的原因重组）和FLP（flippase重组酶）催化loxP位（轨迹，穿越x，P1）或首次登记税（flippase识别目标）的网站，分别为1的DNA片段，这些片段两侧切除，反转或易位。应用范围包括基因的激活或沉默和诱导型核糖核酸（RNA）干扰<sup&gt; 2。可以使用的条件表达的荧光或酶如β-半乳糖苷酶或碱性磷酸酶的记者标记神经元，并检查它们的局部组织和连接3。大型诱变项目在北美（ <a href="http://www.norcomm.org/index.htm" target="_blank">http://www.norcomm.org/index.htm</a> ）和欧洲（ <a href="http://www.knockoutmouse.org/about/eucomm" target="_blank">http://www.knockoutmouse.org/about/eucomm</a> ）的小鼠胚胎干细胞克隆库有条件的靶基因和陷阱，最终将覆盖整个小鼠基因组中。鼠标线膨胀的数目，反映根据特定的神经元的选择性基因操作（ <a href="http://nagy.mshri.on.ca/cre_new/index.php" target="_blank">http://nagy.mshri.on.ca/cre_new/index</a>某一特定人群的启动子或基因座的DNA的重组酶，可以划线，从这些克隆产生的小鼠<a href="http://nagy.mshri.on.ca/cre_new/index.php" target="_blank">PHP</a> ）。 <p class=“jove_content”&gt;然而，限制基因操作不同人群的神经元或感兴趣的特定区域可能不实现通过基因单独定位，如果还没有已知的或不表达在该区域的所有神经元到神经元特异性的启动子人口的利益的兴趣。在脊髓中，实验设计，可能需要一个或两个头尾段空间限制的基因操作。立体定位注射病毒载体表达Cre或FLP，允许限制基因重组小鼠脊髓中的DNA片段由loxP位或frt位点两侧的区域，即所谓的两侧装接loxP的或flrted等位基因。与组成DNA重排，这将导致从杂交育种与重组表达小鼠的动物，这种策略还提供了时间控制基因的激活或沉默。病毒载体编码两侧装接loxP或调情转基因提供了一个逆向选择的基因操作在小鼠体内表达的corresponding重组的神经元特异性启动子的下游。重组表达载体亲和力的神经元是4。高容量（无胆）腺病毒，腺相关病毒，单纯疱疹病毒和慢病毒常​​用嗜神经载体。选择合适的病毒用于研究的问题是实验设计的一个重要组成部分。的转基因的大小，送货路线，特异性感染的神经元，而不是神经胶质细胞，感染的疗效，炎性和毒性副作用需要考虑4。 在这里，我们描述了立体的病毒载体注射到脊髓背角，采用的技术，我们的条件在我们的研究对疼痛的神经生物学基因调控。背角接收传入的初级躯体感觉神经元的输入，包括伤害性神经元。当地的interneurons处理信息，然后投射神经元的传递从背角的大脑5。我们展示了一种嗜神经的重组腺相关病毒（rAAV），组成性激活的巨细胞病毒启动子表达增强型绿色荧光蛋白（EGFP）下背角神经元在脊髓节段L4感染。

<table border="1"><tbody><tr><td> 材料名称 </td><td> 公司 </td><td> 目录编号 </td><td></td></tr><tr><td>脊髓底板</td><td>大卫KOPF工具</td><td> 912 </td><td></td></tr><tr><td>小动物立体定位仪</td><td>大卫KOPF工具</td><td> 900 </td><td></td></tr><tr><td>鼠标气体麻醉头支架</td><td>大卫KOPF工具</td><td> 923-B </td><td></td></tr><tr><td>可调底座支架</td><td>大卫KOPF工具</td><td> 982 </td><td></td></tr><tr><td> V型缺口尖峰</td><td>大卫KOPF工具</td><td> 987 </td><td></td></tr><tr><td>小动物的温度控制系统</td><td>大卫KOPF工具</td><td> TCAT-2LV </td><td></td></tr><tr><td> ADSON钳</td><td>精细科学工具</td><td> 11006-12 </td><td></td></tr><tr><td>椎板切除术钳</td><td>精细科学工具</td><td> 11223-20 </td><td></td></tr><tr><td> UltraMicroPump（一）SYS-MICRO4控制器</td><td>世界精密仪器</td><td> UMP3-1 </td><td></td></tr><tr><td>微量，65RN </td><td>汉密尔顿</td><td> 7633-01 </td><td></td></tr><tr><td> RN压缩接头，1毫米</td><td>汉密尔顿</td><td> 55750-01 </td><td></td></tr><tr><td>高硼硅玻璃毛细管</td><td>世界精密仪器</td><td> 1B100F-4 </td><td></td></tr><tr><td> Microgrinder </td><td>成重</td><td> EG-44 </td><td></td></tr></tbody></table>

stereotaxic injection of a viral vector for conditional gene manipulation in the mouse spinal cord

实质内注射病毒载体使在不同人群的中枢神经系统的神经元或特定区域的条件的基因操作。我们展示了一个立体的注射技术，可以使靶基因的表达或沉默的小鼠脊髓背角。手术是短暂的。它需要一个单一的椎椎板切除术，提供快速恢复脊柱的动物和不受影响的活力。在低速和使用微量的斜角玻璃套管控喷射的小矢量悬浮液体积减少组织损伤。局部免疫反应的矢量取决于采用的病毒的内在特性;根据我们的经验，这是轻微和短暂的，使用时的重组腺相关病毒。如增强型绿色荧光蛋白报告基因的方便的载体，空间分布监测和疗效和蜂窝指定的转染ficity。

成功转染产生强大的基因的表达，不惜背角对侧，腹角和背根神经节神经元的注入背角（ 图1）。 <img src="/files/ftp_upload/50313/50313fig1.jpg" alt="图1" /> 图1。 （A）表达的荧光记者左背角L4脊髓，两个星期后的rAAV-EGFP的立体定向注射（血清型AAV2 / 8，9个基因组拷贝/ EGFP（绿色） 转染背角神经元。微升）。神经元进行免疫染色的神经?...

Watch this Scientific Journal Video about Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord at JoVE.com

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

病毒载体可以进行有针对性的基因操作。我们提出了一个条件性基因的表达或消融方法在小鼠脊髓，采用立体的病毒载体注射到脊髓背角，初级躯体感觉传入中枢神经系统的神经元之间的突触接触的一个突出的部位。

立体定向注射病毒载体的基因操作在小鼠脊髓条件

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

Research

JoVE Journal

Neuroscience

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

脊髓电二：外吸电极的制作

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

科研

神经科学

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

在体内脊髓的鼠标线使用双光子显微镜成像

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

扩展胚胎及成体神经干细胞<em&gt;在子宫内</em电穿孔或病毒立体定向注射

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

新生小鼠脑侧脑室注射病毒的持续和广泛的神经元传导

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

一个程序，植入脊柱商会纵<em&gt;在体内</em&gt;鼠标脊髓成像

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

细胞动力学在小鼠脊髓双光子成像

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

新生小鼠脑巴细胞基因调控的立体定向手术

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

拉米内切手术和脊髓窗口植入小鼠

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord

直接注射慢病毒载体突出大鼠脊髓中的多种运动途径

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

神经干细胞向大鼠和小鼠大脑的动脉内传递：对脑缺血的应用

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...