Name: efficient gene delivery into multiple cns territories using in utero electroporation
Uploaded: 2011-06-23T13:00:00
Description: Watch this Scientific Journal Video about Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation at JoVE.com

作者想感谢他们最初的工作建立在子宫内电穿孔技术在CS实验室的伊娃Hadzimova，皮埃尔Mattar和Christopher Kovach。这项工作是由加拿大卫生研究院的研究所（CIHR）授予（澳门币44094）和CIHR /基金会战斗失明（FFB）新兴队格兰特（00933-000）CS和亚省儿童医院研究基金会的资助，以DMK。路是由CIHR加拿大希望奖学金的支持下，RC是一个FFB的助学金支持和领汇是在遗传学CIHR训练津贴及儿童发展的支持。

1。设置<ol><li>设置手术区，如图所示。 1A，A“。设置的关键组件包括微量的Eppendorf Femtojet，Narishige显微持针器，徕卡steromicroscope，光纤照明系统，ECM的830方波电极电穿孔系统，电热垫和异氟醚麻醉蒸发器。 </li><li>使用超声外科手术工具Metriclean2低发泡的解决方案与Bransonic超声波清洗机清洗所有的工具。通过高压灭菌消毒的工具。消毒工具，然后存放在Germex。 </li><li>准备温暖的无菌生理盐水的?...

<ol>
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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>Garcia-Frigola, C., Carreres, M. 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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+hedgehog+signaling+controls+thalamic+progenitor+identity+and+nuclei+specification+in+mice.">Sonic hedgehog signaling controls thalamic progenitor identity and nuclei specification in mice.</a> J Neurosci. 29, 4484-4497 (2009).</li><li>Tsuchiya, R., Takahashi, K., Liu, F. C., Takahashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aberrant+axonal+projections+from+mammillary+bodies+in+Pax6+mutant+mice:+possible+roles+of+Netrin-1+and+Slit+2+in+mammillary+projections.">Aberrant axonal projections from mammillary bodies in Pax6 mutant mice: possible roles of Netrin-1 and Slit 2 in mammillary projections.</a> J Neurosci Res. 87, 1620-1633 (2009).</li><li>Buffo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+pattern+of+the+transcription+factor+Olig2+in+response+to+brain+injuries:+implications+for+neuronal+repair.">Expression pattern of the transcription factor Olig2 in response to brain injuries: implications for neuronal repair.</a> Proc Natl Acad Sci U S A. 102, 18183-18188 (2005).</li></ol>

在子宫内的电，可用于分析的发育过程中的多种。例如，记者如GFP，mCherry或碱性磷酸酶基因转染可以用来进行谱系跟踪和神经细胞迁移实验。另外，Cre重组酶，可瞬时表达有选择性地消除了在一个空间和/或暂时控制方式floxed等位基因。此外，shRNA的或显性负构造可以被电击倒靶基因的功能。最后，有针对性的过表达或错误表达的关键基因在野生型和/或基因突变的鼠标线可以用来研究细胞?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>分类</th></tr><tr><td>精细剪刀</td><td>精细科学工具公司</td><td> 14078-10 </td><td> 手术工具 </td></tr><tr><td>虹膜剪刀，弯</td><td>精细科学工具公司</td><td> 14061-10 </td><td> 手术工具 </td></tr><tr><td>奥尔森Hegar前精致的持针器</td><td>精细科学工具公司</td><td> 12002-12 </td><td> 手术工具 </td></tr><tr><td>环钳，9毫米</td><td>精细科学工具公司</td><td> 11103-09 </td><td> 手术工具 </td></tr><tr><td>眼敷料Forcep </td><td>精细科学工具公司</td><td> 11051-10 </td><td> 手术工具 </td></tr><tr><td>杜蒙＃7的DMX Forcep </td><td>精细科学工具公司</td><td> 11271-30 </td><td> 手术工具 </td></tr><tr><td>杜蒙＃5 DMX Forcep </td><td>精细科学工具公司</td><td> 11251-30 </td><td> 手术工具 </td></tr><tr><td>组织forcep - Adson </td><td>精细科学工具公司</td><td> 11027-12 </td><td> 手术工具 </td></tr><tr><td>弹性贴面剪辑应用方</td><td>世界精密仪器</td><td> 500343 </td><td> 手术工具 </td></tr><tr><td>穿孔勺，直径15毫米</td><td>精细科学工具公司</td><td> 10370-18 </td><td> 手术工具 </td></tr><tr><td> Autoclip卸妆</td><td>米克朗</td><td> 427637 </td><td> 手术工具 </td></tr><tr><td>丝绸黑色编织缝合</td><td>爱惜康公司</td><td> K871 </td><td> 手术工具 </td></tr><tr><td>反射皮肤缝合不锈钢伤口剪辑</td><td>世界精密仪器</td><td> 500346 </td><td> 手术工具 </td></tr><tr><td>流脑830方波电穿孔系统</td><td> VWR - CanLab </td><td> 58018-004 </td><td> 仪器 </td></tr><tr><td>镊子W /变间隙2回合5毫米白金板电极</td><td>保得国际公司</td><td> CUY650P5 </td><td> 仪器 </td></tr><tr><td>镊子W /变间隙2回合7毫米白金板电极</td><td>保得国际公司</td><td> CUY650P7 </td><td> 仪器 </td></tr><tr><td> Eppendorf公司Femtojet微量</td><td> VWR CanLab </td><td> CA62111 - 488 </td><td> 仪器 </td></tr><tr><td>脚踏控制的Eppendorf Femtojet微量</td><td> VWR CanLab </td><td> CAACCESS（杂） </td><td> 仪器 </td></tr><tr><td> Bransonic超声波清洗机型号1510R - DTH </td><td> VWR CanLab </td><td> CA33995 - 534驻地网- 952 - 118 </td><td> 仪器 </td></tr><tr><td>萨特P97 Micropipet拖轮</td><td>萨特仪器，Carsen集团股份有限公司</td><td> P - 97 </td><td> 仪器 </td></tr><tr><td>微量移液器 - 高硼硅丝外径为1mm，编号：0.78毫米，长10厘米的</td><td>萨特仪器</td><td> BF100 - 78 - 10 </td><td> 仪器 </td></tr><tr><td> 3轴粗臂</td><td>卡尔蔡司加拿大公司</td><td> M - 152 </td><td> 仪器 </td></tr><tr><td>磁显微夹持装置</td><td>世界精密仪器</td><td>货币供应量M1 </td><td> 仪器 </td></tr><tr><td>钢的显微操纵器底座</td><td>世界精密仪器</td><td> 5052 </td><td> 仪器 </td></tr><tr><td>微管架</td><td>世界镨ecision仪器</td><td> MPH3 </td><td> 仪器 </td></tr><tr><td>微管拉手</td><td>世界精密仪器</td><td> 5444 </td><td> 仪器 </td></tr><tr><td>体视显微镜</td><td>徕卡</td><td> MZ6 </td><td> 仪器 </td></tr><tr><td>汽化器为异氟醚麻醉</td><td>波特仪器公司</td><td> 100 - F型</td><td>仪器</td></tr><tr><td> Metriclean2低发泡超声外科手术工具的解决方案</td><td> Metrex研究公司</td><td> 10-8100 </td><td> 手术试剂 </td></tr><tr><td>庆大霉素0.2克亚甲蓝缝合后抗生素喷雾40mg/ml </td><td>西格玛爱秩序</td><td> G1264 </td><td> 手术试剂 </td></tr><tr><td> Germex为消毒外科手术工具</td><td> Vétoquinol </td><td> DIN＃00141569 </td><td> 手术试剂 </td></tr><tr><td>法国眼科软膏</td><td> Vétoquinol </td><td> DIN＃00516414 </td><td> 手术试剂 </td></tr><tr><td>奈尔® </td><td>分布由教会和德怀特 - 有限责任公司</td><td>市售</td><td> 手术试剂 </td></tr><tr><td> Stanhexidine 4％W / V皮肤清洁</td><td>欧米茄实验室公司</td><td> 01938983 </td><td> 手术试剂 </td></tr><tr><td>丁丙诺啡（Temgesic）镇痛</td><td>先灵葆雅</td><td> 531-535 </td><td> 手术试剂 </td></tr><tr><td> Sulpha“25”sulphamethazine口服抗生素</td><td>专业兽医实验室</td><td> DIN＃00308218 </td><td> 手术试剂 </td></tr><tr><td>乳酸钠林格解决方案</td><td>百特公司</td><td> DIN＃0061085 </td><td> 手术试剂 </td></tr><tr><td>盐水 - 0.9％氯化钠</td><td> B - 百灵医药公司</td><td> DIN＃01924303 </td><td> 手术试剂 </td></tr><tr><td>吸入麻醉药 - 异氟醚的USP </td><td>加拿大公司的制药合作伙伴</td><td> DIN＃02237518 </td><td> 手术试剂 </td></tr><tr><td>快绿FCF </td><td> Sigma - Aldrich公司</td><td> F7252 </td><td> 手术试剂 </td></tr></tbody></table>

efficient gene delivery into multiple cns territories using in utero electroporation

操纵基因表达的能力是现代实验胚胎学的基石，从而澄清了多种发展途径。几个功能强大和完善的转基因技术操纵在小鼠的基因表达水平，从而损俱损，增益功能模型的生成。然而，转基因小鼠的一代，是既费钱又费时。因此，基因操作的替代方法得到了广泛的追捧。 电是在子宫内的基因传递到活老鼠胚胎1,2，我们已经成功地适应了3,4方法。它主要是在OVO电通常使用的技术在鸡5，成功的基础上。简单地说，DNA注入开放心室发育中的大脑和应用的电气目前形成细胞膜中的瞬态毛孔成因，允许进入细胞内的DNA吸收。在我们的手中，胚胎可有效电穿孔早期胚胎一天（五）11.5，而针对年轻的胚胎将需要超声引导下的显微注射的协议，如前面描述6。相反，E15.5，是最新的阶段，我们可以很容易地electroporate，由于顶叶和额骨分化，阻碍微量注射入脑的发病。相比之下，视网膜是通过胚胎发育的结束访问。整个胚胎或产后早期胚胎可以在任何时间点收集。注射记者构造有利于转染细胞的鉴定。 迄今为止，在子宫内的电已被最广泛使用的新皮层发展 1,2,3,4的分析。最近的研究有针对性的胚胎视网膜7,8,9和10,11,12丘脑。在这里，我们提出了一个在子宫内电协议，可以很容易地适应目标的胚胎中枢神经系统的不同域的修改。我们提供的证据表明，使用这种技术，我们可以针对胚胎的端脑，间脑和视网膜。代表性的成果是，首先使用这种技术的引入侧脑室DNA的表达结构，使我们能够监测胚胎端脑祖细胞成熟，分化和迁移。我们还表明，这种技术可用于靶DNA周围的3 路心室间脑领土，允许分化成间脑核神经元被监视的迁徙路线。最后，我们证明的micromanipulators使用，使我们能够准确地引入小目标的地区，包括视网膜下腔的DNA结构，让我们来分析视网膜发育操纵基因表达的影响。

Watch this Scientific Journal Video about Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation at JoVE.com

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

在子宫内电允许在一个空间和时间控制在发展中国家的中枢神经系统（CNS）的方式进行快速的基因传递。在这里，我们描述了一种高适应的在子宫内电的协议，可用于提供成多个胚胎中枢神经系统领域，包括端脑，间脑和视网膜的表达结构。

高效的基因传递到多个中枢神经系统使用新界在子宫内电穿孔

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

Research

JoVE Journal

Neuroscience

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

超声引导下显微注射到小鼠前脑在子宫内在E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development.  However, because the uterine wall is opaque ...

科研

神经科学

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

在子宫内的电穿孔其次原发性神经文化为研究基因的功能在皮层神经元的一个子集

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

基因传递到产后大鼠脑非心室质粒注射和电穿孔

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

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 ...

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 ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

外用转基因大鼠通过代<em&gt;在子宫内</em&gt;电穿孔和<em&gt;在体内</em&gt;生物发光筛选

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

遗传操纵鼠标的发展下丘脑通过<em&gt;在子宫内</em&gt;电穿孔

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

谷氨酸和GABA快速微离子导入：一个有用​​的工具，调查突触整合

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

鼠标显微注水技术进行有针对性的物质传递到中枢神经系统<i&gt;通过</i&gt;的颈内动脉

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

延期共聚焦显像移植神经元在器官性切片培养胚胎小鼠脑使用<em&gt;在Utero</em&gt;电穿孔

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...