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注射器和无菌乳酸林格氏液。 </li></ol> 2。麻醉<ol><li>将动物里面的小（12厘米x 20厘米），麻醉室。使用喷雾器，提供5％的异氟醚麻醉和氧气在1L/min时（图1A，A"）。值得注意的是，重要的是要收集异氟醚废气清除系统。 </li><li>从动物室时，呼吸变得深和定期。脚趾捏和眨眼反射测试，以确保充分麻醉。 </li><li>孕鼠在它的后面放置在一个加热垫（37℃）之上。加热垫是先用70％乙醇擦拭消毒，然后消毒，lintless纱布覆盖。使用呼吸器管和面罩仪器提供异氟醚（2.25％）在手术过程中。 </li><li>颈背皮肤下注射丁丙诺啡（为0.1mg/kg体重）皮下。 </li><li>监控整个手术深和定期的呼吸的动物。动物不应该喘气，呼吸浅或迅速。浅，呼吸急促，可能是浅麻醉的标志，并应增加异氟醚的流动。如果动物开始喘息，降低异氟醚的流动。在一个呼吸骤停的情况下，关闭异氟醚关闭，并增加氧流量，直至动物复苏。重新启动在低浓度的异氟醚。 </li></ol> 3。动物准备手术<ol><li>磁带前肢动物加热垫，并再次检查了收缩反应钝钳捏后足。 </li><li>用消毒棉签拭子，蔓延到腹部的脱毛霜厚层（即奈尔）。在坚定的笔触，确保underfur是完全包覆。留在一个三分钟的平均的间歇性脱毛干净，用无菌棉签，检查。湿一个无菌纱布，用无菌水，抹在腹部，直到所有的痕迹都不见了。重复与洗必泰（即Stanhexidine）皮肤清洁，多饮水。干燥与干净，无菌纱布腹部。 </li><li>更多的无菌水（即Stanhexidine）遵循一个洗必泰消毒切口部位。干燥与干净，无菌纱布腹部。重复此步骤，使用70％乙醇和新鲜，消毒棉签。 </li><li>放置在腹部将在手术过程中通过子宫角拉到中间的狭缝，用无菌纱布垫。 </li></ol> 4。切口和子宫喇叭曝光<ol><li>为了揭露子宫角，首先找到白线作为中线的皮肤下隐隐线。使用镊子，拔离腹壁皮肤切一小，从中期腹部向下与皮肤剪刀（约1厘米的长度）的直切口。 </li><li>用弯钳和剪刀，与腹膜重复的过程。 </li><li>一个小切口垂直缝无菌纱布浸湿温热无菌生理盐水。 </li><li>滑入切口钳，找到子宫。保持开放与钳切口，同时取出子宫角。 </li><li>认真掌握之间的第一次和第二次的可见胚胎的子宫和拉胚胎上的纱布。取出子宫角两侧，放置在无菌纱布上，数胚胎数，并指出任何再吸收或"不健康"（如车身尺寸较小）胚胎。用生理盐水湿子宫角，并仔细地返回到腹部子宫的一半。 </li></ol> 5。 DNA注射入脑室<ol><li>手术针准备一个萨特火焰山与毛细血管的微管采用高硼硅微量拖轮（计划:热量= 750，拉= 30，VEL = 90，时间= 150） 。用消毒的手术刀刀片，针尖轻轻地剃掉了一个45度角。 </li><li>要加载针，巴斯德吸液管拉至下一个火焰的直径缩小。甲口片连接到加载吸管制订〜10μL3mg/ml内毒素的DNA混合，用0.01％甲基绿（注:绿色染料甲基允许DNA注射到可视化）。充满针，然后装在一个显微的立场，并连接到Femtojet喷油。 </li><li>最接近卵巢的胚胎开始，圆形钳轻轻转动蜕膜内的胚胎，使定位"面对面"，使注射发生在一个喙尾鳍方向。光纤光纤照明光源是用来帮助胚胎的大脑，这是由延髓地区的大型横向脑室（图1B，B"）两侧可视化的中线。 </li><li>胚胎是使用圆形钳的位置举行。针的上方，使子宫进入大脑的角度约45度。使用的显微操纵器，和一个快速和稳定的的议案，针尖插入子宫壁，进入胚胎的侧脑室。针对侧脑室，第三脑室或目标端脑，间脑和视网膜，视网膜下腔分别注射。请注意，在早期的DNA 注入侧脑室，第三脑室流入萌芽阶段，胚胎脑囊泡开放。 </li><li>按下脚踏板，注入内毒素的DNA（1-3μL），使用的Eppendorf Femtojet微量。一个成功的注射甲基绿色染料蔓延整个胚胎脑室很容易可视化。然后将针慢慢地删除绘图备份，以避免破损。 </li></ol> 6。电穿孔<ol><li>注射胚胎（仍然子宫内）是放置在无菌纱布覆母亲的腹部和潮湿温热生理盐水彻底。请注意**除膜表面的生理盐水是必不可少的，允许电流效率通过。 </li><li> GenePaddle电极，用来夹住以下的DNA注入子宫。五平方米的40 - 50V和50毫秒的电脉冲，每使用一个ECM8300 BTX电系统（图1C，C"）第二脉冲的速度交付。 </li><li>保护湿纱布盖注入/电穿孔胚胎，并继续到下一个胚胎。 </li><li>所有所需的胚胎注入/电完成后，小心地放回子宫角腹部（图1D）。 </li><li>小心地取出子宫角，与最接近的卵巢胚胎再次开始。 </li></ol> 7。缝合线和装订<ol><li>一旦所有的胚胎的子宫内，添加到腹腔〜2-3毫升温盐水。 </li><li>要关闭伤口，消除上腹部的纱布，并确定腹膜双方开始。开始使用黑色编织真丝线缝合腹膜。这是不建议使用此过程可吸收缝合线，因为它通常较弱，并可能导致手术后的出血和感染。最接近胸骨腹膜锚线和进行针距锚。确保所有缝线严密，没有基本的器官或皮肤已无意中进入腹膜缝。其余的缝合线剪辑并丢弃。 </li><li>拉皮肤封闭在缝合线与钳。使用9毫米反射™皮肤缝合钉书针，夹在伤口的皮肤，小心不要主食下面的缝合线，或皮肤拉得太紧。首选的方法是保持皮肤和远离身体与钳，并在一个90度角，与主食工具。继续，直到伤口关闭（约三个订书钉）。一旦完成，确保订书钉紧轻轻按下缝合工具（图1D"）。 </li></ol> 8。手术后护理<ol><li>监测麻醉复苏，将到她肚子里的女性，关闭异氟醚。呼吸器内饲养动物的脸，打开氧气高达3％，以帮助从麻醉中复苏。动物应被放置在一个干净的干纱布垫，以防止低温加热垫的顶部。 </li><li>而动物是昏昏沉沉的，注入2毫升林格氏液皮下注射动物的颈部皮肤下。 </li><li>氧流量下的动物几分钟，气候变暖纱布，以振奋的动物。从动物的运动短喷，应遵守，因为它返回到一个正常的呼吸模式。观察了好几分钟，直到动物似乎意识和清醒的。 </li><li>喷雾用庆大霉素喷雾女性的腹部，并在无菌纱布包裹动物。 </li><li>转移到无菌笼子里的动物与蒸压床上用品和纸板住房。 </li><li>水瓶应彻底清洗，消毒，用无菌水填充。至500毫升水加2毫升磺胺二甲嘧啶抗生素（25％的股份）。 </li><li>管理0.05毫克/公斤丁丙诺啡上午手术后皮下。 </li><li>密切监测手术后的最初24小时内的女性。如果动物的保存时间超过24小时，女性被转移到新STERILE笼子，第2天。 </li></ol> 9。代表性的成果大脑皮层新皮层是中枢神经系统，负责视觉和感觉处理和较高的认知功能的区域。它是由谷氨酸能投射神经元和GABA能interneurons，背，腹端脑，分别来自。为了满足不同的基因在新皮层发展角色，我们使用在子宫内电要么misexpress或块不同的基因的表达在背侧或腹侧端脑1,3,4（即，通过使用的shRNA结构）。 misexpress基因在胚胎的端脑，我们使用一个双顺反子表达载体（pCIG2），包含一个CMV -βactin子增强和内部核糖体进入位点（IRES），增强型绿色荧光蛋白（EGFP; pCIG2）卡带，允许转epifluorescence（图2A - C），被检测到的细胞。我们使用这项技术的引入端脑的表达结构，从E11.5到E15.5（数据显示，在E12.5，图2）。允许胚胎培养几天后，电，我们能够跟踪的绿色荧光蛋白标记的祖细胞的分化和迁移。分化收益，后3天电GFP +细胞分化和迁移脑室区（VZ），脑室下区（SVZ）和中间区（IZ），到皮质板（CP;图2C）。 ，如果在E12.5电穿孔的大脑24小时后分析，我们可以监视合作心尖祖细胞标记的标签（即，PAX6;图2A）祖细胞的成熟和神经细胞迁移的早期事件，基底祖（即Tbr2;图2B）及有丝分裂（即Tbr1新皮层神经元;图2C）。 间脑下丘脑位于腹面的中枢神经系统和内分泌和植物神经系统之间的网关功能的基础。在发展过程中，多个细胞核，使成熟，下丘脑区分从一个共同的祖区域，行胚胎间脑的腹侧，大部分地区。目前，祖细胞的增殖，神经细胞的分化和形成下丘脑核的分子途径，规范知之甚少。我们在子宫内的目标注入第三脑室E12 embryos.We DNA报告基因的表达，胚胎间脑的电使用，随后GFP的E14，在鉴别神经元的分布。有了这种方法，我们已经成功地有针对性的GFP表达到丘脑，前丘脑和下丘脑（图3A）。冠状切片分析表明，我们已经成功祖细胞有针对性的GFP表达，行第三脑室和神经元的脑室区迁移到地幔区域，形成原子核（图3B） 视网膜视网膜是眼睛的神经层，负责传递给大脑的电脉冲转换成光子。它的发展胚胎间脑outpocketing，因此，从神经管是派生的。视网膜神经从根尖祖视网膜下的空间，从眼头间质细胞的分离，一腔相邻的车厢，在于发展。视网膜祖细胞针对DNA的结构，因此需要注射到一个小的目标区域。使用micromanipulators，我们已成功目标胚胎在这个狭小的地区从E13.5到E15.5（如图所示E15.5 mCherry记者PCIC electroporations;图4。）。如果电穿孔视网膜研究转染后24小时，我们观察到大多数mCherry +细胞分裂祖位于外成神经细胞层（onbl），而不是在视网膜神经节细胞层（GCL;插图形象），有丝分裂细胞本地化。 <img src="/files/ftp_upload/2957/2957fig1.jpg" alt="图1"/> 图1。 在子宫内电设置和方法论介绍 （一）设置手术的关键部件包括一个Femtojet的Eppendorf微量（1），Narishige显微持针器（2），徕卡steromicroscope（3），光纤照明系统（4），流脑830方波电穿孔系统电极（5）和加热垫（6）。 （A&#39;）的动物是使用异氟醚麻醉蒸发器的麻醉。 （B，B&#39;），简单地说，麻醉后，子宫角接触和快绿混杂的DNA注射到胚胎脑囊泡。一个成功的注射液是通过跟踪快速的绿色染料（B&#39;），可视化。 （C，C&#39;）的DNA后成功注入，桨电极置于子宫，positi两侧oning胚胎的DNA，以便将朝着正极被拉入大脑区域的选择。 （D，D&#39;），一旦所有需要的胚胎注入子宫推到腹部（四），腹膜缝合，皮肤装订（D"）。怀孕的女性，然后允许恢复所需的时间点收集的电穿孔幼仔。 <img src="/files/ftp_upload/2957/2957fig2.jpg" alt="图2"/> 图2。代表性的例子背telencephalic电（AC）通过一个E13.5背在E12.5电穿孔与pCIG2端脑冠状切面的显微照片。电穿孔GFP +细胞分析心尖祖细胞标志物的表达（PAX6 +，红色的A），基底祖（Tbr2 +，红色，乙）和有丝分裂的神经元（Tbr1 +，红色C ）。 CP，板皮质; SVZ，脑室下区，VZ;脑室区。 <img src="/files/ftp_upload/2957/2957fig3.jpg" alt="图3"/> 图3。间脑电击的有代表性的例子 。（A）已在E12的E14，脑电穿孔与pCIG2腹面观。绿色荧光蛋白epifluorescence是在丘脑中可见，前丘脑和下丘脑，这表明在整个第三脑室的显微注射pCIG2蔓延质粒。 （二）通过电穿孔脑冠状切面的显微照片，显示迁移的GFP +脑室区和进入地幔区，将形成下丘脑核细胞。 3V，第三脑室，T，丘脑，下丘脑;海兰; MZ，地幔区;电话，端脑; PRET，prethalamus; VZ，脑室区。 <img src="/files/ftp_upload/2957/2957fig4.jpg" alt="图4"/> 图4。视网膜电的有代表性的例子，通过电穿孔PCIC在E15.5，E16.5眼mCherry +细胞的积累外成神经细胞层的冠状切面，而不是在Brn3b +视网膜神经节细胞神经节细胞层。插图是盒装面积的高倍率图像。 GCL，视网膜神经节细胞层;乐，镜头; onbl，外成神经细胞层;重，视网膜。

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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命运的决定。这个实验的高通量是至关重要的，因为它允许许多因素的组合，在很短的时间测试。一个谨慎注意的是，此过程在细胞在基因表达的原因变化，该行针入口网站（即，在伤口上调的基因损伤反应; 我们和其他13观察）。因此，建议把重点放在伤口部位以外的电穿孔细胞。此外，胚胎存活率低时，首先学习这种技术，但迅速崛起与实践&gt; 95％。迄今为止，我们已经成功地使用在子宫内电技术，以确定前神经BHLH在端脑 4转录因子调节的基因。我们也验证了这一技术的使用分析telencephalic顺式调控元件3。

<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 ...

efficient-gene-delivery-into-multiple-cns-territories-using-utero

Nanyang Technological University

Research

JoVE Journal

Neuroscience

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

19.8K 次观看.  University of Calgary. 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。

视频: Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

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

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. However, because the uterine wall is opaque during early embryogenesis, the ability to target specific parts of the embryo for microinjection is greatly limited. Fortunately, high-frequency ultrasound imaging permits the generation of images that can be used in real time to guide a microinjection needle into the embryonic region of interest. Here we describe the use of such imaging to guide the injection of retroviral vectors into the ventricular system of the mouse forebrain at embryonic day (E) 9.5. This method uses a laparotomy to permit access to the uterine horns, and a specially designed plate that permits host embryos to be bathed in saline while they are imaged and injected. Successful surgeries often result in most or all of the injected embryos surviving to any subsequent time point of interest (embryonically or postnatally). The principles described here can be used with slight modifications to perform injections into the amnionic fluid of E8.5 embryos (thereby permitting infection along the anterior posterior extent of the neural tube, which has not yet closed), or into the ventricular system of the brain at E10.5/11.5. Furthermore, at mid-neurogenic ages (~E13.5), ultrasound imaging can be used direct injection into specific brain regions for viral infection or cell transplantation. The use of ultrasound imaging to guide in utero injections in mice is a very powerful technique that permits the molecular and cellular manipulation of mouse embryos in ways that would otherwise be exceptionally difficult if not impossible.

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

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. Here we describe the use of high-frequency ultrasound imaging to guide the injection of retroviral vectors into the mouse brain at embryonic day (E) 9.5.

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 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 1, 2. 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.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

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

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.
The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo 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

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

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

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 this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

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

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

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

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. 
 For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. 
 Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. 
 Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at &ge;6 months after birth.
 We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

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

Genes can be manipulated during development of the cortex or the hippocampus of the rat via in utero electroporation (IUE) at E16, to enable fast and targeted modifications in neuronal connectivity for later studies of behavior or neuropathology in adult animals. Postnatal in vivo imaging for control of IUE success is performed by bioluminescence of activating co-transfected luciferase.

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 modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

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

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

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

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous substances into the brain. These exogenous substances may induce injurious impact or constitute therapeutic strategy. The most common delivery methods of exogenous substances into the brain are based on systemic deliveries, such as subcutaneous or intravenous routes. Although commonly used, these approaches have several limitations, including low delivery efficacy into the brain. In contrast, surgical methods that locally deliver substances into the CNS are more specific and prevent the uptake of the exogenous substances by other organs. Several surgical methods for CNS delivery are available; however, they tend to be very traumatic. Here, we describe a mouse infusion microsurgery technique, which effectively delivers substances into the brain via the internal carotid artery, with minimal trauma and no interference with normal CNS functionality.

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

The present protocol describes a mouse microsurgery infusion technique, which effectively delivers substances directly into the brain 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 ...

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

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

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

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

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, ...