在实验室中，ES的工作是由瑞士国家科学基金会的支持。我们想感谢节拍昆茨拍摄的协助。

1。制备的RNAi质粒DNA的细胞类型特异性基因沉默质粒（ 图1）使用标准的分子克隆技术，如前面描述中详细3,4合成。 1.1克隆到载体:oligonucelotide设计<ol><li>我们使用相同的通用寡核苷酸和克隆的协议中所描述的pRFPRNAiC载体4（ARK-基因组学）提供的产品信息。 <ol class="lalpha"><li>对于克隆到第一发夹站点: 5&#39;引物HP1: 5&#39;-GGCGGGGCTAGCTGGAGAAGATGCCTTCCGGAGAGGTGCTGCTGAGCG 3&#39;引物HP1: 5&#39;-GGGTGGACGCGTAAGAGGGGAAGAAAGCTTCTAACCCCGCTATTCACCACCACTAGGCA </li><li>用于克隆到第二发夹站点: 5&#39;引物HP2: 5&#39;-GGCGGGACGCGTGCTGTGAAGATCCGAAGATGCCTTGCGCTGGTTCCTCCGTGAGCG 3&#39;引物HP2: </li><li> 5&#39;-CGCCGCGCATGCACCAAGCAGAGCAGCCTGAAGACCAGTAGGCA </li></ol></li><li>我们使用金斯瑞的选择特定基因的靶序列的siRNA的目标搜索: <a href="https://www.genscript.com/ssl-bin/app/rnai" target="_blank">https://www.genscript.com/ssl-bin/app/rnai</a> 。 </li><li>引物用于克隆一个基因特异性miRNA的到第一发夹站点: </li></ol>沉默GFP的一个例子如下所示。 靶序列（22nt）:5&#39;-GGCACAAGCTGGAGTACAACTA GFP正向HP1 = 59mer <img alt="序列1" fo:src="/files/ftp_upload/4384/4384seq1highres.jpg" src="/files/ftp_upload/4384/4384seq1.jpg" /> GFP反HP1 = 58mer <img alt="序列2" fo:src="/files/ftp_upload/4384/4384seq2highres.jpg" src="/files/ftp_upload/4384/4384seq2.jpg" /> 常见的有在这些寡核苷酸序列的组成部分miRNA的侧翼序列（鸡），和常见的循环/的干序列（从人类miRNA30），。的基因特异的靶序列用下划线标出。需要注意的是时ismatch的正链（以粗体显示; G→A在这个例子中）在这个位置上，以模仿自然不匹配的miRNA30在5&#39;基地。 <ol start="4"><li>一个基因特异性miRNA的到所述第二发夹站点中克隆用引物: </li></ol> LacZ基因沉默的一个例子如下所示。 靶序列（22nt）:5&#39;-CGCGCTGTATCGCTGGATCAAA LacZ的前锋HP2: <img alt="序列3" fo:src="/files/ftp_upload/4384/4384seq3highres.jpg" src="/files/ftp_upload/4384/4384seq3.jpg" /> LacZ的反向HP2: <img alt="序列4" fo:src="/files/ftp_upload/4384/4384seq4highres.jpg" src="/files/ftp_upload/4384/4384seq4.jpg" /> 请注意，再次，在正向链的靶序列的5&#39;的基础上已改变（以粗体显示，C→A在这个例子中），以便它不匹配的反义序列，模仿miRNA30。 1.2 PCR反应及亚克隆<ol><li>基因特异的寡核苷酸是我们连同普遍的寡聚物在PCR反应中，与鸡产生miRNA30状发夹miRNA的侧翼序列。 </li></ol><table border="1"><tbody><tr><td fo:width="2in">克隆到第一发夹网站: 1微升 - 10纳克GFP的正向引物HP1 1微升 - 100毫微克的5&#39;引物HP1 1微升 - 100毫微克的3&#39;引物HP1 1微升的dNTPs（10mM的） 5微升10倍Pfu酶反应缓冲液 1μl的Pfu DNA聚合酶（Promega）中 39μLPCR级水</td><td fo:width=".5in">或</td><td fo:width="2in">克隆到第二簪网站: 1微升 - 10毫微克LacZ的正向引物HP2 1微升HP2 - 100毫微克的5&#39;引物 1微升 - 100毫微克的3&#39;引物HP2 1微升的dNTPs（10mM的） 5微升10倍Pfu酶反应缓冲液 1μl的Pfu DNA聚合酶（Promega）中 39μLPCR级水</td></tr></tbody></table> 周期: <table border="1"><tbody><tr><td fo:width=".5in"> 94°C </td><td fo:width=".5in"> 94°C </td><td fo:width=".5in"> 55°C </td><td fo:width=".5in"> 72°C </td><td fo:width=".5in"> 72°C </td><td fo:width=".5in"> 4°C </td></tr><tr><td> 1分钟</td><td> 30秒</td><td> 30秒</td><td> 1分钟</td><td> 9分钟</td><td>持有</td></tr><tr><td></td><td colspan="3"> 30个循环</td><td></td><td></td></tr></tbody></table><ol start="2"><li>纯化的PCR产物，消化与限制性内切酶和亚克隆到载体: <ol class="lalpha"><li>第一个发夹网站:用NheI位和MluI </li><li>第二个发夹网站使用MluI位和Sph </li></ol></li><li>按照标准的技术能力的细菌细胞转化的。板细胞的的质粒midipreparation（ 如 Nucleobond Xtra的试剂盒，Machery-NAGEL）的LB培养基（含氨苄青霉素）和收获DNA。 </li><li>暂停集中的质粒DNA在无菌DDH 2 0的测定分光光度法和储存在-20°C </li></ol> 1.3测序的miRNA质粒在标准条件下的测序反应往往失败，因为强大的二次结构的发夹。为了提高这7: <ol class="lalpha"><li>进行测序反应在10mM Tris-氯与0.01毫摩尔EDTA（pH8.0）中，而不是水。这增加转换超螺旋DNA的单链DNA，这是更适合用于测序。 </li><li>添加的热变性步骤（98℃，5分钟）测序之前。这种转换到单链DNA超螺旋质粒DNA。 </li></ol> 2。电穿孔 2.1蛋的处理<ol><li>将鸡蛋打入的孵化器设置为38.5℃和〜45％的湿度。 </li><li>孵蛋，直到胚胎已经达到了预期的发展阶段。为了学习连合神经元的轴突指导，典型地Ÿelectroporate胚胎时，他们已经达到了汉堡和汉密尔顿阶段（HH）,17-18（约3天的潜伏期后）8。但是，注射和电穿孔需要做的感兴趣的蛋白质已积累之前，使击倒高效。 </li><li>从培养箱中取出鸡蛋，并把它们在一个稳定的水平位置为20分钟，以重新定位在蛋的上侧之上蛋黄胚胎。在此期间返回的鸡蛋孵化器。 </li></ol> 2.2试剂和设备的下游<ol><li>准备的磷酸盐缓冲盐水（PBS），并通过高压灭菌或通过将溶液通过一个0.2微米的过滤器消毒。 </li><li>不断玻璃micropipettes通过拉动毛细血管（世界精密仪器1B120F-4; 1.2 / 0.68外径/内径（mm））在一个合适的牵引装置（如成茂PC-10）。折断的微量吸移管的前端，得到的前端直径为〜5微米，并把它连接到一个管件与适当的直径。 </li><li>挂载的铂电极（0.5厘米长）牢固地在一个手持式的帧，与电极间的距离为0.5厘米。将电极连接到一个方波脉冲发生器（BTX ECM 830）。 </li><li>设置的脉冲发生器与以下参数: <ol class="lalpha"><li>对于单方面电:电压:25 V的脉冲数:5，脉冲长度:50毫秒，脉冲间隔:1秒</li><li>对于双边电:电压:18 V的脉冲数:5，脉冲长度:50毫秒，脉冲间隔:1秒</li></ol></li><li>准备的注射器用18G针，手术刀，斯普茨瓶的70％乙醇和磁带。 </li><li>熔融石蜡中的加热板的烧杯中在80℃下</li></ol> 2.3窗口<ol><li>使用的组织浸泡在70％乙醇擦拭的鸡蛋。 </li><li>放置一个带状体沿长轴的蛋。 </li><li>做两个小的孔，的蛋壳用手术刀;一次钝的一端鸡蛋和其他区窗口的角落。 </li><li>使用注射器，取出约。 3毫升从每个蛋蛋白到蛋的钝端处的孔中，在以45°的角度通过插入针。小心，以免损坏的蛋黄。 </li><li>开一个窗口，使用小剪刀，保持水平，以避免破坏胚胎的蛋壳。如果需要的话，可以验证和位置的胚胎，用铅笔标记，持有强烈的光线对先有蛋的钝端。 1.5〜2厘米见方的一个窗口，对于大多数应用程序是非常有用的。 </li><li>密封的孔先有蛋的钝端，任何裂缝，蛋壳融化的石蜡，用刷子。 </li><li>密封的窗口，用透明胶带（ 例如苏格兰魔术）。 </li><li>返回蛋的孵化器。 </li></ol> 2.4电穿孔<ol><li>准备无菌的工具，并用70％乙醇擦拭工作区域。 </li><li>准备的我njection的解决方案。的适当的DNA浓度，必须由用户确定的，并根据用于驱动表达的增强子/启动会有所不同。作为指导，我们通常使用0.2-2.0微克/微升（见讨论）。以总共20微升，注射混合物应包含: </li></ol><table border="1"><tbody><tr><td fo:width="2in"> RNAi的质粒DNA（在H 2 O中 ） </td><td fo:width=".5in">所述微升</td></tr><tr><td> 20倍的PBS </td><td> 1微升</td></tr><tr><td> 0.4％台盼蓝</td><td> 2微升</td></tr><tr><td>无菌DDH 2 O，最终体积为</td><td> 20微升</td></tr></tbody></table> ，使用轻柔吸加载的DNA混合物到玻璃微细管连接到油管。 <ol start="3"><li>从窗口鸡蛋，汉堡和汉密尔顿8阶段的胚胎中取出磁带。 </li><li>使用镊子和春风似剪刀小心地将extraembryonic膜的胚胎尾部的一半。膜可以很容易地从胚胎抬离的区域中主要的右和左卵黄静脉输入中继线。轻轻地撕掉或切膜，拉他们对尾。如果需要的话，可在胚胎可视化视频由Boulland和他的同事9所示，通过注射的溶液的印度油墨"或"快速"绿色胚胎光盘和蛋黄之间更好。 </li><li> DNA溶液注入到中央管的神经管，只是后肢以上。控制注射量经口。蓝色染料蔓延，从尾巴尖的心室发育中的大脑。 </li><li>添加几滴无菌PBS的胚胎的顶部。 </li><li>将平行于前侧 - 后侧的胚胎轴的电极。避免接触的胚胎或血管。 </li><li>保持稳定的电极，electroporate。 </li><li>小心地取出电极和冲洗无菌水从蛋清中删除变性的蛋白质。 <ol class="lalpha"><li>对于双边的电穿孔，电极的极性切换，重新定位胚胎平行的电极，并重复电穿孔。在无菌水中冲洗电极。 </li></ol></li><li>上掉落一些无菌PBS的胚胎。蛋用胶带重新密封，并返回它的孵化器，直到达到所需的发育阶段。适当的密封是关键的胚胎，以避免脱水。 </li></ol> 3。脊髓的准备工作 3.1胚胎解剖<ol><li> HH25-26（孵化第5天），取出胚胎从卵中，使用产钳或一小汤匙。将它放置在PBS中，在涂有聚硅氧烷（Sylgard的弹性体）的陪替氏培养皿。 </li><li>删除的胚外膜，奠定了胚胎在它的后面。稳定它通过颈部和尾（使用0.20毫米虫引脚），通过固定板，用轻柔的小号tretching。 <ol class="lalpha"><li>所有的胚胎（切片购买）可以固定在这里，取代用4％的多聚甲醛（PFA）的PBS，在室温下孵育1小时。为了使开卷制剂，继续与未定影的组织（如下文所述）的协议。 </li></ol></li></ol> 3.2从胚胎中分离脊髓<ol><li>引脚四肢，确保插针插入远离胚胎的角度，以便它们不干扰剥离。胚胎应被照射从下面启用在下面的步骤被感知的组织密度。 </li><li>取出的心脏和内脏春风似剪刀，并用钳子轻轻地刮。分段椎骨和脊髓应该是可见的，如果所有的器官已全部拆迁完毕。 </li><li>使用春风似剪刀，通过覆盖在颈部脊髓的椎体浅伤口。胚胎180°旋转，使两个短任一侧上的脊髓的椎骨，纵向通过削减从颈部朝向尾部。使用镊子掀开远离脊髓和剥离组织（包含所有的椎骨）在一个单一的条对尾椎骨。 </li><li>轻轻地伸展和引脚通过尾巴和四肢的胚胎。 </li><li>使用精细的显微外科手术刀（ 如 Grieshaber 68101）或钨针切掉脊髓脑膜覆盖。看一个黑暗的，致密的组织之间的神经管和背根神经节。调整照明角度，如果必要的话。沿着这条线的每一侧上的脊髓纵向轻轻切，从颈部到尾部。由于温和的伸展固定的胚胎脊髓脑膜分开。 </li><li>通过脊髓水平的翅芽和尾鳍肢芽，切，解除脊髓的胚胎在一个平滑的吻端到尾鳍<html>运动，使用产钳。浸入在PBS中，在此步骤中，应保持的分离的脊髓。不要抬出来的菜。 </li></ol> 3.3固定开放图书<ol><li>传播孤立的脊髓到锅铲，并将其转移到一个新的有机硅成荫的培养皿中含4％多聚甲醛的PBS。 </li><li>生产一台安装准备，仔细地钉扎脊髓中的6个职位（吻侧，中部和尾部的每一侧，使用0.10毫米的昆虫针）。我们每个标签准备一个小的"标志"，不仅确定了胚胎，但也表明了前端开卷准备。 </li><li>在室温下孵育30分钟至1小时。开书不应该被固定的DiI扩散10，因为这会降低效率并增加背景。 </li><li>小心地倒出4％PFA，取而代之的是与PBS。在4°C，直到准备注射用DiI或安装，保持菜肴。 </li></ol>e_step"&gt; 3.4的DiI荧光注入到连合神经元<ol><li>观察开放式荧光miscroscopy的书籍，并选择合适的侧开卷注射用DiI。 </li><li>准备:极速DiI荧光（5毫克/毫升在乙醇中），并绘制成的溶液的玻璃微吸管连接到塑料管。折断尽可能精确的微量吸移管的端部，得到一个非常小的直径的小费。将针头插入一盘PBS和检查，DII不漏针。如果的DiI荧光渗漏，的针直径过大。在这种情况下，准备新的针头。 </li><li>从下面照亮开卷准备。查找其它组织，从制剂的横向边缘一个hemibook宽度的约1/5位于更致密的纵向条纹。这对应的连合神经元，而被发现的顶板只是腹侧的细胞体。在开卷的一端开始，插入玻璃针进入组织，并作为针我小号撤回，粉扑少量用口吸管的直接投资收益。 </li><li>工作迅速，使沿开卷在定期的时间间隔大约为0.5毫米的长度的几个注射。如果变得堵塞针头，用钳子小心地清除。如果笔尖变得过大（和DiI泄漏），更换针。 </li><li>当你完成每一个打开的书，用移液管吸走，并丢弃任何多余的，泄露DII。如果不这样做将导致高背景。 </li><li>留下约三天的准备工作，在4°C，使DII沿轴突传播。 </li></ol> 3.5安装成像<ol><li>使用18 G针的注射器传播薄的，不间断的边界真空油脂（ 如道康宁976V）围绕着一个24毫米×24毫米盖玻片的边缘。添加数滴无菌PBS井。请注意，不可以共同安装介质含有甘油与n-丙基没食子儿茶素没食子酸酯mpatible与DiI荧光11。同样，真空润滑脂的粘度低，可能与PBS混合，导致高背景。 </li><li>取下标签从开卷，并将其转移到在PBS液滴。沉浸在开卷，并将其定位在中间的孔。 </li><li>轻轻将另外24毫米x 24毫米盖玻片之上，，确保开卷保持开放。如果有必要，可以删除，盖玻片开卷重新定位。轻轻按压周围的边缘准备建立一个完整的密封油脂。被挤掉多余的PBS将在此步骤中。避免气泡。 </li><li>在黑暗中保持准备在4°C，直到准备用荧光显微镜检查和文件。这项工作应尽快（一周内），以确保筹备工作干不出来。 </li></ol> 4。代表性的成果电穿孔和表达的质粒在上述条件下，荧光蛋白应该清楚地检测到在适当的细胞类型，而不需要额外的放大的信号通过抗体标记。的荧光蛋白质只应被检测出，在所需的细胞类型/ s的。开卷筹备和与不同的质粒电穿孔的胚胎的横截面在图2中所示的代表性例子。 人工miRNA的效率必须先进行筛选，对一种新的感兴趣的基因的人工的miRNA其击倒效应的效率和特异性。我们发现，β-肌动蛋白启动子驱动的结构，电穿孔0.25微克/微升，这3适当的。击倒在体内可通过免疫组化或原位杂交检测。 DiI荧光标记 DiI荧光注入到适当的有针对性的野生型胚胎产量的80％以上，注射部位有理想，有典型的轨迹， 如图3所示。动物变异的动物要低。 <img alt="图1" src="/files/ftp_upload/4384/4384fig1.jpg" /> 图1广义miRNA的表达质粒载体的示意图。不同的RNA聚合酶II启动子/增强子的使用使细胞类型的特异性表达。转染的细胞识别所表达的荧光记者直接链接（在一个单一的成绩单），以一个或两个人工miRNA中，敲除基因的表达。粗体字表示一个人工的miRNA对LacZ基因的有义链，如文中所述。 <img alt="图2" src="/files/ftp_upload/4384/4384fig2.jpg" /> 代表性的例子Ø 图2。f荧光蛋白的表达模​​式的指示的质粒载体电穿孔后得到的。横截面和开放的书是从鸡胚电HH18 HH25-26。 β-肌动蛋白启动子驱动无处不在表达，MATH1增强表达在DI1神经元和Hoxa1基因的增强表达，特别是在地板上。 CN，连合神经元; FP，楼承板。 <img alt="图3" src="/files/ftp_upload/4384/4384fig3.jpg" /> 图3。本打开的书准备的DiI注射部位的应用和分析。 DiI荧光应注入中的点状图案，靠近外侧缘打开的书页上，将电穿孔的侧（确定的荧光蛋白的表达）。经过3天的扩散，连合轴突轨迹应该是能够在荧光显微镜下观察。正常的的轴突轨迹将增长向地面p晚，穿过底板，，然后吻侧转身增长。基因敲下来所产生的异常表型可比较这个原型的轨迹。在这个例子中，一些神经轴突在底板档或错误将决定对侧。

<ol>
	<li>Ch&#233;dotal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Further+tales+of+the+midline.">Further tales of the midline.</a> Current Opinion in Neurobiology. 21, 68-75 (2011).</li><li>Avraham, O., Hadas, Y., Vald, L., Zisman, S., Schejter, A., Visel, A., Klar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+control+of+axonal+guidance+and+sorting+in+dorsal+interneurons+by+the+Lim-HD+proteins+Lhx9+and+Lhx1.">Transcriptional control of axonal guidance and sorting in dorsal interneurons by the Lim-HD proteins Lhx9 and Lhx1.</a> Neural Development. 4, 21 (2009).</li><li>Wilson, N. H., Stoeckli, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type+specific,+traceable+gene+silencing+for+functional+gene+analysis+during+vertebrate+neural+development.">Cell type specific, traceable gene silencing for functional gene analysis during vertebrate neural development.</a> Nucleic Acids Research. 39, e133 (2011).</li><li>Das, R. M., van Hateren, N. J., Howell, G. R., Farrell, E. R., Bangs, F. K., Porteous, V. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+robust+system+for+RNA+interference+in+the+chicken+using+a+modified+microRNA+operon.">A robust system for RNA interference in the chicken using a modified microRNA operon.</a> Developmental Biology. 294, 554-563 (2006).</li><li>Perrin, F. E., Stoeckli, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+lipophilic+dyes+in+studies+of+axonal+pathfinding+in+vivo.">Use of lipophilic dyes in studies of axonal pathfinding in vivo.</a> Microscopy Research and Technique. 48, 25-31 (2000).</li><li>Timmer, J., Johnson, J., Niswander, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+in+ovo+electroporation+for+the+rapid+analysis+of+neural-specific+murine+enhancers.">The use of in ovo electroporation for the rapid analysis of neural-specific murine enhancers.</a> Genesis. 29, 123-132 (2001).</li><li>Kieleczawa, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+modifications+of+the+standard+DNA+sequencing+protocol+allow+for+sequencing+through+siRNA+hairpins+and+other+repeats.">Simple modifications of the standard DNA sequencing protocol allow for sequencing through siRNA hairpins and other repeats.</a> Journal of Biomolecular Techniques. 16, 220-223 (2005).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> Journal of Morphology. 88, 49-92 (1951).</li><li>Boulland, J., Halasi, G., Kasumacic, N., Glover, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenotransplantation+of+Human+Stem+Cells+into+the+Chicken+Embryo.">Xenotransplantation of Human Stem Cells into the Chicken Embryo.</a> J. Vis. Exp. (41), e2071 (2010).</li><li>Kim, B. G., Dai, H. -. N., McAtee, M., Vicini, S., Bregman, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labeling+of+dendritic+spines+with+the+carbocyanine+dye+DiI+for+confocal+microscopic+imaging+in+lightly+fixed+cortical+slices.">Labeling of dendritic spines with the carbocyanine dye DiI for confocal microscopic imaging in lightly fixed cortical slices.</a> Journal of Neuroscience Methods. 162, 237-243 (2007).</li><li>Murphy, M. C., Fox, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anterograde+tracing+method+using+DiI+to+label+vagal+innervation+of+the+embryonic+and+early+postnatal+mouse+gastrointestinal+tract.">Anterograde tracing method using DiI to label vagal innervation of the embryonic and early postnatal mouse gastrointestinal tract.</a> Journal of Neuroscience Methods. 163, 213-225 (2007).</li><li>Helms, A. W., Abney, A. L., Ben-Arie, N., Zoghbi, H. Y., Johnson, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoregulation+and+multiple+enhancers+control+Math1+expression+in+the+developing+nervous+system.">Autoregulation and multiple enhancers control Math1 expression in the developing nervous system.</a> Development. 127, 1185-1196 (2000).</li><li>Li, X., Lufkin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cre+recombinase+expression+in+the+floorplate,+notochord+and+gut+epithelium+in+transgenic+embryos+driven+by+the+Hoxa-1+enhancer+III.">Cre recombinase expression in the floorplate, notochord and gut epithelium in transgenic embryos driven by the Hoxa-1 enhancer III.</a> Genesis. 26, 121-122 (2000).</li><li>Mauti, O., Baeriswyl, T., Stoeckli, E. T. G. e. n. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+by+Injection+and+Electroporation+of+dsRNA+in+Avian+Embryos.">Silencing by Injection and Electroporation of dsRNA in Avian Embryos.</a> Cold Spring Harbor Protocols. , (2008).</li><li>Krull, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+primer+on+using+in+ovo+electroporation+to+analyze+gene+function.">A primer on using in ovo electroporation to analyze gene function.</a> Developmental Dynamics. 229, 433-439 (2004).</li><li>Cullen, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+and+confirming+the+specificity+of+RNAi+experiments.">Enhancing and confirming the specificity of RNAi experiments.</a> Nature Methods. 3, 677-681 (2006).</li></ol>

没有利益冲突的声明。

这个简单的，基于矢量的人工miRNA的表达策略可用于敲除内源​​性基因表达的鸡神经管。这些功能的工具提供了多种基因沉默，时间控制和特异性的细胞类型，以方便阐明复杂的发育途径。特别是，我们已经证明了这些质粒中的实用程序在连合轴突引导，因为质粒可用于连合神经元的不同的基因敲除，或在它们的中间目标中，floorplate 3。 miRNA的表达质粒（因此，沉?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> 0.5毫米的玻璃毛细管</td><td>世界精密仪器</td><td> 1B120F-4 </td></tr><tr><td>玻璃针拉马</td><td>成重</td><td> PC-10 </td></tr><tr><td>电穿孔</td><td> BTX </td><td> ECM 830 </td></tr><tr><td> Sylgard的有机硅弹性体</td><td>世界精密仪器</td><td> SYLG184 </td></tr><tr><td>钨丝，0.075毫米; </td><td>世界精密仪器</td><td> TGW0325 </td></tr><tr><td>昆虫引脚，0.20毫米</td><td>精细科学工具</td><td> 26002-20 </td></tr><tr><td>昆虫引脚，0.10毫米</td><td>精细科学工具</td><td> 26002-10 </td></tr><tr><td>春风似剪刀</td><td>精细科学OLS </td><td> 15003-08 </td></tr><tr><td>杜蒙＃5镊子</td><td>精细科学工具</td><td> 11252-20 </td></tr><tr><td>杜蒙＃55镊子</td><td>精细科学工具</td><td> 11255-20 </td></tr><tr><td>快速DiI荧光</td><td>分子探针</td><td> D-7756 </td></tr><tr><td>荧光显微镜</td><td>奥林巴斯</td><td> SZX12，BX51 </td></tr></tbody></table>

in ovo electroporation of mirna-based plasmids in the developing neural tube and assessment of phenotypes by dii injection in open-book preparations

，连合DI1神经元已被广泛研究，阐明在开发过程中1,2轴突导向机制。这些神经元位于脊髓背侧，并发送它们的轴突沿着千篇一律的轨迹。最初连合轴突投射腹侧向，然后整个floorplate的。这些轴突穿越中线后，锋利的喙转，纵向对大脑的项目。受这些步骤中的每一步的协调活动的吸引力和排斥力的指导线索。这些线索的正确解释轴突的他们的划定途径上的指导是至关重要的。因此，一个特定的分子的生理贡献连合轴突引导理想地调查的上下文中的生活胚胎。因此，必须精确控制体内的基因敲除，以仔细分辨轴突导向活动的基因，可以播放多种在开发过程中的角色。 在这里，我们描述的方法敲除基因表达的鸡神经管在细胞类型特异性的，可追溯的方式。我们使用3窝藏细胞类型特异性的启动子/增强子驱动表达的荧光蛋白的标志，然后直接由miR30的RNAi誊4（位于内的3&#39;-UTR的cDNA编码的荧光蛋白质）（新的质粒载体图1）。神经管电转化时，这些载体引起有效的基因表达下调，表达明亮的荧光标记蛋白，能直接跟踪的细胞经历击倒3。混合不同的RNAi载体电穿孔之前，允许在独立的区域的脊髓中的两个或多个基因的同时击倒。这使得复杂的细胞和分子间的相互作用，在开发过程中，要检查的方式，速度快，S实施，精确和便宜。用DiI开卷准备5连合轴突轨迹跟踪的结合，这种方法是一种有用的工具， 体内的细胞和分子机制的研究，连合轴突生长和指导。原则上，任何启动子/增强子可以被使用，可能使该技术更广泛地适用于在体内研究6在开发过程中的基因功能。 此视频演示了如何处理和窗口鸡蛋，成神经管和注射的DNA质粒，电穿孔程序。连合轴突导向探讨，从胚胎中取出脊髓为开卷编制，固定，注射用DiI，使轴突途径可以追溯到。脊髓之间安装的盖玻片和可视化，利用共聚焦显微镜。

Watch this Scientific Journal Video about In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations at JoVE.c

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

可以下调基因的表达在神经管的方法是，在特定的细胞类型，可跟踪的方式进行描述。我们演示了如何<em在OVO</em&gt;电的小分子RNA的质粒，引起时空控制的RNA干扰可用于研究在发展中的神经管连合轴突导向。

基于miRNA的质粒在发展中的神经管和表型的评估DiI荧光注射开卷准备在卵的电

Commissural dI1 neurons have been extensively studied to elucidate the mechanisms underlying axon guidance during development1,2. These neurons are ...

in-ovo-electroporation-mirna-based-plasmids-developing-neural-tube

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

16.4K 次观看.  University of Zurich. 可以下调基因的表达在神经管的方法是，在特定的细胞类型，可跟踪的方式进行描述。我们演示了如何

视频: In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos1,2 that we have successfully adapted3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick5. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described6. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.
To date, in utero electroporation has been most widely used for the analysis of neocortical development1,2,3,4. More recent studies have targeted the embryonic retina7,8,9 and thalamus10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.

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 ...

科研

神经科学

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 ...

In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos

The rhombic lip is an embryonic neuroepithelium located in the hindbrain at the junction between the neural tube and the roofplate of the fourth ventricle (reviewed in 1). The rhombic lip can be subdivided into the upper rhombic lip (URL) which encompasses rhombomere 1 (r1) and generates neurons of the cerebellum and the lower rhombic lip (LRL) which gives rise to diverse neuronal brainstem lineages 2-4. LRL derivatives include the auditory neurons of the cochlear nuclei and those of the precerebellar nuclei that are involved in regulating balance and motor control 5-8. Neurogenesis from the LRL occurs over a large temporal window that encompasses embryonic days (E) 9.5-16.55, 9. Different neuronal lineages emerge from the LRL as postmitotic cells (or are born) during distinct developmental days during this neurogenic window. 
Electroporation of gene expression constructs can be used to manipulate gene expression in LRL progenitors and can potentially change the fate of the neurons produced from this region 10-12. Altering gene expression of LRL progenitors in the mouse via in utero electroporation has been highly successful for manipulating lineages born on embryonic day E12.5 or later 10, 12-14. In utero electroporations prior to E12.5 have been unsuccessful primarily due to the lethality associated with puncturing the fourth ventricle roofplate, a necessary step in delivering exogenous DNA that is electroporated into the LRL. However, many LRL derived lineages arise from the LRL earlier than E12.5 9. These earlier born lineages include the neurons that comprise the lateral reticular, external cuneate, and inferior olivary nuclei of the precerebellar system which function to connect inputs from the spinal cord and cortex to the cerebellum 5. In order to manipulate expression in the LRL of embryos younger than E12.5, we developed an in vitro system in which embryos are placed into culture following electroporation.
This study presents an efficient and effective method for manipulating the gene expression of LRL progenitors at E11.5. Embryos electroporated with green fluorescent protein (GFP) driven from the broadly active CAG promoter reproducibly expressed GFP after 24 hours of culture. A critical aspect of this assay is that gene expression is only altered because of the expression of the exogenous gene and not because of secondary effects that result from the electroporation and culturing techniques. It was determined that the endogenous gene expression patterns remain undisturbed in electroporated and cultured embryos. This assay can be utilized to alter the fate of cells emerging from the LRL of embryos younger than E12.5 through the introduction of plasmids for overexpression or knock down (through RNAi) of different pro-neural transcription factors.

In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos

This study describes the development of an in vitro electroporation technique that allows for the manipulation of gene expression in the lower rhombic lip of midgestation embryos.

菱形唇下的Midgestation小鼠胚胎在体外电穿孔

The rhombic lip is an embryonic neuroepithelium located in the hindbrain at the junction between the neural tube and the roofplate of the fourth ventricle ...

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

在OVO在鸡胚中脑多巴胺神经元发育中的基因功能研究电穿孔

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial and temporal control of ectopic microRNA expression. However, ventral brain structures like ventral midbrain are rather difficult to reach for any manipulations. Here, we demonstrate an efficient way to electroporate miRNA into ventral midbrain using thin platinum electrodes. This method offers a reliable way to transfect specific areas of the midbrain and a useful tool for in vivo studies.

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Ectopic expression is one technique to elucidate the microRNAs role in brain development. However, targeting specific areas using in ovo electroporation is challenging. Here, we show an efficient way to selectively electroporate ventral and dorsal midbrain regions.

在微RNA在腹鸡中脑大毛表达

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial ...

Murine Neural Plate Targeting by In Utero Nano-Injection (NEPTUNE) at Embryonic Day 7.5

Manipulating gene expression in the developing mouse brain in utero holds great potential for functional genetics studies. However, it has previously largely been restricted to the manipulation of embryonic stages post-neurulation. A protocol was developed to inject the amniotic cavity at embryonic day (E)7.5 and deliver lentivirus, encoding cDNA or shRNA, targeting &gt;95% of the neural plate and neural crest cells, contributing to the future brain, spinal cord, and peripheral nervous system. This protocol describes the steps necessary to achieve successful transduction, including grinding of the glass capillary needles, pregnancy verification, developmental staging using ultrasound imaging, and optimal injection volumes matched to embryonic stages. 
Following this protocol, it is possible to achieve transduction of &gt;95% of the developing brain with high-titer lentivirus and thus perform whole-brain genetic manipulation. In contrast, it is possible to achieve mosaic transduction using lower viral titers, allowing for genetic screening or lineage tracing. Injection at E7.5 also targets ectoderm and neural crest contributing to distinct compartments of the eye, tongue, and peripheral nervous system. This technique thus offers the possibility to manipulate gene expression in mouse neural-plate- and ectoderm-derived tissues from preneurulation stages, with the benefit of reducing the number of mice used in experiments.

Murine Neural Plate Targeting by In Utero Nano-Injection NEPTUNE at Embryonic Day 7.5

In this protocol, we describe how to inject the mouse amniotic cavity at E7.5 with lentivirus, leading to uniform transduction of the entire neural plate, with minimal detrimental effects on survival or embryonic development.

在胚胎第7.5天通过子宫内纳米注射（NEPTUNE）靶向小鼠神经板

Manipulating gene expression in the developing mouse brain in utero holds great potential for functional genetics studies. However, it has previously ...

发育生物学

Loss-of-Function Approach in the Embryonic Chick Retina by Using Tol2 Transposon-Mediated Transgenic Expression of Artificial microRNAs

The chick retina has long been an important model system in developmental neurobiology, with advantages including its large size, rapid development, and accessibility for visualization and experimental manipulations. However, its major technical limitation had been the lack of robust loss-of-function approaches for gene function analyses. This protocol describes a methodology of gene silencing in the developing chick retina that involves transgenic expression of artificial microRNAs (miRNAs) by using the Tol2 transposon system. In this approach, a Tol2 transposon plasmid that contains an expression cassette for the EmGFP (emerald green fluorescent protein) marker and artificial pre-miRNA sequences against a target gene is introduced into the embryonic chick retina with a Tol2 transposase expression construct by in ovo electroporation. In the transfected retinal cells, the transposase catalyzes the excision of the expression cassette from the transposon vector and its integration into host chromosomes, leading to the stable expression of miRNAs and the EmGFP protein. In our previous study, we have demonstrated that the expression of Nel, a glycoprotein that exerts multiple functions in neural development, can be significantly suppressed in the developing chick retina by using this technique. Our results indicate that this methodology induces a stable and robust suppression of gene expression and thus provides an efficient loss-of-function approach for studies of retinal development.

We have developed a novel loss-of-function approach that involves the introduction and genomic integration of artificial micro-RNA sequences into chick embryos by using in ovo electroporation and the Tol2 transposon system. This technique provides a robust and stable gene knockdown methodology for studies of gene function during development.

使用Tol2转座子介导的人工microRNA的转基因表达在胚胎雏鸡视网膜中实现功能丧失的方法

The chick retina has long been an important model system in developmental neurobiology, with advantages including its large size, rapid development, and ...

Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation

Fragile X mental retardation protein (FMRP) is an mRNA-binding protein that regulates local protein translation. FMRP loss or dysfunction leads to aberrant neuronal and synaptic activities in fragile X syndrome (FXS), which is characterized by intellectual disability, sensory abnormalities, and social communication problems. Studies of FMRP function and FXS pathogenesis have primarily been conducted with Fmr1 (the gene encoding FMRP) knockout in transgenic animals. Here we report an in vivo method for determining the cell-autonomous function of FMRP during the period of circuit assembly and synaptic formation using chicken embryos. This method employs stage-, site-, and direction-specific electroporation of a drug-inducible vector system containing Fmr1 small hairpin RNA (shRNA) and an EGFP reporter. With this method, we achieved selective FMRP knockdown in the auditory ganglion (AG) and in one of its brainstem targets, the nucleus magnocellularis (NM), thus providing a component-specific manipulation within the AG-NM circuit. Additionally, the mosaic pattern of the transfection allows within-animal controls and neighboring neuron/fiber comparisons for enhanced reliability and sensitivity in data analyzing. The inducible vector system provides temporal control of gene editing onset to minimize accumulating developmental effects. The combination of these strategies provides an innovative tool to dissect the cell-autonomous function of FMRP in synaptic and circuit development.

Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation

Using in ovo electroporation, we devised a method to selectively transfect the auditory inner ear and cochlear nucleus in chicken embryos to achieve a cell-group-specific knockdown of fragile X mental retardation protein during discrete periods of circuit assembly.

通过产 卵电 穿孔解剖听觉回路中脆性X智力迟钝蛋白的细胞自主功能

Fragile X mental retardation protein (FMRP) is an mRNA-binding protein that regulates local protein translation. FMRP loss or dysfunction leads to ...

A Pipeline using Bilateral In Utero Electroporation to Interrogate Genetic Influences on Rodent Behavior

As genome-wide association studies shed light on the heterogeneous genetic underpinnings of many neurological diseases, the need to study the contribution of specific genes to brain development and function increases. Relying on mouse models to study the role of specific genetic manipulations is not always feasible since transgenic mouse lines are quite costly and many novel disease-associated genes do not yet have commercially available genetic lines. Additionally, it can take years of development and validation to create a mouse line. In utero electroporation offers a relatively quick and easy method to manipulate gene expression in a cell-type specific manner in vivo that only requires developing a DNA plasmid to achieve a particular genetic manipulation. Bilateral in utero electroporation can be used to target large populations of frontal cortex pyramidal neurons. Combining this gene transfer method with behavioral approaches allows one to study the effects of genetic manipulations on the function of prefrontal cortex networks and the social behavior of juvenile and adult mice.

The role of recently discovered disease-associated genes in the pathogenesis of neuropsychiatric disorders remains obscure. A modified bilateral in utero electroporation technique allows for the gene transfer in large populations of neurons and examination of the causative effects of gene expression changes on social behavior.

使用双边子宫内电渗透的管道，以询问遗传对啮齿动物行为的影响

As genome-wide association studies shed light on the heterogeneous genetic underpinnings of many neurological diseases, the need to study the contribution ...

生物学

Murine In Utero Electroporation

In utero electroporation is an important technique for studying the molecular mechanisms that guide the proliferation, differentiation, migration, and maturation of cells during neural development. Electroporation enables the rapid and targeted delivery of material into cells by utilizing electrical pulses to create transient pores in cell membranes. Although electroporation has traditionally been used in in vitro studies, scientific advancements have now broadened its utilization to intact organs, such as those found in mouse embryos developing in utero. This video will introduce the key principles behind in utero electroporation in addition to reviewing the basic surgical techniques required to access developing embryos within a pregnant rodent. Details of the injection and electroporation steps are provided along with important considerations for directing gene delivery to specific brain regions. Finally, neurobiological applications of in utero electroporation are presented, such as investigating how specific genes contribute to neural development and how connections form between developing neurons.

Murine In Utero Electroporation

小鼠子宫内电穿孔

In utero electroporation is an important technique for studying the molecular mechanisms that guide the proliferation, differentiation, migration, and ...

基于miRNA的质粒在发展中的神经管和表型的评估DiI荧光注射开卷准备在卵的电

摘要

探索更多视频

此视频中的章节

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

在子宫内电穿孔：鼠标控制时空基因Transefection

菱形唇下的Midgestation小鼠胚胎在体外电穿孔

在OVO在鸡胚中脑多巴胺神经元发育中的基因功能研究电穿孔

在微RNA在腹鸡中脑大毛表达

在胚胎第7.5天通过子宫内纳米注射（NEPTUNE）靶向小鼠神经板

使用Tol2转座子介导的人工microRNA的转基因表达在胚胎雏鸡视网膜中实现功能丧失的方法

通过产卵电穿孔解剖听觉回路中脆性X智力迟钝蛋白的细胞自主功能

使用双边子宫内电渗透的管道，以询问遗传对啮齿动物行为的影响

小鼠子宫内电穿孔

基于miRNA的质粒在发展中的神经管和表型的评估DiI荧光注射开卷准备在卵的电

摘要

探索更多视频

此视频中的章节

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

在子宫内电穿孔：鼠标控制时空基因Transefection

菱形唇下的Midgestation小鼠胚胎在体外电穿孔

在OVO在鸡胚中脑多巴胺神经元发育中的基因功能研究电穿孔

在微RNA在腹鸡中脑大毛表达

在胚胎第7.5天通过子宫内纳米注射（NEPTUNE）靶向小鼠神经板

使用Tol2转座子介导的人工microRNA的转基因表达在胚胎雏鸡视网膜中实现功能丧失的方法

通过产 卵电 穿孔解剖听觉回路中脆性X智力迟钝蛋白的细胞自主功能

使用双边子宫内电渗透的管道，以询问遗传对啮齿动物行为的影响

小鼠子宫内电穿孔

通过产卵电穿孔解剖听觉回路中脆性X智力迟钝蛋白的细胞自主功能