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. 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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

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

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

从胚胎脊髓​​连合神经的解剖和文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

科研

神经科学

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in rodents have shown a direct link between testosterone and aggressive behavior1 and folk wisdom adapts these findings to humans, suggesting that testosterone induces antisocial, egoistic or even aggressive behavior2. However, many researchers doubt a direct testosterone-aggression link in humans, arguing instead that testosterone is primarily involved in status-related behavior3,4. As a high status can also be achieved by aggressive and antisocial means it can be difficult to distinguish between anti-social and status seeking behavior.
We therefore set up an experimental environment, in which status can only be achieved by prosocial means. In a double-blind and placebo-controlled experiment, we administered a single sublingual dose of 0.5 mg of testosterone (with a hydroxypropyl-&beta;-cyclodextrin carrier) to 121 women and investigated their social interaction behavior in an economic bargaining paradigm. Real monetary incentives are at stake in this paradigm; every player A receives a certain amount of money and has to make an offer to another player B on how to share the money. If B accepts, she gets what was offered and player A keeps the rest. If B refuses the offer, nobody gets anything. A status seeking player A is expected to avoid being rejected by behaving in a prosocial way, i.e. by making higher offers.
The results show that if expectations about the hormone are controlled for, testosterone administration leads to a significant increase in fair bargaining offers compared to placebo. The role of expectations is reflected in the fact that subjects who report that they believe to have received testosterone make lower offers than those who say they believe that they were treated with a placebo. These findings suggest that the experimental economics approach is sensitive for detecting neurobiological effects as subtle as those achieved by administration of hormones. Moreover, the findings point towards the importance of both psychosocial as well as neuroendocrine factors in determining the influence of testosterone on human social behavior.

The procedure described in this protocol shows that testosterone administration and folk beliefs about testosterone may be associated with directly opposed social behaviors.

睾酮和社会决策，结合行为内分泌和实验经济学:

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in ...

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

在发展中国家斑马鱼的前脑的神经祖细胞克隆相关的定时实时成像

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

在发展中国家小鼠视网膜体内电穿孔

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

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

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing 1. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth 2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes 4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development6.
The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos7. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells8-10. 
We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol11. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.

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

The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques.

转基因小鼠内耳的发展<em&gt;在体内</em&gt;电穿孔

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

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 ...

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

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

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

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

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 ...

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 ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

完整的犁鼻器和副嗅球的体外制剂

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...