We are indebted to Pierre Mattar and Freda Miller for their assistance with the in utero electroporation and to Noel Ghanem and Bensun Fong for creating diagrams. We thank Linda Jui and Jason G. MacLaurin for excellent technical assistance. Equipment was supported by the Centre for Stroke Recovery.
This work was supported by a scholarship from HSFO and OGS to D.S.S. and a CIHR grant to R.S.S.

注：所有的实验由渥太华动物保健伦理委员会的大学被批准，秉承加拿大委员会动物护理的指引。 1.预外科制备和材料<ol><li>质粒制备： <ol><li>准备根据制造商的方案使用Qiagen MegaPrep质粒。 Innoculate至少400毫升LB肉汤与转化的细菌，以确保高的DNA产量和孵育过夜。 </li><li>重悬的质粒在100-200微升的TE到至少5微克/微升的最终浓度。等分试样的DNA，并储存于-20℃。如果该浓度太低，不沉淀，重悬，因为这可以增加杂质。开始一个新的准备。 </li><li>在手术前，稀的单一质粒（酶Cre-GFP例如）至2微克/微升无菌水以1微升的0.1％（重量/体积）的快速，每10或20微升稀释的质粒溶液绿色染料。如果GFP和感兴趣的基因都可以共同电穿孔在单独的质粒，注入的GFP质粒和质粒携带感兴趣的基因以1：4的比例，以最大限度地提高的可能性，标有GFP的所有细胞被共同电穿孔与两个质粒。 </li></ol></li><li>拉玻璃微毛细管（1毫米外径的 - 以下称为针）上的标准针拔出器，使用单一步骤拉力在62℃。 </li><li>高压灭菌器包含以下内容的手术包：持针，伤口拉钩，镊子，剪刀手术，剪刀针，钉书机，钉书钉，纱布，伤口盖，手术单（ 图1A，B，材料表 ）。 </li><li>收取其他所需的工具：电穿孔，Femtojet Microinjecter，Tweezertrodes（或7mm），生理盐水，缝合5毫升注射器（ 材料表 ）。 </li></ol> 2.准备鼠标外科手术<ol><li>对疼痛管理，注入定时 - 对统治小鼠用丁丙诺啡（0.05毫克/公斤体重）前一小时的手术。应用此协议从胚胎天12.5（E12.5）定时随时随地小鼠E17.5。 </li><li>使用微加载枪头备份负载拉针与质粒的解决方案。不一路填充到针的尖端还因为这会导致针变得堵塞。 </li><li>用2-5％异氟烷气体以1升/分钟的O 2麻醉小鼠。一旦不动，放置在脸部面膜，然后敷药膏眼睛，以防止干燥。注射1毫升生理盐水（0.9％氯化钠）皮下回来。 注意：当用异氟烷气体工作，可使用的气体清除剂夺回过量气体，并防止其漏出到房间。 </li><li>剃腹部。与Chlorohexadine擦洗腹部洗净，用清水冲洗，并应用chlorohexadine和70％乙醇的最终准备的解决方案。盖鼠标用无菌的悬垂性或纱布覆盖皮毛，但留下的腹部暴露（ 图1C，D）。 </li></ol>在子宫内电<ol><li>使在皮肤上的下奶嘴之间的腹部切口。通过撕开结缔组织拉扯皮肤远离下面的肌肉层，然后通过腹膜切成沿白线的腹腔。插入伤口收器拉伤口边缘分开。 </li><li>从腹腔中取出子宫打下出来，如图（ 图1E）。 <ol><li>如果不同幼仔是用不同的质粒（如GFP 相对于 GFP与感兴趣的基因）被注入，计数在每一侧幼仔并决定多少与每个质粒注入哪些和。更换子宫的一侧回腹部，以保持胚胎温暖和润滑。立即润湿用生理盐水露出子宫（优选加热到体温）。 </li><li>如果注射用相同的质粒所有幼仔（注入的Cre-GFP成转基因胚胎时如）ONLý除去子宫1角的时间。 </li></ol></li><li>把一个装针注射棒的抓地力头和精心切割的尖端与一对小的手术剪刀或罚款钳。切割尖端，以便将针保持尽可能长的时间，同时仍允许流体穿过（ 图1Ji-ⅳ）。 <ol><li>调整注入的长度（以秒为单位）和注射压力的微量，使得当脚桨压0.1-0.2微升小但容易看得见的液滴出现在针的尖端。保持这种液滴的容量为一致的不同的针之间的可能。 注：此步骤是为胚胎存活（见讨论）是至关重要的。 </li><li>使用以下注射参数Femtojet：注射压力（PI）= 250-300百帕;注射时间（TI）= 0.8〜1.2秒;补偿压力= 10-50百帕。如果需要的话，调整这些参数以适应针之间的变化。 </li></ol></li><li> S选择一个胚胎，并在它的后面，头向上倾斜（ 图1F）轻轻地放置。 注意：这可能需要转弯或转移胚胎;要小心，不要施加太大的压力，避免了羊膜囊破裂，而操纵胚胎。 </li><li>一旦胚就位，定位心室，其表现为阴影平行于矢状窦月牙形。碰到针到心室上述子宫的前端以一个很小的角度，并通过子宫壁引导针。所需的压力的量取决于针的锐度。通过皮质进入心室推针。 注意：虽然很少有任何额外的可检测性，胚胎的头部往往推开略微然后反冲当针穿过皮质进入心室。 </li><li>泵脚桨注入质粒溶液。继续泵，直到绿色区域是可见的，并符合心室（ 图1G）的弧形。在年轻的胚胎（E12.5至E14.5）的染料往往会扩散到对侧心室。 注：泵的数量取决于针尖的直径，胚胎（和心室的所得大小）和所需的注射体积的年龄。不断注入量尽可能一致胚胎之间。 </li><li>注入三个或四个胚胎。保留所有暴露胚胎湿润整个过程生理盐水。 </li><li>回到喷射出的第一胚胎并放置桨叶使得正电极将以此质粒进入脑（ 图1H）的所希望的区域。角的负电极，使得电极之间的最短路径直接通过针对电穿孔的结构（ 图2）。 </li><li>一旦桨到位，申请的震撼。根据胚胎的年龄和大小，可使用5个脉冲的35-50 V（ 见 T能1）。使用下面的电参数：5毫秒脉冲长度为950毫秒的脉冲间隔。重复所有注射胚胎。 </li><li>切的新的预加载的针尖端作为在步骤3.3作为最后的针可能干燥并在步骤3.9堵塞。注入的下一组三个或四个胚胎或者相同质粒或不同质粒，然后应用休克如在步骤3.9。当在子宫一侧的所有幼仔被注入，从女性的腹部取出子宫的第二侧，然后更换完成侧保持温暖。 </li><li>返回子宫的两侧，女小心翼翼地保持表面湿润用生理盐水，不要施加太大的压力，以防止创伤羊膜囊。缝合使用简单的连续缝合关闭肌肉层。主食皮肤封闭。如果订书钉不可用，缝合用一个简单的中断缝皮肤。 （ 图1I） </li><li>虽然suturiNG，逐步调低异氟醚，让怀孕的女性的麻醉来减轻。 注：这将加快大坝觉醒完成手术后，保持对麻醉尽可能短的时间尽可能将提高胚胎存活率的女性。总手术时间，从当阴首先麻醉开始，应该是大约30分钟。 </li></ol> 4.善后<ol><li>放置缝合女性在培养箱或加热垫从麻醉中恢复。床她干净的被褥，并放置在笼子里的标准恢复凝胶保持水分。对疼痛管理手术后，给阴三个剂量丁丙诺啡（0.05毫克/公斤体重）在8小时的时间间隔手术后，然后加入另外的4剂量在12小时的时间间隔。 </li></ol> 5.收获脑注：大脑可以在任何年龄高达成年收获。以下内容适用出生前，大脑收集。需要注意的是，一旦幼崽出生时，子宫内的订单丢失，所以无论是在每个垃圾小狗必须用相同的质粒注射，或对照组和实验动物之间的区别的另一种方法，必须制定。 <ol><li>制备冷（4℃），过滤，4％多聚甲醛（PFA）之前实施安乐死女性。 </li><li>用腹膜内注射致死剂量的戊巴比妥钠（100mg / kg的体重）的安乐死阴和切开腹部。拉出子宫的两侧，布置为当幼仔最初注入。 <ol><li>在所有幼仔接受相同的质粒，注射的Cre成转基因垫料当如情况下，胚胎顺序并不重要，因此切子宫出女性和地点在PBS中。 </li><li>如果幼鼠注射不同的质粒，一定要找到每一个小狗，并指出任何死的小狗，并确定哪些注射控制和experimen从阴取出子宫之前TAL质粒。小心地跟踪哪些小狗属于哪个组后子宫是PBS。 </li></ol></li><li>分别卸下每个小狗，斩首用锋利的剪刀或刀片，然后将头PBS。在转基因小鼠中的情况下，收集组织样品用于基因分型，并保持每个头分离的，如在一个12孔板中。 </li><li>根据解剖范围，从头骨移除大脑，小心不要尼克皮质或撕裂中线。用荧光显微镜检查，看看是否大脑有一个GFP +补丁，这表明他们已成功电。 </li><li>固定大脑通过浸入的PFA在4℃下过夜。 注意：检查大脑的GFP之前的固定表达，因为PFA通常淬火信号。 </li><li>通过浸没在20％蔗糖至少三天之前冷冻Cryoprotect大脑。使用10％的蔗糖梯度，15％和20％（24小时在每种溶液）吨Ø防止组织损伤，由于渗透压。储存冷冻大脑在-80℃下和上切割一低温恒温器在10-14微米厚的切片。 </li></ol> 6.联合染色抗GFP 注意：如果抗原修复是必要的，以便共同染色用抗GFP，它使用一个温和的抗原检索协议，这并不损害GFP信号是重要的。有些抗原修复协议过于苛刻与GFP染色一起使用（甚至对GFP抗体）。一个基本的柠檬酸预处理之前申请的第一抗体是成功的大多数表位24。 <ol><li>使在双蒸2 O 0.01M的溶液中的柠檬酸使用校准pH计的pH至6。 </li><li>热柠檬酸染色室中微波或水浴直至柠檬酸达到75-80℃。放幻灯片染色室10-15分钟。保持温度尽可能稳定。 </li><li>在PBS洗幻灯片3×3分钟，在室温下进行。应用第一抗体并继续按照标准协议进行免疫染色。 </li><li>验证通过共标记的GFP和感兴趣的基因的Cre介导的切除，以确认击倒（ 图3A）。 </li></ol>

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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+contribution+of+nestin-expressing+stem+cells+to+adult+neurogenesis.">Dynamic contribution of nestin-expressing stem cells to adult neurogenesis.</a> J. Neurosci. 27 (46), 12623-12629 (2007).</li><li>Andrusiak, M. G., Vandenbosch, R., Park, D. S., Slack, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+retinoblastoma+protein+is+essential+for+survival+of+postmitotic+neurons.">The retinoblastoma protein is essential for survival of postmitotic neurons.</a> J. Neurosci. 32 (42), 14809-14814 (2012).</li><li>Shan, B., Chang, C. Y., Jones, D., Lee, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcription+factor+E2F-1+mediates+the+autoregulation+of+RB+gene+expression.">The transcription factor E2F-1 mediates the autoregulation of RB gene expression.</a> Mol. Cell. Biol. 14 (1), 299-309 .</li><li>Ferland, R. J., Cherry, T. J., Preware, P. O., Morrisey, E. E., Walsh, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Characterization of Foxp2 and Foxp1 mRNA and protein in the developing and mature brain. 460 (2), 266-279 (2003).</li></ol>

The authors have nothing to disclose

两个在执行宫内电穿孔的主要挑战是：1）防止胚胎致死; 2）降低电穿孔之间的差异。一种在预防致死的最重要因素是针质量，因为钝针头造成的胚胎更大的危害。当切割针，保持针尖尽可能长，同时仍然允许0.1-0.2微升流体的可见液滴出现以下脚焊盘（图1J）的泵。如果针太钝或轴太短，将所得的组织损伤可以杀死胚胎。相反，如果针尖太细，会花很长时间来注入足够量质粒;离...

径向迁移是一个核心进程早期皮质发育。这取决于多种细胞自主因素，如正确的有丝分裂退出和神经元分化，细胞极性，细胞骨架动力学和跨膜受体的表达的调控，以及非细胞自主因素，如形成放射状胶质支架和洄游指导分泌分子1-3。由于任何这些机制的破坏会损害神经细胞迁移的转基因小鼠模型4-8，确定迁移缺陷的根本原因是一个复杂而艰难的过程。 在子宫内电（IUE）可用于补充和简化的基因敲除模式的表型，从而解释阐明所需的放射状迁移7,9-11重要机制。 在子宫内电（IUE）的过程，其中一个质粒carryi<html>纳克感兴趣两者的基因和记者注入脑室在小鼠或大鼠胚胎的大脑，然后吸入到细胞衬里通过使用电流12-14心室。这使得研究者分析的向上或向下的发展或电穿孔神经元的功能感兴趣的基因的调节的影响。下面IUE，大脑可以处理的免疫组化7,9-11，电15或细胞培养16,17。 IUE的主要优点是允许基因表达的高度特异性操纵。此外，IUE可用于通过定向电流（ 图2）为目标的显影大脑的特定区域。 IUE也可用于靶向通过注射质粒下多种启动子或质粒活化系统的控制携带基因的特定细胞类型（四环素诱导的基因表达是一个例子）10,18-21。 jove_content“&gt; IUE可以与酶Cre-LOX介导的基因切除系统7-9,11,22一起使用。含有编码该蛋白质的Cre消息的基因的质粒可以被电穿孔入胚胎纯合的两侧装接loxP的等位基因（在该基因或特定的DNA区通过两个loxP位点的Cre介导的DNA重组）侧翼。Cre重组然后将诱导的感兴趣的基因的重组特别在电穿孔神经前体，产生的敲除的两侧装接loxP allele.The效果蛋白质拦截神经迁移和单个神经元内的发展可以被研究。酶Cre的电穿孔诱导重组中只有一小人口受到影响的细胞，留下支撑环境不变。相反，酶Cre的下特定的细胞类型的控制组织特异性表达子，发生在整个组织使两个迁移成神经细胞和周围环境可能会受到影响，因此，jux这两种方法的taposition可以确定一个给定的迁移的缺陷是否是由于细胞中自主或细胞非自主的机制。在这两个实验系统缺陷的迁移表明从一个细胞中自主机构所观察到的表型的结果;正常迁移下列酶Cre的电穿孔以在组织特异性的Cre模型缺陷的迁移表明感兴趣的基因是通过非细胞自主机制作用。 IUE也可以使用通过电穿孔潜在相互作用的基因插入敲除动物7,9,10执行救援实验。例如，研究者可以尝试通过电穿孔的下游靶标，并确定是否迁移缺陷是在将电穿孔的神经元校正营救在转基因模型中的迁移表型。这具有额外的好处，一个成功的救援表示正常的迁移可以通过一根SPE操纵蛋白质表达可以恢复cific神经元，尽管周围的环境仍存在不足的靶基因。再次，这种方法是有更多的时间和比横过或产生的转基因系，与确定的缺陷机制​​是否是细胞中自主的额外好处成本效益。 IUE可用于跟踪通过报道质粒的电迁移神经元到敲除胚胎（ 图4C）。因为只有衬心室在手术时的神经元被电穿孔17，IUE可用于遵循出生在特定的时间与它们在体内的形态的可视化的优点的神经元。 最后，它有可能使用IUE靶向脑区如内侧或背侧端脑，海马或神经节隆起（ 图2）。这使研究人员在一个特定的区域独立的并发症的结果，调查基因功能的电源荷兰国际集团的蛋白敲除邻近结构。 视网膜母细胞瘤蛋白（Rb蛋白），P107和P130包括口袋蛋白家族，并建立良好的细胞周期退出监管。然而，有越来越多的证据表明，这些蛋白质也调节神经发育的细胞周期独立方面。正如我们以前已经表明，Rb蛋白和P107两种切4,23和径向迁移6起到至关重要的作用。在这里，我们展示了口袋蛋白家族成员Rb蛋白P107及神经移植6的作用，体现在酶Cre的子宫内电使用的转基因模型。总之，IUE提供分析基因缺失（或过表达）的细胞自主作用的有力方式。当与具体的淘汰机型组织相结合，IUE可以提供关于控制神经迁移机制的更多信息。

<table border="0" cellpadding="0" cellspacing="0" width="538">
	<tbody>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:233px;">Table 1: Equipment and Materials</td>
			<td style="width:64px;"></td>
			<td style="width:64px;"></td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:119px;">Product Name</td>
			<td style="width:115px;">Company</td>
			<td colspan="2" style="width:128px;">Catalog Number</td>
			<td style="width:177px;">Description</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:119px;">ECM 830 Square Wave Electroporator</td>
			<td style="width:115px;">BTX Harvard Apparatus</td>
			<td colspan="2" style="width:128px;">45-0052</td>
			<td style="width:177px;">Electroporator only &#8211; buy cables separately (45-0204)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">1250FS Footswitch for ECM 830</td>
			<td style="width:115px;">BTX Harvard Apparatus</td>
			<td colspan="2" style="width:128px;">45-0211</td>
			<td style="width:177px;">Foot paddle is necessary for hands-free electroporation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Tweezertrodes</td>
			<td style="width:115px;">BTX Harvard Apparatus</td>
			<td colspan="2" style="width:128px;">45-0489 (5mm)&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 45-0488 (7mm)</td>
			<td style="width:177px;">Platinum forcep style electrodes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Femtojet</td>
			<td style="width:115px;">Eppendorf</td>
			<td colspan="2" style="width:128px;">920010504</td>
			<td style="width:177px;">Microinjector</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Griphead 0</td>
			<td style="width:115px;">Eppendorf</td>
			<td colspan="2" style="width:128px;">920007414</td>
			<td style="width:177px;">Holds the pulled needle</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Universal Capillary Holder</td>
			<td style="width:115px;">Eppendorf</td>
			<td colspan="2" style="width:128px;">920007392</td>
			<td style="width:177px;">Connects griphead to tube</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Injection Tube</td>
			<td style="width:115px;">Eppendorf</td>
			<td colspan="2" style="width:128px;">920007431</td>
			<td style="width:177px;">Connects capillary holder to Femtojet</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Foot Control</td>
			<td style="width:115px;">Eppendorf</td>
			<td colspan="2" style="width:128px;">920005098</td>
			<td style="width:177px;">Foot paddle for hands-free injection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Microloader Tips</td>
			<td style="width:115px;">Eppendorf</td>
			<td colspan="2" style="width:128px;">930001007</td>
			<td style="width:177px;">Tips for loading the pulled capillary tubes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Plasmid Mega Kit</td>
			<td style="width:115px;">Qiagen</td>
			<td colspan="2" style="width:128px;">12181</td>
			<td style="width:177px;">5 QIAGEN-tip 2500, Reagents, Buffers</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Fast Green FCF</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td colspan="2" style="width:128px;">F7252-5G</td>
			<td style="width:177px;">Non-toxic dye</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Extra Fine Bonn Scissors</td>
			<td style="width:115px;">Fine Science Tools</td>
			<td colspan="2" style="width:128px;">14084-08</td>
			<td style="width:177px;">For trimming the needles</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Bonn Strabismus Scissors</td>
			<td style="width:115px;">Fine Science Tools</td>
			<td colspan="2" style="width:128px;">14084-09</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Graefe Forceps</td>
			<td style="width:115px;">Fine Science Tools</td>
			<td colspan="2" style="width:128px;">11050-10</td>
			<td style="width:177px;">Straight</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Colibri Retractors</td>
			<td style="width:115px;">Fine Science Tools</td>
			<td colspan="2" style="width:128px;">17000-03</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Stapler</td>
			<td style="width:115px;">Fine Science Tools</td>
			<td colspan="2" style="width:128px;">12031-07</td>
			<td style="width:177px;">For 7mm clips</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Castroviejo Needle Holder</td>
			<td style="width:115px;">Medical Tools</td>
			<td colspan="2" style="width:128px;">SNH-6737</td>
			<td style="width:177px;">Straight, with lock</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Needle Puller</td>
			<td style="width:115px;">Narishige</td>
			<td colspan="2" style="width:128px;">PC-10</td>
			<td style="width:177px;">For pulling microcappillaries</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Microcapillaries</td>
			<td style="width:115px;">Drummond</td>
			<td colspan="2" style="width:128px;">1-000-800</td>
			<td style="width:177px;">0.4 mm inner diameter glass capillary tubes</td>
		</tr>
	</tbody>
</table>

induction of protein deletion through in utero electroporation to define deficits in neuronal migration in transgenic models

使用的Cre-lox系统中的转基因小鼠系基因缺失是用于研究蛋白质功能的有力工具。然而，除了在非常特殊的Cre模型，缺失整个组织或细胞群的蛋白质常常导致从多个相互作用机制导致复杂的表型。确定是否从一个细胞中自主机制，这是固有的小区中的问题，或由非细胞自主机制，这将导致从该小区的环境受损破坏的表型的效果，可以是难以辨别。深入了解蛋白质功能在体内情况下， 在子宫内的电穿孔（IUE）使基因缺失的细胞中的显影皮质或一些其它选定的脑区域内的一小部分。 IUE可以用来针对特定的大脑区域，包括背端脑，内侧端脑，海马，或神经节隆起。这有利于观察Ø的F细胞中自主基因缺失中的一个健康的环境的上下文中的后果。该协议的目的是要表明IUE如何可用于分析在径向迁移的缺陷在一个两侧装接loxP的转基因小鼠品系，并强调蛋白缺失的细胞中自主和非细胞自主效应区分。由表型的基因缺失，整个皮质与IUE介导的基因缺失在有限的细胞群中产生的，更深入的了解比较成蛋白质功能的大脑发育比可以通过使用技术孤立地获得。

Different regions of the brain can be targeted for electroporation by varying the orientation of the paddle electrodes over the embryo&#8217;s head (Figure 2). For all parts of the forebrain, the plasmid is injected into the lateral ventricle (the hind brain can be targeted by injecting into the fourth ventricle). In general, place the electrodes so that the straight line between them passes through the region of interest, with DNA being drawn toward the positive electrode. To target the lateral cortex (...

Watch this Scientific Journal Video about Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models at JoVE.com

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

Neuron migration is regulated by numerous cell autonomous and non-cell autonomous factors. This protocol shows how in utero electroporation can be used to determine whether a phenotype in a transgenic mouse is due to disruption of a cell intrinsic mechanism or impairment of interaction between the neuron and its environment.

诱导蛋白缺失的通过<em&gt;在子宫内</em&gt;电穿孔来定义赤字的神经细胞迁移的转基因模型

Genetic deletion using the Cre-Lox system in transgenic mouse lines is a powerful tool used to study protein function. However, except in very specific ...

induction-protein-deletion-through-utero-electroporation-to-define

Research

JoVE Journal

Developmental Biology

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

10.1K 次观看.  University of Ottawa. Neuron migration is regulated by numerous cell autonomous and non-cell autonomous factors. This protocol shows how in utero electroporation can be used to determine whether a phenotype in a transgenic mouse is due to disruption of a cell intrinsic mechanism or impairment of interaction between the neuron and its environment. 

视频: Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

评定神经连接的形成在共培养体系的光遗传学方法

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

科研

发育生物学

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

小鼠海马组织的前子宫内电和器官切片文化

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

技术目标显微注射到显影<em&gt;爪</em&gt;肾

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

人多能干细胞对原始和最终造血祖细胞定向分化的研究

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Generation of Genetically Modified Mice through the Microinjection of Oocytes

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear injection of DNA expression constructs into fertilized oocytes remains the most commonly used technique to generate transgenic mice for overexpression. With the introduction of CRISPR technology for gene targeting, pronuclear injection into fertilized oocytes has been extended to the generation of both knockout and knockin mice. This work describes the preparation of DNA for injection and the generation of CRISPR guides for gene targeting, with a particular emphasis on quality control. The genotyping procedures required for the identification of potential founders are critical. Innovative genotyping strategies that take advantage of the "multiplexing" capabilities of CRISPR are presented herein. Surgical procedures are also outlined. Together, the steps of the protocol will allow for the generation of genetically modified mice and for the subsequent establishment of mouse colonies for a plethora of research fields, including immunology, neuroscience, cancer, physiology, development, and others.

The microinjection of mouse oocytes is commonly used for both classic transgenesis (i.e., the random integration of transgenes) and CRISPR-mediated gene targeting. This protocol reviews the latest developments in microinjection, with a particular emphasis on quality control and genotyping strategies.

通过显微注射卵母细胞生成转基因小鼠

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

小鼠第二腭融合的实时成像

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

rna 干扰和热休克诱导转录因子表达在重编程生殖细胞到神经元中的应用C. 线虫

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. Because of the overlapping expression domains of Notch and its ligands in many cell types and the existence of feedback mechanisms, studying the effects of a given post-translational modification on trans- versus cis-interactions of Notch and its ligands in vivo is difficult. Here, we describe a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of &#62;6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands.

细胞聚集法评价果蝇切口与反式配体的结合及其对顺式配体的抑制作用

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...