Name: induction of protein deletion through in utero electroporation to define deficits in neuronal migration in transgenic models
Uploaded: 2015-01-12T12:30:00.000+00:00
Duration: 12 min 1 s
Description: 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

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><tbody><tr><td>ECM 830 Square Wave Electroporator</td><td>BTX Harvard Apparatus</td><td>45-0052</td><td>Electroporator only &amp;ndash; buy cables separately (45-0204)</td></tr><tr><td>1250FS Footswitch for ECM 830</td><td>BTX Harvard Apparatus</td><td>45-0211</td><td>Foot paddle is necessary for hands-free electroporation</td></tr><tr><td>Tweezertrodes</td><td>BTX Harvard Apparatus</td><td>45-0489 (5mm)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 45-0488 (7mm)</td><td>Platinum forcep style electrodes</td></tr><tr><td>Femtojet</td><td>Eppendorf</td><td>920010504</td><td>Microinjector</td></tr><tr><td>Griphead 0</td><td>Eppendorf</td><td>920007414</td><td>Holds the pulled needle</td></tr><tr><td>Universal Capillary Holder</td><td>Eppendorf</td><td>920007392</td><td>Connects griphead to tube</td></tr><tr><td>Injection Tube</td><td>Eppendorf</td><td>920007431</td><td>Connects capillary holder to Femtojet</td></tr><tr><td>Foot Control</td><td>Eppendorf</td><td>920005098</td><td>Foot paddle for hands-free injection</td></tr><tr><td>Microloader Tips</td><td>Eppendorf</td><td>930001007</td><td>Tips for loading the pulled capillary tubes</td></tr><tr><td>Plasmid Mega Kit</td><td>Qiagen</td><td>12181</td><td>5 QIAGEN-tip 2500, Reagents, Buffers</td></tr><tr><td>Fast Green FCF</td><td>Sigma-Aldrich</td><td>F7252-5G</td><td>Non-toxic dye</td></tr><tr><td>Extra Fine Bonn Scissors</td><td>Fine Science Tools</td><td>14084-08</td><td>For trimming the needles</td></tr><tr><td>Bonn Strabismus Scissors</td><td>Fine Science Tools</td><td>14084-09</td><td></td></tr><tr><td>Graefe Forceps</td><td>Fine Science Tools</td><td>11050-10</td><td>Straight</td></tr><tr><td>Colibri Retractors</td><td>Fine Science Tools</td><td>17000-03</td><td></td></tr><tr><td>Stapler</td><td>Fine Science Tools</td><td>12031-07</td><td>For 7mm clips</td></tr><tr><td>Castroviejo Needle Holder</td><td>Medical Tools</td><td>SNH-6737</td><td>Straight, with lock</td></tr><tr><td>Needle Puller</td><td>Narishige</td><td>PC-10</td><td>For pulling microcappillaries</td></tr><tr><td>Microcapillaries</td><td>Drummond</td><td>1-000-800</td><td>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

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

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

10.2K 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

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

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

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

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

在子宫内的电穿孔其次原发性神经文化为研究基因的功能在皮层神经元的一个子集

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

科研

神经科学

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 ...

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 ...

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

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous system. Overexpression of desired genes is just as possible as shRNA mediated loss-of-function studies. Therefore it offers a wide range of applications. The feasibility to target particular cells in a distinct area further increases the range of potential applications of this very useful method. For efficiently targeting specific regions knowledge about the subtleties, such as the embryonic stage, the voltage to apply and most importantly the position of the electrodes, is indispensable.
Here, we provide a detailed protocol that allows for specific and efficient in utero electroporation of several regions of the C57BL/6 mouse central nervous system. In particular it is shown how to transfect regions the develop into the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus. For this information about the appropriate embryonic stage, the appropriate voltage for the corresponding embryonic stage is provided. Most importantly an angle-map, which indicates the appropriate position of the positive pole, is depicted. This standardized protocol helps to facilitate efficient in utero electroporation, which might also lead to a reduced number of animals.

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

This protocol describes in detail how to specifically transfect different regions in the C57BL/6 central nervous system via in utero electroporation. Included in this protocol are detailed instructions for transfections of regions that develop into the cortex, hippocampus, thalamus, hypothalamus, lateral septal nucleus and striatum.

Cortex-，Hippocampus-，Thalamus-，下丘脑 - ，横向隔核 - 纹状体专用<em&gt;在子宫内</em&gt;电穿孔的C57BL / 6小鼠

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous ...

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Birth defects that involve the cerebral cortex &#8212; also known as malformations of cortical development (MCD) &#8212; are important causes of intellectual disability and account for 20-40% of drug-resistant epilepsy in childhood. High-resolution brain imaging has facilitated in vivo identification of a large group of MCD phenotypes. Despite the advances in brain imaging, genomic analysis and generation of animal models, a straightforward workflow to systematically prioritize candidate genes and to test functional effects of putative mutations is missing. To overcome this problem, an experimental strategy enabling the identification of novel causative genes for MCD was developed and validated. This strategy is based on identifying candidate genomic regions or genes via array-CGH or whole-exome sequencing and characterizing the effects of their inactivation or of overexpression of specific mutations in developing rodent brains via in utero electroporation. This approach led to the identification of the C6orf70 gene, encoding for a putative vesicular protein, to the pathogenesis of periventricular nodular heterotopia, a MCD caused by defective neuronal migration.

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Periventricular nodular heterotopia (PNH) is the most common form of malformation of cortical development (MCD) in adulthood but its genetic basis remains unknown in most sporadic cases. We have recently developed a strategy to identify novel candidate genes for MCDs and to directly confirm their causative role in vivo.

一种新的结合阵列全息、全外测序和宫内电穿孔的方法来识别脑畸形的致病基因

Birth defects that involve the cerebral cortex &#8212; also known as malformations of cortical development (MCD) &#8212; are important causes of ...

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

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

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

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

在子宫内电途径研究神经元亚群的兴奋性和单细胞连接

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

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

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

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

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

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

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

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To do so requires generating a mosaic in which neurons with loss or gain of function of a gene are surrounded by genetically unperturbed tissue. Here, we combine the Cre-lox recombination system with in utero electroporation in order to generate mosaic brain tissue that can be used to study the cell-autonomous function of genes in neurons. DNA constructs (available through repositories), coding for a fluorescent label and Cre recombinase, are introduced into developing cortical neurons containing genes flanked with loxP sites in the brains of mouse embryos using in utero electroporation. Additionally, we describe various adaptations to the in utero electroporation method that increase survivability and reproducibility. This method also involves establishing a titer for Cre-mediated recombination in a sparse or dense population of neurons. Histological preparations of labeled brain tissue do not require (but can be adapted to) immunohistochemistry. The constructs used guarantee that fluorescently labeled neurons carry the gene for Cre recombinase. Histological preparations allow morphological analysis of neurons through confocal imaging of dendritic and axonal arbors and dendritic spines. Because loss or gain of function is achieved in sparse mosaic tissue, this method permits the study of cell-autonomous necessity and sufficiency of gene products in vivo.

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous functions of genes in the brain can be studied by inducing loss or gain of function in sparse populations of cells. Here, we describe in utero electroporation to deliver Cre recombinase into sparse populations of developing cortical neurons with floxed genes to cause loss of function in vivo.

宫内电穿孔诱导小鼠大脑皮质中氧合酶的重组

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

前子宫内胚胎小鼠脑电穿孔和脊髓切片培养迁移 GABAergic 中间神经元的活体成像

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...

诱导蛋白缺失的通过

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