作者要感谢支持，从塞缪尔Oschin全面癌症研究所癌症研究论坛奖，以及资金的雪松，西奈半岛的再生医学研究所和卡介苗家庭。描述该项目由美国国家研究资源中心，格兰特UL1RR033176资助的CTSI核心券的形式得到了支持，并且现在正处于国家中心推进转化科学，格兰特UL1TR000124。内容完全是作者的责任，并不一定代表美国国立卫生研究院的官方意见。

这个程序是按照雪松 - 西奈IACUC要求。调查人员应当确保机构IACUC符合前程序。所有工具和试剂应当在使用前灭菌。 1，工具的准备，解决方案和DNA混合物<ol><li>将100毫米火抛光的高硼硅玻璃毛细管成微量拉马。设置加热，以允许标准加权拉以形成约17.5毫米枪头。用锋利的手术剪刀的技巧在距离约8-9毫米的尖端开始，以创造大约100微米直径的开口。 </li><li>做一只股票1％w / v的快速绿色解决方案，用干燥快绿混合过滤，无核酸酶水（利用20微米注射器过滤器）。 </li><li>分离纯化的无内毒素的质粒DNA是高度浓缩的（ 即 &gt; 3微克/微升），稀释在Tris-EDTA（TE）缓冲液中。质粒加入所需量的向混合物稀释于TE缓冲液中，并加入1:10的比例在1％固绿溶液（使0.1％w / v的快速绿色最终浓度）。使用质粒的0.5-2.0微克/微升终浓度为转基因的强劲表现。 </li></ol> 2，动物麻醉，移液器加载和软脑膜表面质粒注射<ol><li>通过将它们放在一个培养皿中被置于冰上5-8分钟（时间取决于小狗年龄/体重）诱导低温麻醉在1-2天出生后的小鼠。一旦体温是由缺乏从脚趾捏和/或尾部反射运动的证实，老鼠是准备注入。将动物背冰上如果疼痛反射是显而易见的。注射过程和电需要不到2-3分钟的总时间如此之低体温，注射和电穿孔程序，从而保持适当的麻醉。 </li><li>在麻醉过程中，使用的是标准的移液管，移液管仔细所需plasmi量ð混合拟注入（0.5-2微升）到一块石蜡膜以供参考。这里，0.5微升质粒混合物的总体积被注入。接着，用微型注射器，小心地插入装配玻璃毛细吸管到含有管质粒混合物。采取额外的预防措施，以防止尖端的断裂通过确保它不触及管的边缘。通过慢慢地拨打背面转印拨盘吸出溶液进入吸管。一旦足够量的已被加载到移液管，转盘前进，直到压力恢复到0帕斯卡中立位置。 </li><li>使用300-450百帕压力注射，以确保适当的压力，即使填充软脑膜表面/脑膜空间，将附近的移液石蜡电影基准注射液（2.2）的尖端，并使用脚踏板从微量进样器弹出的一个卷到石蜡膜。相应地调整压力，直到排出量等于先前吸管referencË量。 </li><li>一旦前端已平衡和麻醉已被证实，幼仔已准备好进行注射。 </li><li>该实验的目的是在颅骨和上面的软膜表面的质粒混合物注入到向下允许用于DNA电穿孔进入软脑膜表面祖细胞（ 图1A）。对于皮质注射，用拇指和食指少占主导地位的手按住小狗下来。皮质半球和其他表面结构，如优良丘是P0和P2之间的可见光，允许这些结构（ 图1B）的轻便定位。使用皮质的惯用手和目标所需的区域（ 即电机，体感等），插入吸管过去的皮肤和颅骨照顾，以避免脑动脉。一定要防止过去的头骨（这是刚刚刺入颅骨后，表示因缺乏进一步阻力）尖端的进一步渗透。 </li><li>血浆注入使用ID解决方案的微量进样器的脚踏板，并确保其均匀地分散在整个组织中（ 图2A）。 注意:根据经验确定最大化质粒递送，同时避免血肿由于流体压力的方法是非常重要的。这可以通过调整喷射的持续时间，角度，体积，精确的靶位点，以避免血管破​​裂来完成。 </li><li>一定要尝试，并从蜡纸或塑料薄膜石蜡先前作出的注射将尖端测试液滴保持尖端的注射之间的干燥。这是必需的，以防止蒸发和crystalized DNA溶液，从而导致在针尖和不一致的交货量的堵塞的末端积累。 </li></ol> 3，电穿孔<ol><li>设定电穿孔至135-150 V为5个脉冲，持续50毫秒，以950毫秒的间隔中的每个脉冲之间。为了提高电导率和防止皮肤的烧灼，盖3毫米铂tweezertrodes与电穿孔凝胶。检查脚趾捏和尾部反射在这一点上，以确保麻醉。如果响应，放置在冰上几分钟至麻醉。 </li><li>为皮层（和上丘）注射液，采用3毫米电极（ 图2B），以增加幼仔的存活率（与7相比-和10 -毫米的电极，这是用于脑室区电穿孔）。将带负电荷的探针上的电极在注入区和带正电的探针在眼部周围，例如下面的对侧区域，该电极取向是在45-60°的角度相对于所述中线（ 图2C）一个。 </li><li>占曲率时，使用电穿孔的踏板触发电流和保持tweezertrodes到位3-5脉冲，根据小狗的年龄和体重，有效地靶向DNA插入下层软脑膜细胞表面半球的。 注意:该负极可以扫过喷射区域中的脉冲或一个较大的电极可以被用来增加电穿孔区域之间。 </li><li>立即电后，仔细擦去凝胶从幼崽，并将其放置在约5分钟加热灯。 </li><li>幼仔会敏锐地失去其自然的红色，粉红色的色调在电由于后发绀分钟。他们回到自己的笼子前，观察仔兔的自然颜色和正常运动的开始复苏。 </li><li>确保与母亲再社会化，通过观察她是否带回巢中的幼崽，并开始培养他们。如果她变得咄咄逼人，确保幼崽没有凝胶的残余，并培育他们与一些床上用品的气味转移到幼崽。 </li></ol> 4，修改为瞄准高级丘 目标: <ol><li>麟蹄克拉上丘中相同的方式皮层但是注意，目标区域位于正后方和侧方的λ和大致在中线（ 图3A和3B）。与成功的注射剂，所述质粒组合自然填充上丘的轮廓。一旦注射已成功，动物是准备好电。 </li></ol> 电穿孔: <ol><li>在一个非常类似的方式，对上丘注射，将带负电荷的探针上的电极在注入区和眼部周围，吻或颏带正电的探头，驱动源DNA插入上丘的浅表区域（ 图3C） 。在这种情况下，电极的方向是在25-40°的角度相对于所述中线一个。 </li></ol>

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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enhanced+monomeric+blue+fluorescent+protein+with+the+high+chemical+stability+of+the+chromophore.">An enhanced monomeric blue fluorescent protein with the high chemical stability of the chromophore.</a> PLoS One. 6 (12), (2011).</li></ol>

作者宣称，他们有没有竞争的财务权益。

对软脑膜表面祖成功电穿孔的最关键方面是:1）靶向质粒组合到软脑膜表面; 2）避免血肿​​的发生在注射部位; 3）避免与电脑相关的死亡率。 适当地靶向软脑膜表面由头骨测量和小心刺入完成，以避免软膜表面的渗透。 1）位移后轻轻揉组织（ 即快绿是在皮肤和结缔组织头骨上面的本地化）的快速绿色染料与皮肤:针对不当上覆皮肤或基础脑室或脑实质将由证明或2）由快速绿色染料进入大脑，表明该质粒混合物已进入心室和/或软组织的消失。质粒组合的正常软脑膜表面定位将染料积累的头骨日下证明在不能用温柔的皮肤位移受到干扰。然而，实践和灵巧是需要这种成功的目标。血肿形成可能是由血管质粒注射破坏引起的。血凝块会在注射部位有以下电穿孔当组织被收集的几天和几周内被观察到。为了避免血肿，调节流体注入的脉冲长度和/或采取的脑血管的一般模式注意避免容器破裂。在一般情况下，上丘，不容易受到凝结。死亡，由于电流通过tweezertrodes交付是很容易通过使用3毫米tweezertrodes更灶性提供电流，并通过引导电极远离腹侧中脑的表现（ 图2C和3C）避免。 该技术的主要缺点是有针对性的区域是由质粒溶液价差的大小限制以及电极的大小。甲交付0.5微升质粒生成的大约3mm的区域，因此，是良好匹配的3毫米电极尺寸。取措施是较小的（溶液铺展面积或电极桨区）将决定电穿孔细胞的修补程序的最终大小。此外，细胞的一个子集被标记，由于需要对有丝分裂进行的转基因表达24。然而，这是有利的，对于研究干细胞或祖细胞群和细胞分化。 软膜表面电穿孔是一种方法，允许对软脑膜表面祖细胞及其后代的强劲遗传操作。这种技术是快速，简便，高效，提供了更具体的，更容易在与其他唯一可比较的方法，逆转录病毒转导相比，靶向这些细胞的手段。高滴度的病毒生产需要技巧，周的时间，并带来安全风险 - 尤其是在泛嗜性pseudot的情况下，yped逆转录病毒。此外，电穿孔是较为灵活的，因为它不需要病毒克隆和生产。任何真核表达载体可以采用，包括病毒质粒。此外，大量制备，海关质粒可以混合和匹配，可轻松共同电击。典型地，有一个〜90-95％的共表达对于任何给定的两个质粒具有类似的启动子。中应注意的是，虽然我们只采用这靶向技术在皮层和上丘，该方法应服从于中枢神经系统的最软膜或表面的区域提供了之前的电流输送有质粒DNA的局部积累空间。 电穿孔的性质往往有利于有丝分裂细胞24的转导。因此，成年动物的电穿孔应该是可行的，但可能会导致大量减少细胞中表达，由于减少与衰老细胞增殖活性的转基因。但是，如最近由Jaubodin详述和同事，有丝分裂后的细胞在这个区域中的定位是可能通过采用反式-环己烷1,2 -二醇，它由permeablilizing核膜25允许核质粒连接。此外，电穿孔是与中铁快运的技术或其他设计的鼠标类型31非常兼容。最后，转座子技术将允许对这个群体29，30的稳定的体细胞转基因。总之，软脑膜表面电代表了这些独特的祖域的遗传操作一个优秀的和灵活的工具。

神经干细胞和祖细胞是存在于整个哺乳动物CNS 1，2。它们的性质和在周围的脑和脊髓的脑室区域胚胎和成年生发区属性已被广泛地记载在过去的十年中1-3。在很大程度上，这是由于越来越精确的遗传工具，如两侧装接loxP等位基因或逆转录病毒谱系追踪4的神经系统特异性表达Cre重组的发展。然而，一个祖区域软脑膜表面祖区，最近才在任何细节5-7被描述并等待全面的检查。 脑的软膜表面被定义为在脑表面和周围脑膜8之间的界面。在发育过程中，神经上皮，以及后来的放射状胶质年底脚连接到这个表面9,10。有些杉木ST的神经元在人脑和神经细胞的多丝分裂在此区域11的观察。后来，在胚胎神经，皮质的interneurons被称为遍历软脑膜地区，除了在中间区和脑室下区12-14它们的迁徙路线。在此期间，干细胞可以从该区域中进行培养，它似乎是神经和gliogenesis 5的活性位点。在成人大脑，它已经报道的interneurons可以从软脑膜表面祖细胞缺氧的挑战7诞生。然而，该地区对他的胚胎和出生后发育过程中genensis的贡献由于专门调查这一区域6的难度依然晦涩的部分。在上丘和在大脑皮质，表面（或层我在皮层）的interneurons可以调节下层兴奋性神经元群体的电路的输出，从而有助于significantly这些结构的功能。特别是，第1层的interneurons在首要的位置，调节神经元的整定其广泛的连接性皮质列15,16的浅表和深部层大脑皮层的上层射击。以类似的方式，水平的interneurons接受兴奋性输入从皮 ​​质和视网膜纤维，项目在相对 ​​宽的区域，并推测介导的抑制神经元群体响应远程视觉刺激17,18的。另外，其形态是非常适合于在显影视觉系统19中的图案化波活动中发挥潜在作用。有趣的是，interneuron发育和成熟发生很大程度出生后。另外，该成熟过程中已经发现，通过神经细胞的活动进行调节，因此对电路功能20,21终身后果发育可塑性的基板。值得注意的是，正Ø促销员的描述，可以专门针对这些细胞植入基因。除以祖细胞可以靶向与逆转录病毒7，但病毒产量是耗时并需要技巧，得到所需要的细胞转导的高滴度。 电穿孔导致了文艺复兴时期的神经发育的研究，因为它允许在神经祖细胞4，22，23信号通路的快速，高效的基因审讯。电穿孔涉及到注射的质粒DNA，随后递送电脉冲的头部的外侧，以单向驱动DNA导入周围的脑室4，22，23的增殖祖细胞。电显示通过细胞周期进行质粒的转基因24的表达M期，要求细胞的转运。具体而言，已经发现，只细胞通过内质粒电穿孔的8小时M期会表达外源基因，尽管他们有效地提供心室壁24的所有细胞内〜160微米。据推测，这是由于需要为核膜破裂在允许的附加 ​​型质粒的核设施，如化学品导致的核通透性能诱导后有丝分裂细胞25质粒的表达。最初受聘于胚胎22，电穿孔，是深受26后，27适于用在出生后大脑。最近，我们已经适应了电穿孔的在软脑膜表面祖6遗传操作使用。此外，使用这种方法，我们已经表明，有明显的祖细胞的两个不同细胞系中此区域的元间和星形细胞6。该协议详细介绍了简单，快速，强大的方式来针对这些细胞进行审讯这些细胞的机制调节发展。

<table border="0" cellpadding="0" cellspacing="0" style="width:695px;" width="694">
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		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Item Name</td>
			<td style="width:269px;">Vendor</td>
			<td style="width:177px;">Catalog Number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fire Polished Borosilicate Tubing</td>
			<td>World Precision Instruments, Inc.</td>
			<td>1B100F-4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micropipette Puller</td>
			<td>Sutter Instruments Company</td>
			<td>P-30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fast Green FCF</td>
			<td>Sigma Aldrich, Inc.</td>
			<td>F7528</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">XenoWorks Digital Microinjector</td>
			<td>Sutter Instruments Company</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ECM 830 Generator</td>
			<td><a name="RANGE!B6">Harvard Apparatus, BTX Instrument Div</a></td>
			<td>45-0052</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3mm Platinum Tweezertrodes</td>
			<td>Harvard Apparatus, BTX Instrument Div</td>
			<td>45-0487</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SignaGel Electrode Gel</td>
			<td>Cardinal Health</td>
			<td>70315-025</td>
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		<tr height="22">
			<td height="22" style="height:22px;">Tris-EDTA Buffer, pH 8.0</td>
			<td>Integrated DNA Technologies, Inc.</td>
			<td>11-01-02-05</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Infrared Heat Lamp</td>
			<td>VWR</td>
			<td>36547-009</td>
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			<td height="21" style="height:21px;">Fine Scissors Sharp</td>
			<td>Fine Science Tools</td>
			<td>14060-09</td>
		</tr>
	</tbody>
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neonatal pial surface electroporation

在过去的几年中软脑膜表面已被确定为重要的生发利基胚胎，围产期和成年神经和gliogenesis期间，包括伤后。然而，对于基因询问这些祖人口和跟踪他们的谱系方法已经由于缺乏特异性或耗时的生产病毒的限制。因此，在该区域的进展一直只有少数这个位置的调查相对较慢。在出生后的大脑电已经使用了十多年，研究在胚胎神经干细胞的特性，以及最近。这里我们描述了一种高效，快速和简单的技术的基础上适合电穿孔的方法软膜表面祖细胞的遗传操作。软膜表面电允许这些祖细胞的浅显遗传标记和操纵，从而代表为研究这些细胞的一个省时省力又经济的方法。

软膜表面电穿孔结果中质粒DNA的细胞中，主要是祖细胞，在室温或接近软脑膜表面6的表达。更具体地，所述电极的定向是在听写质粒运动和随后的表达式的方向是至关重要的。因此，在双电极配置，该质粒被定向在负和正电极之间的大致的直矢量。因此，如果负极放置在注射部位和正极是腹侧向负极，质粒将被压入软膜表面。假定定向的电极以产生电流矢量垂直于表面将是非常理想的。用适当的电极取向和使用更小，更灶性定位的电极，生存是100％。然而，在一些地区，如中脑，小心，注意避免电流输送，可能停止的心脏，并由此喧嚣Ë降低电穿孔小鼠幼崽的生存力。刚描述的三电极结构或其它修饰可有助于避免参与生命功能，如心脏速率28的结构定位较困难的区域。它来观看幼仔进行适当的再升温到其自然的身体的温度，这是由运动和颜色明显的是重要的。当他们返回到彩色，它们可以被放回巢。从小鼠，我们最经常使用的CD1应变母亲立即开始培育的幼崽回来。然而，其他菌株可以更变，变得具攻击性或疏忽。转让床上用品的气味给幼崽通常可以防止这些问题。在那里株疏忽或侵略是严重的，促进幼崽从CD1应变母亲是最好的解决方案。 标记的细胞电穿孔后6通常观察到的许多个月。然而，与任何埃尔ectroporation方法，质粒DNA稀释细胞分裂29。质粒稀释见于快骑自行车的人群（ 如 SVZ祖细胞）的相同程度不在我们的软脑膜表面观察到的人口，提示少增殖6。然而，我们现在使用最近描述的换位技术（ 例如 piggyBac转），以允许对转座子的稳定插入基因组DNA的29，30完全缓解这一稀释的问题。另外，Cre重组酶表达质粒可电转化酶Cre报告小鼠来调解这些细胞31的稳定的遗传跟踪。 我们的初步结果表明，我国多数细胞的有丝分裂是在电6时，符合在胚胎中枢神经系统，具体地描述数据，即电穿孔标签的集合S和细胞周期的2 M期的增殖细胞4，但是，需要进一步调查，以确定电穿孔细胞的身份和血统的软脑膜表面，以及难以准确确定其增殖的性格，有文献4杂散化学和细胞遗传标记的许多例子。此外，我们注意到局部注射部位血肿，特别是在皮层护理时不采取提供小批量或当不遵循适当的预防措施，以避免血管破​​裂。 我们以前的表征显示单元中的至少两个谱系-星形胶质细胞前体和interneuron祖细胞6。星形胶质细胞倾向于保持在软膜表面（ 图4A）和各细胞形成致密云周星形细胞的处理6。的interneurons大多保持在软膜表面，但有些细胞能在几乎所有皮质层在上丘6或更深找到。钍ESE神经元表现出广泛的树突，可以是只要几百微米（ 图4B）。我们并没有观察到的证据，少突胶质细胞或神经元兴奋性的皮层6的标签。多数电穿孔的细胞似乎通过有丝分裂在接近的时间上的EP程序皮质6通过。具体来说，电穿孔EGFP +细胞是双标与DNA复制的BrdU标记时，与前此胸苷类似物（ 图4C-4C 3）脉冲2小时。然而，值得注意的是，我们已经看到了在这些地区6 pericytic和/或内皮细胞标记的证据是非常重要的。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51319/51319fig1highres.jpg" src="/files/ftp_upload/51319/51319fig1.jpg" /> 图电穿孔1。新生儿软膜表面（A） 的冠状切面示意图，显示是在注射靶向脑皮层的区域。沿皮层的层显示细胞电穿孔的细胞的实际观测到的混合种群，包括元间祖细胞和astrocystic前体的例子。 （B）的产后第2天鼠注射前，概述软脑膜表面兴趣的区域图像，皮质（CTX）和上丘（SC）。 <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/51319/51319fig2highres.jpg" src="/files/ftp_upload/51319/51319fig2.jpg" /> 图2。浅表皮层 （A） 的实施例注入背侧皮层的定位之前，电穿孔眼和lambda之间的中点，后的生后电穿孔 。 （B）根据幼仔和感兴趣区域，灵活性不同大小的铂tweezertrodes的年龄，可用于电穿孔（3毫米，7毫米，9毫米）。直径3毫米的电极通常用于软膜表面电穿孔（见文讨论）。皮质（C） 的电穿孔。注意，放置在tweezertrodes的，放置在正探头眼睛下方，使电流被驱动到腹侧。 <img alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/51319/51319fig3highres.jpg" src="/files/ftp_upload/51319/51319fig3.jpg" /> 图3:上丘针对性产后电（A）注射的2日龄小狗在上丘，显示了麻醉的小狗和安置枪头只是超出了头骨的手工克制。 （ 二）注射液，显示分散浆在上丘（SC）的区域ID DNA溶液。该tweezertrodes为电的资深大律师，开车前往SC注射部位的背侧表面的DNA（ 三）配售。 <a href="https://www.jove.com/files/ftp_upload/51319/51319fig3highres.jpg" target="_blank">请点击这里查看这个数字的放大版本。</a> <img alt="图4" fo:content-width="5in" fo:src="/files/ftp_upload/51319/51319fig4highres.jpg" src="/files/ftp_upload/51319/51319fig4.jpg" /> 图4。上丘靶向产后电穿孔 （A） 的 连接面 ，共焦图象的最大投影堆叠从背外侧皮层的表达控制荧光报告质粒电穿孔后〜2周膜标签的三叶草32（绿色）和TagBFP2 33核蛋白（蓝色荧光PSeudocolored红色）。皮质interneuron大约两月后，电穿孔（二）冠状切面，显示大量的树突。比例尺测量（A，C）100微米和40微米（B）。 （CC 3）上丘表明大多数的EGFP +细胞是将的BrdU 2小时的电穿孔前的单脉冲标记的软膜表面的矢状切面。

Watch this Scientific Journal Video about Neonatal Pial Surface Electroporation at JoVE.com

Neonatal Pial Surface Electroporation

软脑膜表面是受到越来越多的关注，在中枢神经系统的唯一祖区。在此，我们详细为这个祖区使用改进的电穿孔法快速基因操作的方法。此过程可用于细胞和分子细胞谱系及参与细胞分化信号通路的调查，并阐明子细胞的命运和属性。

新生儿软脑膜表面电穿孔

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and ...

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

13.8K 次观看.  Cedars-Sinai Medical Center. 软脑膜表面是受到越来越多的关注，在中枢神经系统的唯一祖区。在此，我们详细为这个祖区使用改进的电穿孔法快速基因操作的方法。此过程可用于细胞和分子细胞谱系及参与细胞分化信号通路的调查，并阐明子细胞的命运和属性。 

视频: Neonatal Pial Surface Electroporation

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 ...

科研

神经科学

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 ...

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 ...

Neonatal Subventricular Zone Electroporation

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal cells1. Understanding the molecular pathways responsible for NSC self-renewal, commitment, and differentiation is critical for harnessing their unique potential to repair the brain and better understand central nervous system disorders. Previous methods for the manipulation of mammalian systems required the time consuming and expensive endeavor of genetic engineering at the whole animal level2. Thus, the vast majority of studies have explored the functions of NSC molecules in vitro or in invertebrates.
Here, we demonstrate the simple and rapid technique to manipulate neonatal NPCs that is referred to as neonatal subventricular zone (SVZ) electroporation. Similar techniques were developed a decade ago to study embryonic NSCs and have aided studies on cortical development3,4 . More recently this was applied to study the postnatal rodent forebrain5-7. This technique results in robust labeling of SVZ NSCs and their progeny. Thus, postnatal SVZ electroporation provides a cost and time effective alternative for mammalian NSC genetic engineering.

We demonstrate a minimally invasive technique referred to as neonatal subventricular zone electroporation. The technique consists of injecting plasmid DNA into the lateral ventricles of neonatal pups and applying electrical current to deliver and genetically manipulate neural stem cells

新生儿脑室下区电穿孔

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal ...

Subtype-selective Electroporation of Cortical Interneurons

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression of genes of interest to specific neuronal subtypes has proven remarkably challenging due to the relative scarcity of specific promoter elements. GABAergic interneurons constitute a neuronal population with extensive genetic and morphological diversity. Indeed, more than 11 different subtypes of GABAergic interneurons have been characterized in the mouse cortex1. Here we present an adapted protocol for selective targeting of GABAergic populations. We achieved subtype selective targeting of GABAergic interneurons by using the enhancer element of the homeobox transcription factors Dlx5 and Dlx6, homologues of the Drosophila distal-less (Dll) gene2,3, to drive the expression of specific genes through in utero electroporation.

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

皮质的interneurons的亚型选择性电穿孔

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such as epilepsy and neurodegenerative disorders. However, it is technically difficult to obtain EEG recordings in neonates as it requires specialized handling and great care. Here, we present a novel method to record EEG in neonatal rat pups (P8-P15). We designed a simple and reliable electrode using computer pin loci; it can be easily implanted into the skull of a rat pup to record high-quality EEG signals in the normal and epileptic brain. Pups were given an intraperitoneal (i.p.) injection of the neurotoxin kainic acid (KA) to induce epileptic seizures. The surgical implantation performed in this procedure is less expensive than other EEG procedures for neonates. This method allows one to record high-quality and stable EEG signals for more than 1 week. Furthermore, this procedure can also be applied to adult rats and mice to study epilepsy or other neurological disorders.

Here, we introduce a novel technique designed to record electroencephalography (EEG) in freely moving neonatal epileptic pups and describe its procedures, features, and applications. This method allows one to record EEG for more than 1 week.

使用新颖的方法在自由移动的新生大鼠中记录脑电图

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

脊髓神经元从新生小鼠中分离和培养

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

新生小鼠脑巴细胞基因调控的立体定向手术

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

体内新生小鼠皮层神经元的双光子成像

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

Cochlear Surface Preparation in the Adult Mouse

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous etiologies such as exposure to excessive noise, the use of ototoxic medications, bacterial or viral ear infections, head injuries, and the aging process. Loss of sensory hair cells is a common pathological feature of the varieties of acquired hearing loss. Additionally, the inner hair cell synapse can be damaged by mild insults. Therefore, surface preparations of cochlear epithelia, in combination with immunolabeling techniques and confocal imagery, are a very useful tool for the investigation of cochlear pathologies, including losses of ribbon synapses and sensory hair cells, changes in protein levels in hair cells and supporting cells, hair cell regeneration, and determination of report gene expression (i.e., GFP) for verification of successful transduction and identification of transduced cell types. The cochlea, a bony spiral-shaped structure in the inner ear, holds the auditory sensory end organ, the organ of Corti (OC). Sensory hair cells and surrounding supporting cells in the OC are contained in the cochlear duct and rest on the basilar membrane, organized in a tonotopic fashion with high-frequency detection occurring in the base and low-frequency in the apex. With the availability of molecular and genetic information and the ability to manipulate genes by knockout and knock-in techniques, mice have been widely used in biological research, including in hearing science. However, the adult mouse cochlea is miniscule, and the cochlear epithelium is encapsulated in a bony labyrinth, making microdissection difficult. Although dissection techniques have been developed and used in many laboratories, this modified microdissection method using cell and tissue adhesive is easier and more convenient. It can be used in all types of adult mouse cochleae following decalcification.

This article presents a modified cochlear surface preparation method that requires decalcification and use of a cell and tissue adhesive to adhere the pieces of cochlear epithelia to 10 mm round cover slips for immunohistochemistry in adult mouse cochleae.

成年小鼠中的科利耳表面制备

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous ...