Name: a pipeline using bilateral in utero electroporation to interrogate genetic influences on rodent behavior
Uploaded: 2020-05-21T14:45:00.000+00:00
Duration: 6 min 59 s
Description: Watch this Scientific Journal Video about A Pipeline using Bilateral In Utero Electroporation to Interrogate Genetic Influences on Rodent Behavior at JoVE.com

我们感谢丽莎·克雷茨格对手稿的批判性反馈和编辑。我们感谢克鲁兹-马丁实验室的所有研究助理，他们在帮助进行注水和行为大脑细胞计数方面非常宝贵。我们感谢安德烈·克韦奇对三极电极设计的意见，以及托德·布鲁特和波士顿大学生物成像核心的使用。这项工作得到了NARSAD青年研究员赠款（AC-M，#27202）、布伦顿R.卢茨奖（ALC）、I.奥尔登·马奇奖（ALC）、NSF NRT UTB:神经光子学国家研究奖学金（ALC，#DGE1633516）和波士顿大学本科研究机会项目（WWY）的支持。资助者在研究设计、数据收集和分析、决定出版或编写手稿方面没有作用。

所有实验方案均根据美国国家卫生研究院（NIH）动物研究指南执行，并经波士顿大学机构动物护理和使用委员会（IACUC）批准。
1. DNA溶液准备
<ol><li>购买商业质粒或子克隆感兴趣的基因到质粒与所需的促进剂。在这里，使用了 CAG 推广器 （pCAG-EGFP） 下含有 EGFP 的质粒。 注:根据所需的表达水平确定所需的推广人。一般来说，CAG促进剂下的质粒可用于达到高水平的转基因，而细胞型特异性促进剂（例如神经元突触素）往往不太活跃。实验者应该从经验上确定每个质粒的适当表达水平。</li>
	<li>用适当的抗生素转化细菌，在细菌介质中增加库存。<ol>
<li>从-80°C中取出称职细胞（DH5+细胞），在冰上解冻20分钟。</li>
		<li>将 100 pg -100 ng 的质粒 DNA 与 30μL 的称职细胞混合。在冰上孵化混合物20分钟。</li>
		<li>在 42 °C 的水浴中孵育 45 秒，然后将管子还回冰层 2 分钟，以进行热冲击。</li>
		<li>将 200 - 1，000 μL 的 LB 介质添加到经过改造的称职细胞中，并在 37 °C 下在摇动孵化器上生长 45 分钟。</li>
		<li>转化细胞的200 μL板变成含有适当抗生素的LB agar板，并在37°C下将板过夜孵育。</li>
		<li>第二天，在200毫升LB汤中孵育一个细菌菌群，在37°C下用适当的抗生素在摇晃的孵化器上过夜。</li>
	</ol>
</li>
	<li>使用最大预备套件净化质粒DNA。<ol>
<li>按照已获得的 maxiprep 套件中提供的说明操作。对于精英步骤，不要将DNA插入精英缓冲区。相反，使用 200 μL 的无菌 1x PBS 或分子级水来分流 DNA。</li>
		<li>确保DNA的最终浓度大于1微克/μL。如果含有感兴趣的基因的质粒中不含记者基因，那么还准备一个质粒与记者分子（如绿色荧光蛋白（GFP）共同传染，以允许变异细胞的可视化。</li>
	</ol>
</li>
	<li>通过稀释质粒DNA到1倍PBS到1微克/μL每个质粒的最终浓度，为手术准备DNA解决方案。在DNA溶液中加入快速绿色染料，最终浓度为0.1%。对于双边注射，准备每个大坝60μL的溶液（约10只幼崽）。 注:如果质粒不含记者基因，与GFP共用电波。共电率通常为 95% 或更高。在我们手中，输血效率不受共输的影响。所有电冲压质粒应稀释至 1μg/μL。</li>
</ol>2. 订购或繁殖定时怀孕小鼠
<ol><li>如果订购定时怀孕的小鼠，命令小鼠在胚胎日（E）13日或更早到达，以便水坝有足够的时间来适应动物的住房。 在此协议中，CD-1外种小鼠用于所有实验。 注:提前几天订购将减轻动物压力，提高幼崽的存活率。</li>
	<li>如果繁殖超时怀孕的小鼠，每周一次，将雌性小鼠与雄性小鼠配对过夜。检查第二天早上是否有阴道插头（E0.5）。通过监测雌性小鼠的体重来确定怀孕情况。 注意:不同的小鼠菌株在怀孕期间体重增加不同，因此确定所用小鼠菌株的典型体重增加。</li>
	<li>无论是订购还是繁殖老鼠，为了减轻水坝的压力，在笼子里放一个筑巢垫和老鼠屋。减轻压力有助于提高幼崽的存活率。</li>
</ol>3. 三爪电极的设计与组装
<ol><li>使用厚度为 0.063 的 2 级钛板作为电极触点的库存材料。</li>
	<li>使用标准加工技术或精密手工工具，制作具有以下尺寸的电极:20 mm x 5 mm，带有圆形尖端和凹槽背。使用细砂砂纸去除任何粗糙的边缘或毛刺。</li>
	<li>要连接电极触点，将 22 G 搁浅的铜线包裹在电极凹槽周围，并通过焊接固定。使用热收缩管保护此关节。</li>
	<li>然后，使用额外的热收缩管将连接的电极连接到可自动取电的非导电钳上，使两个负电极。将单个正极连接到可自动粘合的非导电材料（如带锯掉头部的牙刷手柄）。使用标准香蕉插头安装电线的开放端。</li>
</ol>4. 手术准备
<ol><li>将怀孕的水坝带到手术区至少30分钟之前，允许减少压力水平后，从动物设施运输。</li>
	<li>使用灭菌杀菌湿巾和 70% 乙醇对整个手术部位进行消毒。手术开始前换手套。</li>
	<li>在玻璃珠消毒器中消毒自动夹式工具。</li>
	<li>将无菌 1 倍 PBS（每坝约 50 mL）转移到 50 mL 圆锥管中，放在热水浴池的管架中加热至 38-40 °C。 使用温度计检查无菌盐水温度。</li>
	<li>打开水加热循环泵，使其在手术开始前加热至37°C。这将在手术期间保持小鼠的体温。</li>
	<li>打开压力喷油器和电镀器，确保手术前正常工作。</li>
	<li>在桌面离心机上短暂旋转质粒DNA溶液（在步骤1.4中获得），并将其放置在冰上。</li>
	<li>将玻璃移液器拉到移液器上，使拉玻璃移液器的尖端直径约为 50μm。</li>
	<li>用 20-40 μL 的 DNA 溶液填充移液器（在步骤 1.4 中获得）。</li>
	<li>设置所有必要的手术项目，包括脱毛化妆水，碘，70%乙醇，棉花交换，眼药膏，缝合线，纱布等。</li>
	<li>准备手术表并填写必要的信息，如鼠标 ID 和体重、手术日期、外科医生姓名等。</li>
</ol>5. 在子宫电镀手术中
<ol><li>在手术前称重鼠标，并在手术纸上注明这一点。</li>
	<li>用4%（v/v）氧异氟混合物在感应室中吸入怀孕小鼠（E16）麻醉。一旦小鼠被诱导，移动到口罩吸入和保持异氟在1-1.5%（v/v）和监测整个手术的呼吸。检查鼠标是否完全麻醉，呼吸速率应为每分钟 55-65 次呼吸。</li>
	<li>施用术前镇痛药:丁丙诺啡（3.25毫克/千克）SC）和甲氧西卡姆（1-5毫克/千克）:SC）最大体积为10-30毫升/千克。</li>
	<li>使用脱毛霜或小心使用剃须刀去除腹部的毛皮。用波维酮碘和70%乙醇擦拭腹部，至少重复3次。使用无菌纱布在腹部周围创建无菌场，也可以使用无菌拖曳。</li>
	<li>在腹部皮肤中做一个中线切口（3-4厘米），一定要用钳子抬起皮肤，以避免穿过肌肉。然后切开肌肉，再次小心抬起肌肉，以避免切割重要的器官。</li>
	<li>使用环钳小心地将子宫角从大坝中拉出，并轻轻地将其放置在无菌场上，确保子宫角有衬垫支撑，不会拖离大坝太远。从此以后，使用预热无菌 1x PBS 在整个手术过程中保持子宫角湿润。</li>
	<li>使用钳子或手指定位胚胎。小心地将拉起的玻璃移液器以 45 度角插入头部的水平平面，并插入侧心室，可在大脑中线和眼睛之间进行视觉识别。将大约 2-3 μL 注入每个横向心室，方法是将移液器插入其中一个，然后插入另一个心室（建议），或将 DNA 溶液注入一个心室，直到它进入两个横向心室。如果注射后存在新月形状，心室已被成功定位。 注意:玻璃移液器的尖端在手术过程中可能会断裂。如果发生这种情况，更换玻璃移液器，确保子宫角保持湿润，同时准备一个新的移液器，并充满DNA溶液。</li>
	<li>要在前额皮层中通过双边方式传染细胞，只需将两个负电极放置在胚胎头部的侧面，然后稍微向侧心室传递，并将正极定位在眼睛之间，就在发育的鼻子前。</li>
	<li>确保胚胎被慷慨滋润。应用四个方形脉冲（脉冲持续时间 = 50 毫秒持续时间，脉冲振幅 = 36 V，插话间隔 = 500 毫秒）。</li>
	<li>注射并电聚所有胚胎，逐一进行，使每个胚胎在DNA溶液注射后立即被电镀。一旦所有胚胎都经过电镀，请小心地将子宫角插入腹腔。在此步骤中，将腹腔涂在无菌 PBS （1x） 中，以帮助子宫角放置。</li>
	<li>用无菌的 1x PBS 填充腹腔，以便在完成配音后不再保留气囊。用可吸收的缝合线缝合肌肉，用丝绸不可吸收的缝合线缝合皮肤。</li>
	<li>允许大坝在加热室中完全恢复至少 1 小时。在接下来的48小时里，定期检查大坝。当大坝从麻醉中恢复过来并恢复知觉时，它会开始移动和搅拌。</li>
	<li>如果水坝出现疼痛迹象，例如将身体向上弹，呼吸迅速，则施用术后镇痛剂。只有当有疼痛的迹象，因为SC注射可以强调大坝，但如果管理给实验组也管理对照组，反之亦然。</li>
</ol>6. 在母亲互动任务中表现早期社会行为
注:此协议是根据以前的出版物5，25改编的。在小鼠从产后天 （P） 18-21 出生后执行此任务。
<ol><li>产妇在产妇互动I（MI1）任务中的母体居所行为。<ol>
<li>确保在执行任务前一周内不会更改笼子床上用品。</li>
		<li>获得或构建一个可轻松清洁的开放领域 （OF） 竞技场（建议使用丙烯酸），尺寸如下:50 x 50 x 30 厘米（长度宽度高度）。行为表现过程中的照明条件可能因实验问题而异，并可能影响觉醒和焦虑行为的水平。在昏暗的光线下（约20勒克斯）在竞技场中心记录行为实验。</li>
		<li>在行为测试前的两天里，将大坝放在竞技场一角的网状钢丝杯（如铅笔杯）下，每天5分钟，让大坝适应竞技场。</li>
		<li>在测试日，从P18-21开始，在老鼠断奶之前，用消毒湿巾和70%的乙醇彻底清洁竞技场。</li>
		<li>设置竞技场与两个对立的角落都包含干净的床上用品和一个角落包含脏巢床上用品从小狗的家庭笼子。</li>
		<li>让每只小狗探索竞技场3分钟，在开始时将每只小狗放在中性空角。 注:以30 fps的摄像机记录行为。彻底清洁每只小狗之间的竞技场，并更换新鲜的床上用品。替代哪个角落是新鲜与巢床上用品作为控制，以避免角偏好由于其他原因（即环境噪音或光线）。如果在 P18-21 开发窗口中运行多个垃圾，则在几天之间同时运行行为实验。还要确保在给定的一天中，控制组和实验组并行运行。</li>
	</ol>
</li>
	<li>产妇互动II（MI2）任务中的母体社会互动<ol>
<li>在 MI1 任务完成后立即执行此任务。</li>
		<li>设置竞技场，使一个角落包含一个空的铁丝网杯，对立的角落包含网状钢丝杯下的大坝。如果老鼠能够移动杯子，用可以贴在杯子顶部的重量来称量杯子，以防止移动。把家里笼子里的脏窝床上用品放在另一个角落里。</li>
		<li>运行 MI2 任务。将小狗放在空角，以 30 fps 记录 5 分钟的行为，让小狗探索。分开运行每只小狗，彻底清洁整个竞技场和每个小狗之间的铁丝网杯。</li>
	</ol>
</li>
</ol>7. 执行成人社会行为任务
<ol><li>在 MI1 和 MI2 任务中运行的相同小鼠中运行成人社交行为，一旦它们长大（P60-P70 或更高）。此处收集的数据是以单独的队列完成的。</li>
	<li>连续3天处理成年小鼠，让实验者适应。确保只有熟悉小鼠的实验者进行行为实验，最好让同一个人执行所有任务。</li>
	<li>习惯老鼠到现场3天，每天5分钟。</li>
	<li>新对象识别任务中的检测行为，以测量新对象的一般运动和兴趣。如果老鼠在社会交往中有特定的缺陷，这将允许对社会行为进行更有意义的解释。<ol>
<li>将老鼠放在竞技场的一角，用新物体（表面光滑、清洁的小塑料玩具）在竞技场的一角放置5分钟。用70%的乙醇彻底清洁老鼠之间的竞技场。</li>
		<li>对于新颖的对象识别，将以前暴露的"小说对象"放在一个角落，将一个新的新事物放在对立的角落。对于所有任务，将试验之间的角切换为控制。</li>
		<li>对于新的社会互动，确保新老鼠的年龄，应变和性别匹配，并适应网状线杯连续2天，每天5分钟。每次试用时，将一只新鼠标放在网状钢丝杯下，在对方角落放置一个空网状钢丝杯。让老鼠在记录行为的同时探索竞技场5分钟。每次试用之间彻底清洁竞技场和网状钢丝杯。</li>
	</ol>
</li>
</ol>8. 分析行为数据
<ol><li>使用深度实验室（https://github.com/AlexEMG/DeepLabCut）执行基本的身体部位跟踪）。有关如何安装和使用 DeepLabCut 的详细说明，可在其 GitHub 页面上找到。此外，还有一个基于自定义的基于巨蛇的库"dlc_utils"（https://github.com/balajisriram/dlc_utils），用于在基本身体部位跟踪完成后进一步分析数据。有关如何使用此库的更多详细信息，请访问 GitHub 页面。<ol>
<li>使用安纳康达安装过程安装深度实验室切割。安装一个GUI功能CPU专用版本的DeepLabCut以及支持GPU的版本，用于培训网络。</li>
		<li>按照下面链接中提供的说明创建跟踪身体部位的项目。请从数据集中选择帧样本，并手动标记这些采样帧中的相关身体部位。训练 DeepLabCut 网络以预测身体部位并验证训练有素的网络是否表现良好。 https://github.com/AlexEMG/DeepLabCut/blob/master/examples/Demo_yourowndata.ipynb</li>
		<li>为了跟踪身体位置和识别开放领域中的简单相互作用，识别动物的中心、头部（操作上定义为耳朵之间的中点）、左右耳以及鼻子和尾巴底部。跟踪多个身体部位允许在框架中由于遮挡而缺少某些身体部位时进行适当的替换。</li>
		<li>除了动物的身体部位，跟踪与环境相关的各种点:如行为框的边缘。这些允许在多个会话中重复估计此类点 - 即使行为设置的位置在会话之间相对于相机略有变化。</li>
		<li>从行为数据中跟踪身体部位后，请注意根据与视频每帧预测相关的信心来筛选预测的身体部位位置。低置信度预测通常与被遮挡的身体部位相关。对于此类预测，请将给定的身体部位替换为另一个（如果此类替换是适当的），或使用其他身体部位的位置来预测相关身体部位可能位于何处。对于大多数开放领域应用，啮齿动物身体的中心机器人很少被遮挡，并且可以高精度和精确度地预测。</li>
		<li>使用中心机器人的预测位置以及环境中跟踪点的位置来估计动物行为的一些特征。例如，在开放领域数据中，位置的时间衍生物可用于计算动物的速度。</li>
	</ol>
</li>
	<li>为了避免偏见，尽可能对实验组进行所有"盲"实验，特别是当评估结果时有任何主观因素时。通过将数据汇集到男性和女性组，测试性别差异对主要实验结果的影响。所有统计测试旨在测试各组之间的动物数量相等。</li>
</ol>9. 细胞计数后，以描述细胞转染的程度
<ol><li>确定每只小鼠转染的细胞数量，因为并非所有小鼠都成功进行了转染，并且转染细胞的数量也会有变化。实现此目的的一种方法是计算交替日冕部分的转染神经元数，然后进行插值以估计转染细胞的总数。为此，图像和计数所有其他日冕部分（50微米）。<ol>
<li>使用带有 4% PFA 的转心输液来修复组织，然后解剖大脑。冷冻保护后，在50微米处在日冕部分切入大脑。</li>
		<li>对于前额皮层，计数细胞在 +2.75 和 +1.35 毫米从布雷格玛。这些坐标包含前额皮质区域，包括部分体瘤敏感皮层 （S1）。 使用这种方法，在更多的胸皮区域或皮下区域没有观察到的转染细胞。</li>
		<li>请务必从右半球表示左半球，例如在剖腹产时用针孔标记一个半球，并在双边计数细胞。使用自动细胞计数软件或手动计数细胞，使用 DAPI 确认细胞体的存在。</li>
	</ol>
</li>
	<li>获得单元格计数后，设置包含阈值。例如，在分析中仅包括双边电镀的小鼠。要进一步分析，将与行为反应发生反应的细胞数量相关，以查看是否有关联。这项技术将针对多个大脑区域，因此有必要提供有关哪些大脑区域被基因操纵的信息。 注:操纵某些基因可能会改变神经元迁移、规范和/或死亡。在细胞计数过程中确保大脑解剖学被检查，每个被感染的神经元都在据称被转染的层内（即L2/3）。毛解剖测量，如皮质厚度也可以量化通过测量从皮亚到皮质L6的距离。</li>
</ol>

<ol>
	<li>Yang, Y., 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=Genetic+association+and+meta-analysis+of+a+schizophrenia+GWAS+variant+rs10489202+in+East+Asian+populations.">Genetic association and meta-analysis of a schizophrenia GWAS variant rs10489202 in East Asian populations.</a> Translational Psychiatry. 8 (1), 144 (2018).</li><li>Schizophrenia Working Group of the Psychiatric Genomics Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+insights+from+108+schizophrenia-associated+genetic+loci.">Biological insights from 108 schizophrenia-associated genetic loci.</a> Nature. 511 (7510), 421-427 (2014).</li><li>Howard, D. M., 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=Genome-wide+meta-analysis+of+depression+identifies+102+independent+variants+and+highlights+the+importance+of+the+prefrontal+brain+regions.">Genome-wide meta-analysis of depression identifies 102 independent variants and highlights the importance of the prefrontal brain regions.</a> Nature Neuroscience. 22 (3), 343-352 (2019).</li><li>Grove, J., 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=Identification+of+common+genetic+risk+variants+for+autism+spectrum+disorder.">Identification of common genetic risk variants for autism spectrum disorder.</a> Nature Genetics. 51 (3), 431-444 (2019).</li><li>Comer, A. L., 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=Increased+expression+of+schizophrenia-associated+gene+C4+leads+to+hypoconnectivity+of+prefrontal+cortex+and+reduced+social+interaction.">Increased expression of schizophrenia-associated gene C4 leads to hypoconnectivity of prefrontal cortex and reduced social interaction.</a> PLoS Biology. 18 (1), 3000604 (2020).</li><li>Chini, M., 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=Resolving+and+Rescuing+Developmental+Miswiring+in+a+Mouse+Model+of+Cognitive+Impairment.">Resolving and Rescuing Developmental Miswiring in a Mouse Model of Cognitive Impairment.</a> Neuron. 105 (1), 60-74 (2020).</li><li>Clifton, N. E., 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+expression+of+genes+associated+with+schizophrenia+and+bipolar+disorder+across+development.">Dynamic expression of genes associated with schizophrenia and bipolar disorder across development.</a> Translational Psychiatry. 9 (1), 74 (2019).</li><li>Oberlander, V. C., Xu, X., Chini, M., Hanganu-Opatz, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+dysfunction+of+prefrontal-hippocampal+networks+in+mouse+models+of+mental+illness.">Developmental dysfunction of prefrontal-hippocampal networks in mouse models of mental illness.</a> European Journal of Neurosciences. 50 (6), 3072-3084 (2019).</li><li>Batista-Brito, R., 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=Developmental+Dysfunction+of+VIP+Interneurons+Impairs+Cortical+Circuits.">Developmental Dysfunction of VIP Interneurons Impairs Cortical Circuits.</a> Neuron. 95 (4), 884-895 (2017).</li><li>Luo, L., Callaway, E. M., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Dissection+of+Neural+Circuits:+A+Decade+of+Progress.">Genetic Dissection of Neural Circuits: A Decade of Progress.</a> Neuron. 98 (2), 256-281 (2018).</li><li>DeNardo, L., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+strategies+to+access+activated+neurons.">Genetic strategies to access activated neurons.</a> Current Opinion in Neurobiology. 45, 121-129 (2017).</li><li>Huang, Z. J., Zeng, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+approaches+to+neural+circuits+in+the+mouse.">Genetic approaches to neural circuits in the mouse.</a> Annual Reviews in Neurosciences. 36, 183-215 (2013).</li><li>Wu, Z., Yang, H., Colosi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+genome+size+on+AAV+vector+packaging.">Effect of genome size on AAV vector packaging.</a> Molecular Therapy. 18 (1), 80-86 (2010).</li><li>Callaway, E. M., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monosynaptic+Circuit+Tracing+with+Glycoprotein-Deleted+Rabies+Viruses.">Monosynaptic Circuit Tracing with Glycoprotein-Deleted Rabies Viruses.</a> Journal of Neurosciences. 35 (24), 8979-8985 (2015).</li><li>Cruz-Mart&#237;n, A., 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=A+dedicated+circuit+links+direction-selective+retinal+ganglion+cells+to+the+primary+visual+cortex.">A dedicated circuit links direction-selective retinal ganglion cells to the primary visual cortex.</a> Nature. 507 (7492), 358-361 (2014).</li><li>Saito, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+electroporation+in+the+embryonic+mouse+central+nervous+system.">In vivo electroporation in the embryonic mouse central nervous system.</a> Nature Protocols. 1 (3), 1552-1558 (2006).</li><li>Cruz-Mart&#237;n, A., Crespo, M., Portera-Cailliau, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+stabilization+of+dendritic+spines+in+fragile+X+mice.">Delayed stabilization of dendritic spines in fragile X mice.</a> Journal of Neurosciences. 30 (23), 7793-7803 (2010).</li><li>Zhang, J. H., Adikaram, P., Pandey, M., Genis, A., Simonds, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+genome+editing+through+CRISPR-Cas9+engineering.">Optimization of genome editing through CRISPR-Cas9 engineering.</a> Bioengineered. 7 (3), 166-174 (2016).</li><li>Strecker, J., 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=RNA-guided+DNA+insertion+with+CRISPR-associated+transposases.">RNA-guided DNA insertion with CRISPR-associated transposases.</a> Science. 365 (6448), 48-53 (2019).</li><li>Cruz-Mart&#237;n, A., Crespo, M., Portera-Cailliau, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+induces+the+elongation+of+early+dendritic+protrusions+via+mGluRs+in+wild+type+mice,+but+not+in+fragile+X+mice.">Glutamate induces the elongation of early dendritic protrusions via mGluRs in wild type mice, but not in fragile X mice.</a> PLoS One. 7 (2), 32446 (2012).</li><li>LoTurco, J., Manent, J. B., Sidiqi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+and+improved+tools+for+in+utero+electroporation+studies+of+developing+cerebral+cortex.">New and improved tools for in utero electroporation studies of developing cerebral cortex.</a> Cerebral Cortex. 19, 120-125 (2009).</li><li>Kolk, S. M., de Mooij-Malsen, A. J., Martens, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+Molecular+Approach+of+in+utero+Electroporation+to+Functionally+Decipher+Endophenotypes+in+Neurodevelopmental+Disorders.">Spatiotemporal Molecular Approach of in utero Electroporation to Functionally Decipher Endophenotypes in Neurodevelopmental Disorders.</a> Frontiers in Molecular Neurosciences. 4, 37 (2011).</li><li>Szczurkowska, J., 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=Targeted+in+vivo+genetic+manipulation+of+the+mouse+or+rat+brain+by+in+utero+electroporation+with+a+triple-electrode+probe.">Targeted in vivo genetic manipulation of the mouse or rat brain by in utero electroporation with a triple-electrode probe.</a> Nature Protocols. 11 (3), 399-412 (2016).</li><li>dal Maschio, M., 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=High-performance+and+site-directed+in+utero+electroporation+by+a+triple-electrode+probe.">High-performance and site-directed in utero electroporation by a triple-electrode probe.</a> Nature Communications. 3, 960 (2012).</li><li>Zhan, Y., 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=Deficient+neuron-microglia+signaling+results+in+impaired+functional+brain+connectivity+and+social+behavior.">Deficient neuron-microglia signaling results in impaired functional brain connectivity and social behavior.</a> Nature Neurosciences. 17 (3), 400-406 (2014).</li><li>Mathis, A., 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=DeepLabCut:+markerless+pose+estimation+of+user-defined+body+parts+with+deep+learning.">DeepLabCut: markerless pose estimation of user-defined body parts with deep learning.</a> Nature Neurosciences. 21 (9), 1281-1289 (2018).</li><li>Bullmann, T., Arendt, T., Frey, U., Hanashima, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transportable,+inexpensive+electroporator+for+in+utero+electroporation.">A transportable, inexpensive electroporator for in utero electroporation.</a> Development Growth and Differentiation. 57 (5), 369-377 (2015).</li><li>Li, Z., 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=Genome-wide+association+analysis+identifies+30+new+susceptibility+loci+for+schizophrenia.">Genome-wide association analysis identifies 30 new susceptibility loci for schizophrenia.</a> Nature Genetics. 49 (11), 1576-1583 (2017).</li><li>Ripke, S., 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=Genome-wide+association+analysis+identifies+13+new+risk+loci+for+schizophrenia.">Genome-wide association analysis identifies 13 new risk loci for schizophrenia.</a> Nature Genetics. 45 (10), 1150-1159 (2013).</li><li>De Marco Garcia, N. V., Fishell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subtype-selective+electroporation+of+cortical+interneurons.">Subtype-selective electroporation of cortical interneurons.</a> Journal of Visualized Experiment. (90), e51518 (2014).</li><li>Borrell, V., Yoshimura, Y., Callaway, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+delivery+to+telencephalic+inhibitory+neurons+by+directional+in+utero+electroporation.">Targeted gene delivery to telencephalic inhibitory neurons by directional in utero electroporation.</a> Journal of Neuroscience Methods. 143 (2), 151-158 (2005).</li><li>Soma, M., 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=Development+of+the+mouse+amygdala+as+revealed+by+enhanced+green+fluorescent+protein+gene+transfer+by+means+of+in+utero+electroporation.">Development of the mouse amygdala as revealed by enhanced green fluorescent protein gene transfer by means of in utero electroporation.</a> Journal of Comparative Neurology. 513 (1), 113-128 (2009).</li></ol>

作者声明没有相互竞争的利益。

这里描述了一条管道，它结合了对大量前皮质神经元感兴趣的新基因的处理与小鼠的行为检测。此外，该管道允许对同一小鼠在产后发育早期和成年期间的行为进行纵向研究。这项技术绕过了依赖遗传动物模型的需要，这种模型在时间和费用方面可能代价高昂。这个协议的优点是，它可以用来研究神经发育和神经精神病，最近GWAS已经发现了新的遗传关联28，29。</sup...

全基因组关联研究（GWAS）推动了与大脑病理学1、2、3、4相关的新候选基因的发现。这些研究在理解精神分裂症（SCZ）等破坏性神经精神病方面特别有益，对新基因的研究成为新研究和治疗干预的起点。在产前和产后早期发育期间，SCZ的基因在前额皮层（PFC）中表现出偏颇的表达，这一区域与几种神经精神病的病理学有关。此外，小鼠模型的精神疾病表现出异常活动在PFC网络6，8，9。这些结果表明，SZC相关基因可能在这个区域的发展布线中发挥作用。需要使用动物模型进行进一步调查，以了解这些候选基因对PFC中建立联系的贡献，并确定这些基因是否在神经精神病的发病机制中具有因果关系。小鼠的基因操纵技术，允许在产前和产后发育期间研究特定神经元回路的基因表达变化，是了解将基因表达变化与PFC功能障碍联系起来的分子机制的有希望的方法。
遗传小鼠系提供了一种研究特定基因对大脑发育和功能的影响的方法。然而，依靠转基因小鼠可能是有限的，因为并不总是有商业上可用的线来检查特定基因对神经回路发展的影响。此外，开发自定义鼠标线可能非常昂贵和耗时。将转基因小鼠与病毒方法相结合的交叉基因操纵策略的使用彻底改变了对大脑10、11、12的理解。尽管取得了很大进展，病毒策略还是有一定的局限性，这取决于病毒载体类型，包括包装能力的限制，可以限制病毒表达13和细胞毒性与病毒表达14。此外，在大多数实验条件下，使用腺相关病毒（AAV）的强健基因表达大约需要2至4周15周，这使得常规的病毒策略在产后早期发育期间无法操纵基因。
在子宫电化（IUE）是一种替代方法，允许快速和廉价的基因转移16，17，当加上荧光标签和药理遗传或光遗传学方法，提供了一个强大的平台，解剖神经元电路的功能。此外，随着CRISPR-Cas9基因组编辑基因的发展，可以通过细胞型特异性敲击或敲除特定基因或通过18、19的调节来过度表达或精确改变。当基因对神经元电路的影响需要在狭窄的发育窗口20中进行测试时，使用IUE的基因操纵方法尤其有利。IUE 是一种多才多艺的技术，通过将基因插入特定促进器下的表达载体，可以轻松实现过度表达。通过使用不同强度的促进剂驱动表达，或使用能够暂时控制基因表达的诱发促进剂21，22，可以达到对基因表达的额外控制。此外，IUE允许在特定皮质层、细胞类型和大脑区域内定位细胞，这并不总是可行的使用其他方法5，17。基于三个电极的IUE配置的最新进展，产生了更高效的电场分布，扩展了这种方法的功能范围，使科学家能够瞄准新的细胞类型，提高可瞄准的23、24个电池的效率、准确性和数量。这项技术最近被用来确定补充组件4A（C4A）的因果作用，这是一种与SCZ相连的基因，在PFC功能和早期认知5中。
这里介绍的是一个实验管道，它结合了基因转移方法，以目标前额皮层（包括PFC）中的大量兴奋神经元为目标，以及行为范式，不仅能够研究细胞和电路水平的变化，而且允许在整个产后发育和成年后对行为进行监测。首先描述的是一种在正面皮质区域双边传染大量层（L）2/3金字塔神经元的方法。接下来，概述了检测幼鼠和成年小鼠社会行为的任务。细胞计数可以在完成行为任务后获得，以量化细胞转染的范围和位置。此外，转染的细胞数量可以与行为数据相关联，以确定更多的转染细胞是否会导致行为更大的扰动。

<table><tbody><tr><td>13mm Silk Black Braided Suture</td><td>Havel&#039;s</td><td>SB77D</td><td>Suture skin</td></tr><tr><td>Adson Forceps</td><td>F.S.T.</td><td>11006-12</td><td>IUE</td></tr><tr><td>C270 Webcam</td><td>Logitech</td><td>N/A</td><td>Record behavior</td></tr><tr><td>Electroporator</td><td>Custom-built</td><td>N/A</td><td>See Figure 1 and 2 and Bullmann et al, 2015</td></tr><tr><td>EZ-500 Spin Column Plasmid DNA Maxi-preps Kit, 20preps</td><td>Bio Basic Inc.</td><td>BS466</td><td>Pladmid preparation</td></tr><tr><td>Fast Green FCF</td><td>Sigma</td><td>F7252-5G</td><td>Dye for DNA solution</td></tr><tr><td>Fine scissors- sharp</td><td>F.S.T.</td><td>14060-09</td><td>IUE</td></tr><tr><td>Fisherbrand Gauze Sponges</td><td>Fisher Scientific</td><td>1376152</td><td>IUE</td></tr><tr><td>Gaymar Heating/Cooling</td><td>Braintree</td><td>TP-700</td><td>Heating Pad</td></tr><tr><td>Glass pipette puller</td><td>Sutter Instrument,</td><td>P-97</td><td>IUE</td></tr><tr><td>Glass pipettes</td><td>Sutter Instrument,</td><td>BF150-117-10</td><td>IUE</td></tr><tr><td>Hair Removal Lotion</td><td>Nair</td><td>N/A</td><td>Hair removal</td></tr><tr><td>Hartman Hemostats</td><td>F.S.T.</td><td>13002-10</td><td>IUE</td></tr><tr><td>Open field maze- homemade acrylic arena</td><td>Custom-built</td><td>N/A</td><td>50 &amp;times; 50 &amp;times; 30 cm length-width-height</td></tr><tr><td>pCAG-GFP</td><td>Addgene</td><td>11150</td><td>Mammalian expression vector for expression of GFP</td></tr><tr><td>Picospritzer III</td><td>Parker Hannifin</td><td>N/A</td><td>pressure injector</td></tr><tr><td>Retractor - 2 Pronged Blunt</td><td>F.S.T.</td><td>17023-13</td><td>IUE</td></tr><tr><td>Ring forceps</td><td>F.S.T.</td><td>11103-09</td><td>IUE</td></tr><tr><td>Sterilizer, dry bead</td><td>Sigma</td><td>Z378569</td><td>sterelize surgical tools</td></tr><tr><td>SUTURE, 3/0 PGA, FS-2, VIOLET FOR VET USE ONLY</td><td>Havel&#039;s</td><td>HJ398</td><td>Suture muscle</td></tr><tr><td>Water bath</td><td>Cole-Parmer</td><td>EW-12105-84</td><td>warming sterile saline</td></tr></tbody></table>

a pipeline using bilateral in utero electroporation to interrogate genetic influences on rodent behavior

随着全基因组关联研究揭示了许多神经系统疾病的异质遗传基础，研究特定基因对大脑发育和功能的贡献的必要性增加。依靠小鼠模型来研究特定基因操纵的作用并不总是可行的，因为转基因小鼠系成本很高，而且许多与疾病相关的新基因还没有商业上可用的基因系。此外，创建鼠标线可能需要多年的开发和验证。在子宫电镀中，一种相对快速和简单的方法，以细胞类型特定的方式在体内操纵基因表达，只需要开发DNA质粒来实现特定的基因操作。子宫内电子化的双边可用于瞄准大量前额皮层金字塔神经元。将这种基因转移方法与行为方法相结合，可以研究基因操纵对前额皮层网络功能以及幼鼠和成年小鼠社会行为的影响。

成功开发并实施了定制电镀器和三爪电极。 对于 IUEs，根据先前描述的设计27（图 1A和图2）构建了廉价的定制电子增压器。一个三爪电极是23，24使用塑料钳子与2个负电极连接到爪尖和正极连接到牙刷手柄的末端（图1B）。对电极和三个爪电极进行了测?...

Watch this Scientific Journal Video about A Pipeline using Bilateral In Utero Electroporation to Interrogate Genetic Influences on Rodent Behavior at JoVE.com

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

最近发现的疾病相关基因在神经精神病发病机制中的作用仍然模糊不清。子宫电化技术的改性双边技术允许在大量神经元中进行基因转移，并检查基因表达变化对社会行为的因果关系。

使用双边子宫内电渗透的管道，以询问遗传对啮齿动物行为的影响

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

a-pipeline-using-bilateral-utero-electroporation-to-interrogate

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

4.0K 次观看.  Boston University. 最近发现的疾病相关基因在神经精神病发病机制中的作用仍然模糊不清。子宫电化技术的改性双边技术允许在大量神经元中进行基因转移，并检查基因表达变化对社会行为的因果关系。

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

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

In Utero Intraventricular Injection and Electroporation of E16 Rat Embryos

In-utero in-vivo injection and electroporation of the embryonic rat neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol, we outline the experimental methodology for preparing rats for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E15-E21 rats, however it is most commonly performed at E16 as shown in this video.

E16大鼠胚胎在子宫内侧脑室注射和电穿孔

In-utero in-vivo injection and electroporation of the embryonic rat neocortex provides a powerful tool for the manipulation of individual progenitors ...

生物学

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol we outline the experimental methodology for preparing mice for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E13-E16 mice, however, it is most commonly performed at E15, as shown in this video. 

E15小鼠胚胎在子宫内侧脑室注射和电穿孔

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

在子宫内电穿孔：鼠标控制时空基因Transefection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

在子宫内和前体内的基因表达在小鼠视网膜神经节细胞的电穿孔

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. 
 For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. 
 Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. 
 Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at &ge;6 months after birth.
 We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Genes can be manipulated during development of the cortex or the hippocampus of the rat via in utero electroporation (IUE) at E16, to enable fast and targeted modifications in neuronal connectivity for later studies of behavior or neuropathology in adult animals. Postnatal in vivo imaging for control of IUE success is performed by bioluminescence of activating co-transfected luciferase.

外用转基因大鼠通过代<em&gt;在子宫内</em&gt;电穿孔和<em&gt;在体内</em&gt;生物发光筛选

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

遗传操纵鼠标的发展下丘脑通过<em&gt;在子宫内</em&gt;电穿孔

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

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 ...

Double In Utero Electroporation to Target Temporally and Spatially Separated Cell Populations

In utero electroporation is an in vivo DNA transfer technique extensively used to study the molecular and cellular mechanisms underlying mammalian corticogenesis. This procedure takes advantage of the brain ventricles to allow the introduction of DNA of interest and uses a pair of electrodes to direct the entrance of the genetic material into the cells lining the ventricle, the neural stem cells. This method allows researchers to label the desired cells and/or manipulate the expression of genes of interest in those cells. It has multiple applications, including assays targeting neuronal migration, lineage tracing, and axonal pathfinding. An important feature of this method is its temporal and regional control, allowing circumvention of potential problems related with embryonic lethality or the lack of specific CRE driver mice. Another relevant aspect of this technique is that it helps to considerably reduce the economic and temporal limitations that involve the generation of new mouse lines, which become particularly important in the study of interactions between cell types that originate in distant areas of the brain at different developmental ages. Here we describe a double electroporation strategy that enables targeting of cell populations that are spatially and temporally separated. With this approach we can label different subtypes of cells in different locations with selected fluorescent proteins to visualize them, and/or we can manipulate genes of interest expressed by these different cells at the appropriate times. This strategy enhances the potential of in utero electroporation and provides a powerful tool to study the behavior of temporally and spatially separated cell populations that migrate to establish close contacts, as well as long-range interactions through axonal projections, reducing temporal and economic costs.

Double in utero electroporation allows targeting cell populations that are spatially and temporally separated. This technique is useful to visualize interactions between those cell populations using fluorescent proteins in normal conditions but also after functional experiments to perturb genes of interest.

使用双边子宫内电渗透的管道，以询问遗传对啮齿动物行为的影响

摘要

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此视频中的章节

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

E16大鼠胚胎在子宫内侧脑室注射和电穿孔

E15小鼠胚胎在子宫内侧脑室注射和电穿孔

在子宫内电穿孔：鼠标控制时空基因Transefection

在子宫内和前体内的基因表达在小鼠视网膜神经节细胞的电穿孔

外用转基因大鼠通过代

遗传操纵鼠标的发展下丘脑通过

Cortex-，Hippocampus-，Thalamus-，下丘脑 - ，横向隔核 - 纹状体专用

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

双电穿孔，以定时和空间分离的细胞群