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>
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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。虽然这种方法提供了激发神经元的细胞型特定转染，但一个限制是，针对其他脑细胞类型（如内科细胞或胶质细胞）不太可行。然而，多项研究表明，一种针对其他脑细胞类型30，31的修改方法。此外，通过改变电极相对于胚胎头部的位置和改变IUE的时间，其他大脑区域可以双边传染，包括海马，杏仁核，小脑，和视觉，体质和运动皮质24，32。此外，不同的皮质层可以通过在不同的发育阶段执行 IUE 来定位。
虽然 IUE 的成功率很高，但有时需要关键步骤和方法的故障排除。质粒必须经过精心设计和细胞系验证。与所有克隆一样，必须小心确保适当的基因表达，例如确认序列在框架中。此外，应确认基因操纵（例如过度表达或沉默）的效果，同时考虑到表达水平可能因小鼠发育过程和 IUE 的发育日期而异。西方的污点和qPCR可以用来确定遗传过度表达的程度5。建议将记者基因（如GFP）在单独的质粒中电镀，因为标记有GFP的蛋白质可能折叠错误或失去功能。或者，如果记者不使用，实验者可以使用原位杂交、qPCR或西印来确定兴趣基因5的表达水平。
如果质粒已被验证，但没有动物似乎对转染呈阳性，彻底检查所有设备，特别是电镀器，以确保适当的功能。当将电压脉冲输送到胚胎时，子宫角应用加热的盐水很好地滋润，电极应在电压脉冲生成时产生气泡。如果电压传递时没有气泡，则电调节器可能会出现问题。 或者，cDNA 可能没有正确注射到心室中。当 cDNA 正确注入横向心室时，快速绿色染料将以新月的形式可见。最后，电极的位置很重要。如果电极定位稍有不正确，则电池可能不会在感兴趣的区域进行转染。因此，在检查成功转染时，保存一些更多的颅脑部分，看看，如果可能，错误的大脑区域被转染。一旦采用这种方法，经验丰富的外科医生有望达到近90%的成功率。此协议可以修改，以针对其他感兴趣的大脑区域。例如，有可能以双边甚至某些皮下区域为目标，包括海马23。也有可能通过建立一个定制的电镀器，这是用于这里提供的数据5，27进一步降低成本。
未来的研究可以利用这种方法来了解新发现的基因候选者在各种神经系统疾病中的作用。所展示的管道提供了一个相对快速的检测，以测试特定的基因操纵对产后早期发育和成年后行为的影响。将来使用这种方法的努力有可能发现哪些基因在某些脑部疾病中起因子作用，包括SCZ和自闭症谱系障碍。

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 border="0" cellpadding="0" cellspacing="0" style="width:664px;" width="663">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">13mm Silk Black Braided Suture</td>
			<td style="width:121px;">Havel&#39;s</td>
			<td style="width:161px;">SB77D</td>
			<td style="width:167px;">Suture skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adson Forceps</td>
			<td style="width:121px;">F.S.T.</td>
			<td style="width:161px;">11006-12</td>
			<td>IUE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">C270 Webcam</td>
			<td style="width:121px;">Logitech</td>
			<td style="width:161px;">N/A</td>
			<td>Record behavior</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Electroporator</td>
			<td style="width:121px;">Custom-built</td>
			<td style="width:161px;">N/A</td>
			<td>See Figure 1 and 2 and Bullmann et al, 2015</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EZ-500 Spin Column Plasmid DNA Maxi-preps Kit, 20preps</td>
			<td>Bio Basic Inc.</td>
			<td style="width:161px;">BS466</td>
			<td>Pladmid preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Fast Green FCF</td>
			<td style="width:121px;">Sigma</td>
			<td>F7252-5G</td>
			<td>Dye for DNA solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Fine scissors- sharp</td>
			<td style="width:121px;">F.S.T.</td>
			<td style="width:161px;">14060-09</td>
			<td>IUE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fisherbrand Gauze Sponges</td>
			<td>Fisher Scientific</td>
			<td>1376152</td>
			<td>IUE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gaymar Heating/Cooling</td>
			<td style="width:121px;">Braintree</td>
			<td style="width:161px;">TP-700</td>
			<td style="width:167px;">Heating Pad</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Glass pipette puller</td>
			<td>Sutter Instrument,</td>
			<td style="width:161px;">P-97</td>
			<td>IUE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Glass pipettes</td>
			<td>Sutter Instrument,</td>
			<td>BF150-117-10</td>
			<td>IUE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Hair Removal Lotion</td>
			<td style="width:121px;">Nair</td>
			<td style="width:161px;">N/A</td>
			<td>Hair removal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hartman Hemostats</td>
			<td style="width:121px;">F.S.T.</td>
			<td style="width:161px;">13002-10</td>
			<td>IUE</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Open field maze- homemade acrylic arena</td>
			<td style="width:121px;">Custom-built</td>
			<td style="width:161px;">N/A</td>
			<td>50 &#215; 50 &#215; 30 cm length-width-height</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">pCAG-GFP</td>
			<td style="width:121px;">Addgene</td>
			<td>11150</td>
			<td>Mammalian expression vector for expression of GFP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Picospritzer III</td>
			<td>Parker Hannifin</td>
			<td style="width:161px;">N/A</td>
			<td>pressure injector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Retractor - 2 Pronged Blunt</td>
			<td style="width:121px;">F.S.T.</td>
			<td style="width:161px;">17023-13</td>
			<td>IUE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Ring forceps</td>
			<td style="width:121px;">F.S.T.</td>
			<td style="width:161px;">11103-09</td>
			<td>IUE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Sterilizer, dry bead</td>
			<td style="width:121px;">Sigma</td>
			<td>Z378569</td>
			<td>sterelize surgical tools</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SUTURE, 3/0 PGA, FS-2, VIOLET FOR VET USE ONLY</td>
			<td>Havel&#39;s</td>
			<td>HJ398</td>
			<td>Suture muscle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Water bath</td>
			<td style="width:121px;">Cole-Parmer</td>
			<td>EW-12105-84</td>
			<td style="width:167px;">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试，以确保其功能正常。IUE通过暴露子宫角、注射质粒DNA和对每个胚胎进行电镀（图1C）进行。如图1B，右图所示，三爪电极可以相当容易地用两只手握住，用爪子稳定胚胎的头部。L2/3 PFC金字塔瘤及其过程通过IUE被贴上GFP标签，从而确认了基因转移实验的成功。
以老鼠前额皮层的大量神经元为目标。 对于幼鼠和成年小鼠，可量化被转染细胞的总数和受感染神经元的分布。使用这种双边 IUE 方法，大约 4000-6000 L2/3 金字塔神经元与 pCAG-GFP 质粒 （图 3A）一起被转染。此外，这些细胞大多被本地化到前皮质区域，包括前额关联皮层、电机关联区域、前肢和非边缘皮层以及轨道和前结晶皮层（图3B）。一个有代表性的例子显示了成人对照小鼠（P60， 图3C）中转染神经元的横向-球形分布和半球之间的分布（图3D）。这证实了双边 IUE 瞄准和基因标记前额皮层中大量 L2/3 金字塔神经元的能力。
青少年和成年小鼠的社会行为。 产妇互动任务的第一部分测试了控制P18转染小鼠（IUE与pCAG-GFP质粒）找到巢床上用品（母体相互作用1[MI1]）的能力。这测试了幼鼠的感应运动能力。对照老鼠花更多的时间探索他们的巢床上用品比探索新鲜的床上用品。因此，建议，如预期的那样，这些小鼠具有完整的感官运动能力和探索行为（图4A和图3B）。任务的第二部分（MI2）利用了老鼠被激励与母亲互动和接近其母亲的倾向。在这项任务中，幼崽大部分时间都在妈妈身边度过，而探索空杯子或巢床上用品的时间则要少得多（图4C，D）。这些结果表明，IUE对照小鼠表现出正常的居家行为。
成年对照小鼠（P60）花了大约35%的时间探索一个新物体（在竞技场上花费的总时间=5分钟，图5A，B）。当呈现一个新颖而熟悉的物体时，成年老鼠花更多的时间探索这个新奇的物体，暗示着对新奇事物的完整兴趣（图5C，D）。在善于交际的任务中，控制成年小鼠花费了类似的时间探索一种新的小鼠和空杯子（图5E，F）。此行为使用免费提供的 DeepLabCut 软件26自动跟踪。示例视频显示在鼠标上成功标记了各种点，包括四肢、心机、头部和耳朵（视频 1）。DeepLabCut用于通过检查老鼠身体的长度来确定老鼠的饲养时间，因为当老鼠后方时，这个距离变短了（视频2）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61350/61350fig1.jpg"> 图1:在子宫内，使用定制的电波器和三爪电极进行电镀。 （A） 定制电镀器（左）及其前面板（右）的图像。1: 功率指示器。2: 电源开关。3: 脉冲指示器。4:测试模式切换。 5:电压选择器。6:脉冲宽度控制。7: 电极 （+）.8: 外部触发器 （TTL）。 9: 电极 （-）.（B） 定制三爪电极（左）的图像，以及在 IUE（右）期间保持三爪电极的推荐方法。（C） 左图: 描绘在 E16 大坝上进行的 IUE 手术的图表。右图:IUE 与 GFP 的代表性 20X 共用图像，目标为 L2/3 mPFC。黄色星号:L2/3 GFP®神经元。左面板刻度条 = 250μm。右面板刻度栏=75微米。数字和数据改编自科默等人，20205. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61350/61350fig1large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61350/61350fig2.jpg"> 图2:定制电镀器的电路图。 根据先前描述的示例27描述定制电镀电路的图表。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61350/61350fig2large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61350/61350fig3.jpg"> 图3:使用 IUE 瞄准大量 L2/3 前额皮层神经元。（A） 青少年对照小鼠的GFP+细胞总数。N=15只老鼠。（B） 成人对照小鼠每区GFP+细胞的百分比。N=22对照小鼠。（C） 代表部分显示前额皮层中转染细胞的横向胸围范围。左面板中的图像是右面板（红色方块）的缩放区域。正面关联皮层:FrA。辅助运动皮层:M2。前肢皮层: PL. 红外皮层: IL. 前结晶皮层: Acc. 中轨道前额皮层: MO. 文特拉轨道前额皮层: VO. 横向轨道前额皮层: LO.前岛皮层:AI。正面皮层区域3:Fr3。 主运动皮层:M1。 主体瘤敏感皮层:S1。皮里形式皮层:皮尔。前嗅觉核:AO。考特 - 普塔门: Cpu 。黑色数字:布雷格玛坐标。左面板刻度条 = 0.5 毫米。右面板刻度条 = 1 mm. 平均± Sem.（D） 数据显示每个鼠标中右半球和左半球电冲的细胞数。显示14只成年小鼠的不间断细胞计数，这些小鼠用pCAG-GFP质粒电聚。配对t测试。 p=0.4757。 数据改编自科默等人，20205.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61350/61350fig3large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>

<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61350/61350fig5.jpg"> 图5:评估成年转染小鼠的社会互动。（A） P60控制成人鼠标在新型对象交互任务中所走路径（黑迹）的代表性示例。粉红色的角落=新事物的位置。（B） 对照小鼠花了大约35%的时间探索这个新物体（总时间5分钟）。在角落里花费的百分比时间与新颖的对象显示。（C） P60控制成年鼠标在新型对象识别任务中所走路径（黑迹）的代表性示例。粉红色的角落:新事物的位置。绿色角落:熟悉对象的位置。（D） 控制小鼠花更多的时间探索新事物，而不是熟悉的物体。显示的歧视指数:DI=（（与新对象的时间-与熟悉的对象的时间）/（与新对象的时间+时间与熟悉的对象）。（E） P60 控制成年鼠标在善于交际任务中旅行路径（黑痕）的代表性示例。粉红色的角落:网状钢丝杯下的新鼠标的位置。绿色角落:空网状钢丝杯的位置。（F） 控制老鼠平均花费同等时间探索一个空杯子和一个装有新老鼠的杯子。图显示DI（（与新鼠标的时间-空杯子的时间）/（与新鼠标的时间+与空杯子的时间）。由于小鼠在完成任务之前没有与社会隔离，因此与新老鼠互动的动力可能已经减弱。然而，仍然有探索的小说鼠标。N=22对照小鼠。数字和数据改编自科默等人，20205.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61350/61350fig5large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
视频1:使用深度实验室切割自动跟踪动物在行为任务中的位置。 使用 DeepLabCut 标记的社交互动任务中成人鼠标的代表性视频。鼠标的各个部分可以标记，如四肢和头部。使用中心机器人是适当的跟踪动物的位置，但其他点可用于识别更复杂的行为，如梳妆或饲养。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61350/Video1.mp4" target="_blank">请点击这里查看此视频。（右键单击下载。</a>
视频2:使用深度实验室切割在行为任务中自动跟踪饲养。 使用 DeepLabCut 标记的社交互动任务中成人鼠标的代表性视频。红色箭头显示鼠标的长度，箭头指向鼠标头部。箭头的长度可用于确定鼠标的饲养情况，因为鼠标和箭头的长度在鼠标后方时变小。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61350/Video2.mp4" target="_blank">请点击这里查看此视频。（右键单击下载。</a>

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

Nanyang Technological University

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 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 ...

科研

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 ...

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

文化早期鸡胚体外（新文化）的方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

双整装原位杂交技术在早期鸡胚

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

整装在鸡的抗体染色方法

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

在鸡胚神经诱导的含量为

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 ...

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

从成年小鼠海马齿状回的剖析

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

人类DNA修复蛋白的遗传研究利用酵母作为模型系统

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...