Name: in utero electroporation of multiaddressable genome-integrating color (magic) markers to individualize cortical mouse astrocytes
Uploaded: 2020-05-21T15:00:00.000+00:00
Duration: 6 min 47 s
Description: Watch this Scientific Journal Video about In Utero Electroporation of Multiaddressable Genome-Integrating Color MAGIC Markers to Individualize Cortical Mouse Astrocytes at JoVE.com

我们感谢 S. Fouquet 以及蒙彼利埃视觉和神经科学协会 （MRI 和 RAM） 的成像和动物核心设施提供的技术援助。这项工作得到了法国雷吉翁·伊勒-德-法兰西基金会和.C癌症康复基金会以及巴黎-萨克莱大学（跨学科博士倡议）到洛杉矶的奖学金的支持，欧洲研究理事会（ERC-SG 336331， PI J. 瓦莱特） 到 E. h.， 由国家重建局根据合同 ANR-10-LABX-65 （实验室生命感官）， ANR-11-EQPX-0029 （Equipex 莫福镜 2）， ANR-10-INBS-04 （法国生物成像），由欧洲研究理事会（ERC-CoG 649117，PI J. Livet）和ATIP-Avenir项目（PI K.Loulier）的雷切·梅迪卡莱基金会（DBI20141231328）。

此处描述的所有动物程序都是按照机构准则进行的。动物协议已得到查尔斯·达尔文动物实验伦理委员会（CEEACD/N+5）的批准。
1. 子宫电镀中魔法标记物的无毒素质粒制备
<ol><li>细菌转化<ol>
<li>在冰上，解冻DH5阿尔法能力细胞存储在-70°C。</li>
		<li>在 37 °C 下加热含有适当抗生素（100μg/mL 氨基林或 50μg/mL 卡纳霉素）的琼脂板。</li>
		<li>在解冻的DH5α能力细胞中加入1 μL的5-50克魔法标记质粒DNA，并在冰上孵育10分钟而不混合。</li>
		<li>对于热冲击转换，将方位放在42°C的水浴中45秒，然后立即放在冰上，等待3-5分钟。</li>
		<li>在无菌条件下，加入 230 μL 的 SOC 介质，并在 37 °C 下孵育 1 小时。</li>
		<li>将方位引用的内容分散在琼脂板上，并在 37 °C 下孵育过夜。</li>
	</ol>
</li>
	<li>普拉斯米德文化<ol>
<li>第二天早上，在无菌条件下，从琼脂板中拾取一个菌落，用适当的抗生素将其放入含有2毫升LB介质的14毫升管中。让它在 300 rpm 的摇晃孵化器中以 37 °C 孵育一天。</li>
		<li>在一天结束时，300 mL 的 LB 种子使用抗生素使用 2 mL 起动机培养从步骤 1.2.1 和孵育过夜在 37 °C 在摇动孵化器在 300 rpm.</li>
	</ol>
</li>
	<li>普拉斯米德DNA制备<ol>
<li>第二天早上，使用制造商的协议，使用无毒素 maxiprep 套件，对魔法标记质粒 DNA（即 PB-CAG-Cytbow 和 Tol2-CAG-Nucbow 以及表达 PB 和 Tol2 转体酶和 Cre 重组酶的 CAG 驱动质粒进行纯化）。</li>
		<li>在200μL的无菌水中排出DNA，并在储存前使用光谱仪估计其浓度-20°C。</li>
	</ol>
</li>
</ol>2. 子宫电镀中魔法标记的准备（MM IUE）
<ol><li>解决方案准备<ol>
<li>在水浴中以 37 °C 加热 30 mL 的 0.9% 盐溶液，并在手术的整个过程中保持温暖。</li>
		<li>准备含有PB-CAG-细胞弓和托尔 2-CAG-努博的质粒混合物（最终浓度， 0.8 微克/μL），PB 和 Tol2 转置酶（最终浓度，每个 0.4 微克/μL），CAG-seCre（最终浓度，0.16μg/μL），和 0.01% 快速绿色染料在 PBS 中无 Ca2+和 Mg2+。 注:出于天体细胞解剖重建的目的，可以省略 托尔2-CAG-努博的 构造。然而，这种质粒是有用的，以区分密切并列的星形细胞的双，当使用魔术标记，以探索天体细胞14之间的克隆关系。</li>
		<li>准备麻醉液，含有100μL氯胺酮（100毫克/mL）和100μL的西拉津（20毫克/mL）稀释在2mL盐溶液中。</li>
		<li>通过稀释盐水溶液中的 0.3 毫克/mL 丁丙诺啡库存溶液 1:10 来准备镇痛溶液。</li>
	</ol>
</li>
	<li>准备手术材料<ol>
<li>在高温下用玻璃珠消毒器或等价物对手术工具（见 材料表）进行消毒。</li>
		<li>将一滴电极凝胶放入 35 mm 盘中。</li>
		<li>将微点插入微喷射器支架中，打破微点的尖端，并渴望DNA溶液。</li>
		<li>放置 1 μL 快速绿色解决方案 （0.01%）在 3 厘米盘的盖子中，并以它作为参考，通过用细钳子打破其尖端来调整微尖端的直径。调整压力参数，使微喷射器产生等量大小的滴落，从而使每次注射可输送大约 1μL 的 DNA 溶液。</li>
	</ol>
</li>
	<li>准备怀孕的雌性小鼠<ol>
<li>称重 Rjorl: 瑞斯怀孕的老鼠。</li>
		<li>用麻醉剂溶液每克体重 （BW） 12.5 μL 进行腹内注射。等待 5 分钟，通过捏脚趾验证鼠标是否睡着了。</li>
		<li>一旦动物没有反应捏，注射1.6 μL/g BW镇痛液皮下。</li>
		<li>在每个眼睛上加入一滴眼凝胶，以防止在手术过程中干燥，并将动物腹部放在加热垫上。</li>
		<li>轻轻剃光腹部，用用碘浸泡的垫子清洁，用酒精垫消毒剃光的区域。</li>
		<li>通过在剃光、清洁和消毒区域周围放置无菌压缩来安排操作字段。</li>
	</ol>
</li>
</ol>3. 子宫内电镀（IUE）
<ol><li>静脉注射<ol>
<li>沿着中线切开一个2厘米垂直的切口，从腹部的下部开始，穿过皮肤，然后穿过底层肌肉。通过轻轻操纵胚胎袋来暴露子宫角，并评估子宫颈的位置和子宫颈两侧的胚胎袋数量。</li>
		<li>将E15.5胚胎的大脑定向到电压中，以观察其位置，很容易识别为与沿着脑裂口运行的三个主要血管的交界处相匹配。</li>
		<li>想象一下，在布雷格玛（通过头骨可见）和眼睛之间有一条虚拟线:引入虚拟线和纵向裂变之间的微点，然后按下喷油器的脚踏板，在目标半球的横向心室中提供 1 μL 的 DNA 溶液。 注:在适当位置注射时，填充的横向心室呈蓝色，表明它已填充 DNA 溶液。</li>
	</ol>
</li>
	<li>穿孔<ol>
<li>将电极（见 材料表）涂抹在注射胚胎的两侧，用覆盖注射半球的节点浸入电极凝胶中。按下电脉冲器的脚踏板，提供 35 V 的四个 50 毫秒脉冲系列，每个脉冲间隔为 950 毫秒。</li>
		<li>用加热盐水溶液抑制胚胎。 注意:在整个手术过程中，胚胎应使用加热盐水溶液保持湿润，子宫不应干涸。</li>
		<li>每个胚胎重复步骤 3.1.2-3.2.2。</li>
		<li>一旦所有目标胚胎都经过电镀，用钳子轻轻将子宫角推回原位，以取代腹部的子宫角。用加热的盐水溶液填充腹腔，防止子宫在缝合时干燥。</li>
		<li>首先用连续的可吸收缝合关闭腹部肌肉，然后用4-0缝合多针（~10）的皮肤。</li>
		<li>将动物放在一个干净的笼子里，用干净的纸巾躺在它的一侧，每5-10分钟把动物转向另一边，直到它醒来，开始自己移动。</li>
		<li>第二天评估动物的状态，特别是如果已经筑巢的话。 注:无需手术后治疗。在没有巢的情况下，任何疼痛的迹象（例如，垂垂、毛茸茸的皮毛）和/或大出血，动物应立即被安乐死。</li>
	</ol>
</li>
</ol>4. 组织收获和剖腹产
<ol><li>组织收集<ol>
<li>在所需的收获时间，在腹内注射酚巴比妥（100毫克/千克BW），进行末期麻醉。在这种情况下，收获时间为产后 （P） 天 P4、P7 和 P21。</li>
		<li>使用冷预制的副醛基溶液进行颅内敷液。</li>
		<li>解剖大脑，将其放置在4°C的准甲醛基溶液中过夜，用于修复后。</li>
	</ol>
</li>
	<li>组织学<ol>
<li>第二天早上用1x PBS冲洗大脑3次10分钟。</li>
		<li>将大脑嵌入3%的阿加罗斯溶解在1x PBS中。</li>
		<li>使用振动刀片微切除术切割 80μm 部分。</li>
		<li>在预填充 1x PBS 的 24 个井板中收集部分。</li>
		<li>在滑梯和盖片之间安装介质中的安装部分（见 材料表）。将安装的幻灯片保持在 -20 °C，以实现最佳荧光蛋白保存和长期存储。</li>
	</ol>
</li>
</ol>5. 多通道心目成像
<ol><li>显微镜设置<ol>
<li>设置胸镜的配置，分别使用440、515和559nm激光线激发mCerulean/m绿松石2、EYFP、tdTomato/mCherry。</li>
		<li>使用 20x 0.8 NA（或更高 NA）目标，并根据奈奎斯特标准调整 XY 采样和 Z 步尺寸。 注:以更高的分辨率（例如，60x 1.4 NA 油目标）和去电算法获得的图像将支持重建星形细胞 arbors 的更精细细节。然而，实验者应该记住，最好的天体细胞过程可能不会通过传统的光学成像来解决。</li>
	</ol>
</li>
	<li>图像采集<ol>
<li>在电镀区找到最亮的星形细胞。</li>
		<li>由于位于靠近表面的标记细胞可能看起来比切片中较深的细胞更亮，因此调整表面单元格上的采集设置以避免图像饱和。</li>
		<li>使用 HiLo LUT 分别调整三个通道中的每个通道的收购设置，同时确保避免像素饱和，HiLo LUT 将零值显示为蓝色，最大像素值显示为红色。 注意:一个足够平衡的图像应该只显示几个蓝色和几乎没有红色像素。</li>
		<li>使用显微镜的电动阶段获取瓷砖 1，024 x 1，024 像素 Z 堆栈，相邻堆栈之间有 10% 的重叠，以便随后对电镀区或感兴趣区域进行马赛克重建。</li>
	</ol>
</li>
</ol>6. 星形细胞区域体积分割
注:这是使用商业软件程序（例如，IMARIS）执行的。
<ol><li>
准备数据集
	<ol>
<li>通过选择完全封闭在图像组织部分的细胞，在 3D 重建（250 x 250 像素）中裁剪标记的星形细胞。 注:如果使用的计算机能够处理它们，也可以直接在更大的容量上工作。</li>
		<li>单击"超越视图"对象工具栏中的蓝色表面按钮，为每个天体细胞创建一个新的表面。</li>
		<li>为了获得更好的可视化对比度，请首先通过单击 "编辑 |调整最小和最大颜色对比度值 显示显示调整 选项并拖动两个句柄。如果需要，请通过直接单击 显示器调整 窗口中的通道名称来更改通道颜色，以便选择新颜色。 注意:最好始终使用相同的彩色显示屏，以防止视觉偏差。</li>
		<li>单击 编辑|在 微米中手动指示voxel大小的图像属性。如果voxel大小不准确，音量计算将不正确。</li>
		<li>保存新设置。</li>
	</ol>
</li>
	<li>
表面分割
	<ol>
<li>使用蓝色图标引入表面。场景列表中将显示表面图标和框。容积框允许查看/隐藏数据集。</li>
		<li>选择表面线并单击 跳过自动创建|手动编辑。</li>
		<li>单击 轮廓|可见性和 单击 "无"。然后移动到 "选择" 视图，然后单击 "绘制 "按钮。</li>
		<li>单击 "模式 "以选择绘图模式。</li>
		<li>使用快速高效的 同位素 半自动绘图工具。通过移动鼠标指头来定义单元格轮廓。如果 同位素 预览与天体细胞轮廓不匹配，则调整鼠标指点位置。要验证预览版，请左键单击选择。要纠正潜在的错误，请使用 板 窗口删除当前的 Z 平面轮廓或按 Ctrl + Z。</li>
		<li>使用 切片|通过 Z 平面导航的位置，通过更改 Z 平面编号移动到下一架飞机，然后启动新的轮廓。优先从细胞中间开始，然后移动到四肢。</li>
		<li>当所有包含天体细胞的 Z 平面绘制轮廓时，请单击 "创建表面"。在创建表面之前，请检查所有组件是否有连接。如果没有，请在导出量数据之前选择最大数据或连接相关数据。</li>
		<li>检查 数据面板 中显示的定量数据和电子表格格式的出口统计数据，从而节省体积值和单位。</li>
		<li>以新名称保存表面以稍后访问它。</li>
	</ol>
</li>
</ol>7. 追踪天体细胞植树
注意:这是使用开放访问软件程序 Vaa3D 完成的。
<ol><li>准备数据集<ol>
<li>加载图像堆栈并搜索显示不同颜色的孤立的星形细胞或附近的星形细胞。 注:可以通过增加目标NA和抽样来改进重建的分辨率。然而，最终，由于结肠显微镜的分辨率限制，无法准确追踪到最好的星体细胞过程。应小心避免超出所获取图像分辨率的过度分解。</li>
		<li>使用斐济，每个星体细胞周围都有 250 x 250 像素的图像堆栈。</li>
		<li>由于 Vaa3D 跟踪仅在一种颜色上执行，请选择一个通道并停用其他通道。</li>
		<li>将图像转换为 RGB 并将其保存 .tiff 格式。</li>
	</ol>
</li>
	<li>阿伯追踪<ol>
<li>在 Vaa3D 中打开 RGB 图像。转到图像|数据|几何然后图像重新采样，并调整X/Y和Z沃克塞尔大小值。 注:在重建星形细胞树骨时，应注意不要比图像提供的分辨率更精细地跟踪细节。</li>
		<li>通过单击 整个图像的可视化| 3D 查看器来打开 3D窗口。右键单击 3D 图像并选择 右键单击以定义标记。用光标指向星体细胞的中心，然后右键单击以设置标记。 注:单击 "逃生" 可再次访问 3D 视图。</li>
		<li>转到 高级|3D跟踪|Vaa3D-神经元2自动追踪。</li>
		<li>在新打开的窗口上，选择应用插件的通道。</li>
		<li>选择背景阈值（通常在 30 到 90 之间）。如果需要，为每个天体细胞调整此值。</li>
		<li>从2D中取消检查半径，并保持自动向下样品和自动重质检查。</li>
		<li>将cnn_type设置为 3，length_thresh设置为 1，SR_ratio设置为 0.1。</li>
		<li>单击 "确定"后，请确保插件根据原始名称自动生成两个文件。将添加后缀-ini.swc和-坐标X-坐标-坐标Z-app2.swc。手动向这些名称文件添加一个独特的标签，以便在继续运行插件时不覆盖它们。</li>
		<li>打开 对象管理器，转到 神经元|线结构，选择跟踪的星体细胞，点击 显示模式，并选择 始终线模式。</li>
		<li>回到3D窗口，在那里出现星体细胞的骨架。 注:如果分割和信号不匹配，重复步骤 7.2.3-7.2.10 并调整阈值直到它们匹配。重新启动插件时，请务必重新输入其他参数，因为它们会重置。</li>
		<li>右键单击 骨架 并选择首选 神经元|行#1.。。APP2_Tracing 访问将出现在新窗口表面上的定量测量 |对象注释。 注:由于光显微镜的分辨率有限，在列出的项目下显示 的测量分支数 和 分叉数 仅提供星形细胞树干复杂性的粗略估计。</li>
	</ol>
</li>
</ol>

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作者没有什么可透露的。

在皮质祖细胞的魔法标记的子宫电冲（IUE）中（图1）使星形细胞在产后大脑皮层的标记在不同的产后阶段（P4-P7-P21，图2）。有趣的是，IUE 的阶段并不关键，因为从 E13.5 到 E15.5 执行的电冲产生与皮质星形细胞14类似的标记模式。然而，标记的金字塔神经元在皮质帕伦奇马的位置随电扑阶段而变化。事实上，IUE执行在E15标记层2-3神经元，而IUE执行在E13标签金字塔神经元在所有皮质层，从第5层到第2-3层6，27。MM IUE 之后皮质金字塔神经元的这种联合标记是此方法的主要局限性，因为它可防止在密集的神经元标记干扰星形细胞过程识别的层中对星形细胞形态进行半自动分割。如果这是一个问题，MM可能会在产后阶段电镀，如在PALE。在P4，在该阶段仍然存在的径向胶质纤维标签也可能干扰Vaa3D树干分割。虽然繁琐，一个解决方案，如果一个人希望在这个阶段进行天体细胞树干重建是手动删除径向胶质纤维，逐步取代径向光纤信号与黑色像素周围的感兴趣的天体使用Adobe Photoshop。
尽管有这种限制，MM IUE 是一种强大的技术，当充分执行。需要小心处理几个关键步骤:1） 胚胎在整个手术过程中必须保持湿润，并仔细操作以增加其存活率:2）使用直径大的玻璃毛细血管或挤压胚胎袋太紧可能导致袋破裂，从而导致胚胎死亡:3）在DNA注射过程中，必须避免血管出血:4） 整个过程不应持续超过40分钟，从麻醉到缝合，以最大限度地提高胚胎的存活率:5） 应激在 IUE 成功中起着至关重要的作用，因此必须避免额外的压力来源，如笼子的改变、交通、噪音和振动，从手术前 5 天到出生后 7 天，以防止流产和吃人。
值得注意的是，希望专门瞄准在给定阶段出生的细胞的实验者可以使用MM工具包，而无需添加小猪包和托尔2转置酶，因此只有电化时出生的细胞才能表达组合标签。该方法的另一个优点是它在标记细胞的密度及其在不同大脑区域的位置方面具有灵活性。事实上，通过增加MM转基因的总浓度，同时保持质粒比不变（Cre重组酶/MM结构的1:10比和转置酶/MM结构的1:2比），可以实现更密集的皮质星形标记。与单色方法相反，例如Ai9小鼠的单色转子电渗透或Cre电冲洗，其中单挑单个星形细胞的能力需要稀疏的标记，MAGIC标记策略提供的颜色对比使星形细胞在广泛的标签密度上个性化。此外，将电极探头定位在不同的方向允许瞄准不同的大脑区域，如前瞻性纹状体（心室位置的节点，与实现大脑皮层电渗透所需的多骨位置相反），或海马（介质位置的节点）28。最后，IUE可以在不同的小鼠菌株中执行，如外生（OF1，瑞士）和近亲繁殖（C57BL/6J或N）小鼠，这为在转基因动物疾病模型中使用MM工具包开辟了道路。然而，要成功地在近亲繁殖小鼠中实现IUE，应调整脉冲数量（C57BL/6小鼠的三个脉冲，瑞士的五个脉冲）、电压（30 V而不是35 V）和镇痛剂量（0.15毫克/mL丁丙诺啡股票溶液和0.8 8 Μlμg BW的注射量）。
与转基因动物育种或PALE和星轨方法相比，这种方法提供了几个优点。首先，与繁殖策略相比，它很少使用动物。它还允许标记皮质星形细胞，因为他们的发展的最初阶段，包括胚胎阶段，不像PALE4，它依赖于产后电冲。此外，与星轨方法8中使用的12个记者结构相比，该策略仅依赖于两种多色转基因，从而简化了DNA组合的制备和成像。此外，MM所表达的不同颜色之间的平衡本质上是由转基因结构决定的，并不取决于实验者对不同成分的混合。此外，这种策略可以超越简单的解剖考虑，可以成功地应用于星形细胞发育的多克隆分析，如先前发表的工作14所示。这项工作利用细胞质和核标记的罕见颜色组合来定义皮质星形细胞克隆，表明它们在生成细胞的空间分布、结构组织、数量和亚型方面表现出广泛的变异性。
由MM标记的皮质祖先产生的皮质星形细胞表现出显著的颜色对比度，并广泛分布在整个大脑皮层（图2）。使用共晶显微镜采集的简单多通道 Z 堆栈用于在几个产后阶段访问关键星形细胞特征，如其地域体积（图 3）及其形态复杂性（图 4）。除了星形细胞之外，这种方法可能被改编为研究其他胶质细胞的形态学，如寡头细胞。然而，应该记住，胸腔显微镜提供的有限分辨率只能提供部分渲染的天体细胞形态复杂性。虽然以更高的分辨率（例如 63x 1.4 NA 油目标）和去燃烧算法获得的图像可用于重建星形细胞 arbors14的更精细细节，但最好的过程无法通过传统的光学成像来解决。然而，这里提出的策略将有兴趣有效地筛选影响皮质星形细胞体积或形态的潜在表型，在神经系统疾病的小鼠模型。

天体细胞在大脑发育和生理学中起着许多重要作用。除了在调节营养吸收和血液流动的血脑屏障中发挥作用外，他们还积极促进突触的形成和功能，同时产生神经调节器，可以改变神经元活动和行为2。此外，天体细胞功能障碍导致各种神经系统疾病3.位于大脑皮层的星形细胞显示一个精心设计的形态，能够广泛接触神经元过程。这些接触，对电路功能至关重要，也通过细胞粘附蛋白4控制星形细胞形态生成和突触发生。神经科学家需要方便和可靠的工具来研究天体细胞发育和形态生成在他们的神经模型的兴趣。然而，由于星形细胞与邻居的紧密对接和均匀的三维平铺，因此，挑出皮质星形细胞并使用免疫标志物全面评估其形态是具有挑战性的。
目前，两种主要的基因工程策略使皮质天体在原位进行标记和个性化:利用记者质粒的电聚化，在转基因小鼠系或体细胞变代中稀疏的记者激活。第一个策略依赖于繁殖一个飞毛腿记者小鼠线与小鼠表示一种诱导形式的Cre重组酶激活专门在天体细胞后塔莫西芬交付（如Aldh1l1-CreERT25）。此策略存在几个缺点。首先，繁殖转基因小鼠需要大量的动物，通常需要多次检测来确定适当剂量的塔莫西芬，以提供皮质星形细胞的足够稀疏标签。分析感兴趣的遗传小鼠模型中的皮质星体表型将需要更多的繁殖和小鼠消费。此外，在子宫塔莫西芬注射已知干扰分体，使这一策略难以应用于研究天体细胞发育的最初阶段。体内DNA电聚是一种替代的无塔莫西芬策略，它依赖于最少数量的动物6。这种方法在胚胎阶段或产后阶段进行，包括在啮齿动物的横向心室中注射报告质粒，然后用电脉冲在细胞膜中产生毛孔，从而允许DNA进入心室内的祖细胞。随后，记者携带的电聚合质粒的转基因由目标细胞机械处理，并表达7。此前曾描述过两种电冲洗方法，为小鼠皮质星形细胞贴上标签:1）产后星形细胞标签（PALE），它依赖于产后早期4-2个单色偶发性报告质粒的电冲洗:2）星轨策略基于在子宫电镀（IUE）的多色综合记者质粒8，9，10。虽然这两种技术在大脑皮层中有效地标记了PrA，但它们也存在一些局限性。在其初始版本中，这两种方法都依赖于胶质纤维酸性蛋白 （GFAP） 促进剂来驱动星形细胞中的表达，这可能将标签偏向径向胶质细胞，以及表达 GFAP 比正常休息 PrA11、12更强烈的皮亚和活性星形细胞。关于PALE，其他缺点是电冲洗的后期阶段，它防止标记早产的PrA（或那些源于早期脱脂的祖先）和分析天体发育的早期阶段，以及使用偶发性病媒，在PrA在第13、14周经历的大规模增殖期间通过连续的分裂被稀释。与 PALE 不同的是，StarTrack 基于集成记者质粒的胚胎电聚，允许跟踪胚胎和产后祖先对 PrA 的贡献。依靠泛素C促进剂（UbC-StarTrack）的更新星轨方案实现了荧光记者在神经祖祖15、16、17的神经元和胶质下降（包括星形细胞）的更广泛表达。然而，在其当前版本中，这种方法的实施是复杂的，因为它依赖于12种不同质粒的等效混合物，表达6种荧光蛋白（FP），部分激发和发射光谱重叠。
这里介绍的是一个直接的子宫电镀基多色标签方法使用综合记者结构驱动的强大和广泛活跃的促进者挑出皮质星形细胞14。此外，还分别提供了使用许可（如 Imaris）和开放访问（Vaa3D18、19、20）图像分析软件的简单图像分析管道，用于分割天体细胞的面积体积和树干。与之前描述的方法相比，此策略仅依赖于 1-2 个多色集成转基因多易处理基因组集成颜色标记（MAGIC 标记或 MM21），这些标记指向细胞质和（可选）核细胞隔间，其表达由由环状体增强剂组成的合成CAG促进器驱动， 鸡β蛋白推广器，兔β-红蛋白拼接接受网站22。这使得皮质星形细胞的标签和跟踪，从胚胎阶段到产后后期阶段，独立于GFAP表达14，23。这些转基因中的每一种都具有以下四种不同的FP:eBFP、绿松石2/mCerulean、EYFP和tdTomato/mCherry，它们显示最小的光谱重叠，可以很容易地绕过1）顺序通道采集:2） 优化激发能力和收集增益:和3）特定的二氯气过滤器，以收集狭窄的FP排放窗口。MM 策略使用Cre/lox重组与自切除的 Cre 重组 （seCre） 来驱动蜂窝群中 FP 的随机表达。MM转基因的单个副本以相互排斥的方式表达FP，而多个转基因则产生FP组合，产生几十种不同的色调。转基因的基因组整合是由小猪巴克（PB）或托尔2转位系统24，25，26驱动的。因此，通过子宫电冲，MM工具包和多色"马赛克"，它产生启用多个相邻的皮质祖先的同步标记和跟踪其胶质下降，包括皮质星形细胞，在很长一段时间内。颜色对比产生于对 PrA 轮廓的明显 FP 允许线的表达，随后提取有关其领土体积（使用 IMARIS）和复杂形态（使用 Vaa3D）的关键信息。这里详细介绍的多色策略是一种方便而可靠的方法，可在各种发育阶段快速、便捷地访问野生型小鼠的皮质星形细胞表面和形态，并且无需使用转基因记者线即可轻松适应研究神经系统疾病小鼠模型中的天体细胞解剖特征。

<table><tbody><tr><td>&lt;strong&gt;1.1 Bacteria transformation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ampicillin</td><td>Euromedex</td><td>EU0400-C</td><td></td></tr><tr><td>DH5 alpha competent cells</td><td>Fisher Scientitic</td><td>11563117</td><td></td></tr><tr><td>Ice box</td><td>Dutscher</td><td>139959</td><td></td></tr><tr><td>Kanamycin</td><td>Sigma</td><td>60615</td><td></td></tr><tr><td>LB Agar</td><td>Sigma</td><td>L2897</td><td></td></tr><tr><td>SOC medium</td><td>Fisher Scientitic</td><td>11563117</td><td></td></tr><tr><td>Sterile petri dish- 10 cm</td><td>Thermo Fisher</td><td>150350</td><td></td></tr><tr><td>Water bath</td><td>VWR</td><td>462-0556H</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;1.2 Plasmid culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>14 ml culture tube</td><td>Dutscher</td><td>187262</td><td></td></tr><tr><td>Glass erlenmeyer- 2L</td><td>Fisher Scientitic</td><td>11383454</td><td></td></tr><tr><td>LB medium</td><td>Sigma</td><td>L3522</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;1.3 Plasmid DNA preparation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>NucleoBond Xtra Maxi Plus EF</td><td>Macherey-Nagel</td><td>740426.10</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;2.1 Preparation of the solutions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>26 G x 1/2 needle</td><td>Terumo</td><td>8AN2613R1</td><td></td></tr><tr><td>30 G x 1/2 needle</td><td>Terumo</td><td>8AN3013R1</td><td></td></tr><tr><td>Fast Green</td><td>Sigma Aldrich</td><td>F7272</td><td></td></tr><tr><td>NaCl</td><td>VWR</td><td>27810.295</td><td></td></tr><tr><td>Single-use polypropylene syringe, 1 mL</td><td>Dutscher</td><td>50002</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;2.2 Preparation of the surgery material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Adson Forceps - DeBakey Pattern- 12.5 cm</td><td>FST</td><td>11617-12</td><td></td></tr><tr><td>Arched tip Forceps- 10 cm</td><td>FST</td><td>11071-10</td><td></td></tr><tr><td>Glass bead sterilizer Steri 250</td><td>Sigma</td><td>Z378569</td><td></td></tr><tr><td>Glass micropipette 1 mm diameter</td><td>FHC</td><td>10-10-L</td><td></td></tr><tr><td>Graefe Forceps - Titanium 1 mm Tips Slight Curve- 10 cm</td><td>FST</td><td>11651-10</td><td></td></tr><tr><td>Graefe Forceps - Titanium 1 mm Tips Straight- 10 cm</td><td>FST</td><td>11650-10</td><td></td></tr><tr><td>Iris Scissors - Delicate Straight- 9 cm</td><td>FST</td><td>14060-09</td><td></td></tr><tr><td>Laboratory tape</td><td>Fisher Scientitic</td><td>11730454</td><td></td></tr><tr><td>Microinjector</td><td>INJECT+MATIC</td><td>No catalog number</td><td></td></tr><tr><td>Olsen-Hegar Needle Holder - 12 cm</td><td>FST</td><td>12002-12</td><td></td></tr><tr><td>Optical fiber</td><td>VWR</td><td>631-1806</td><td></td></tr><tr><td>Plastic-coated white paper</td><td>Distrimed</td><td>700103</td><td></td></tr><tr><td>Signagel electrode gel</td><td>Free-Med</td><td>15-60</td><td></td></tr><tr><td>Sterile Petri dish- 35 mm</td><td>Dutscher</td><td>056714</td><td></td></tr><tr><td>Sterilizer, glass dry bead, Steri 250</td><td>Sigma</td><td>Z378569</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;2.3 Preparation of the pregnant female mouse&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Alcohol pad</td><td>Alcomed</td><td>1731000</td><td></td></tr><tr><td>Buprecare</td><td>Axience</td><td>0.3 mg/ml</td><td></td></tr><tr><td>Compress</td><td>tRAFFIN</td><td>70189</td><td></td></tr><tr><td>Ketamine</td><td>Merial</td><td>Imalgene 1000</td><td></td></tr><tr><td>Ocular gel</td><td>tvm lab</td><td>Ocry-gel</td><td></td></tr><tr><td>RjOrl:SWISS mice</td><td>Janvier Labs</td><td></td><td></td></tr><tr><td>Vetadine, 10% solution</td><td>Vetoquinol</td><td>4337400113B</td><td></td></tr><tr><td>Warming pad</td><td>Harvard Apparatus</td><td>72-0493</td><td></td></tr><tr><td>Xylazine</td><td>Bayer</td><td>Rompun 2%</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;3.2 Electroporation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Absorbable suture Size 4-0 45 cm Suture 1-Needle 19 mm Length 3/8 Circle Reverse</td><td>Novosyn</td><td>C0068220</td><td></td></tr><tr><td>Electroporateur Sonidel</td><td>Sonidel</td><td>NEPA 21</td><td></td></tr><tr><td>Sterile transfer pipets (individually wrapped)</td><td>Dutscher</td><td>043202S</td><td></td></tr><tr><td>Tweezers with 3 mm platinium disk electrodes</td><td>Sonidel</td><td>CUY650P3</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;4.1 Tissue harvesting and sectioning&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>24-well plate</td><td>Falcon</td><td>353047</td><td></td></tr><tr><td>Agarose</td><td>Lonza</td><td>50004</td><td></td></tr><tr><td>Antigenfix</td><td>Microm Microtech</td><td>U/P0014</td><td></td></tr><tr><td>Coverslip</td><td>Dutscher</td><td>100266</td><td></td></tr><tr><td>Dolethal</td><td>Vetoquinol</td><td>DOL202</td><td></td></tr><tr><td>DPBS (10X), no calcium, no magnesium</td><td>Fisher Scientific</td><td>11540486</td><td></td></tr><tr><td>Nail polish</td><td>EMS</td><td>72180</td><td></td></tr><tr><td>Slide</td><td>Dutscher</td><td>100001</td><td></td></tr><tr><td>Vectashield</td><td>Vectorlabs</td><td>H-1000</td><td></td></tr><tr><td>Vibratome</td><td>Leica</td><td>VT1000S</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;5. Multichannel confocal imaging&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>20X oil NA 0.85</td><td>Olympus</td><td></td><td></td></tr><tr><td>Confocal Laser Scanning Microscope</td><td>Carl Zeiss</td><td>LSM880</td><td></td></tr><tr><td>Confocal Laser Scanning Microscope</td><td>Olympus</td><td>FV1000</td><td></td></tr><tr><td>Plan Apochromat 20x/0.8 M27</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;6. Astrocyte territorial volume segmentation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>IMARIS 8.3 and later versions</td><td>Bitplane</td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;7. Astrocyte arborization tracing&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>3D Visualization-Assisted Analysis software suite (Vaa3D)</td><td>HHMI - Janelia Research Campus /Allen Institute for Brain Science</td><td></td><td></td></tr></tbody></table>

in utero electroporation of multiaddressable genome-integrating color (magic) markers to individualize cortical mouse astrocytes

位于小鼠大脑皮层的原质天体细胞（PrA）紧密并列，在成人阶段形成明显连续的三维矩阵。到目前为止，没有任何免疫控制策略可以挑出它们，并在成熟动物和皮质遗传过程中分割它们的形态。皮质PrA起源于位于鼻腔姑息的祖先，在综合载体的子宫电渗透中很容易被瞄准。这里提出了一个协议，为这些细胞贴上多地址基因组整合颜色（MAGIC）标记策略的标签，该策略依赖于小猪/Tol2的转换和Cre/lox 重组，以固态地表达特定的亚细胞隔间的不同荧光蛋白（蓝色、青色、黄色和红色）。这种多色命运映射策略能够在胶质发生之前用颜色标记的组合在附近的皮质祖先进行原位标记，并在单个细胞水平上跟踪其后代，包括星形细胞，从胚胎阶段到成人阶段。通过调整多地址基因组集成颜色标记（MAGIC 标记或 MM）提供的电冲积载体的浓度和颜色对比度实现的半稀疏标记，能够个性化星形细胞，并挑出其区域和复杂的形态，尽管它们具有密集的解剖排列。这里介绍的是一个全面的实验工作流程，包括电化过程的细节，多通道图像堆栈通过胸腔显微镜采集，以及计算机辅助的三维分割，使实验者能够评估单个PrA体积和形态。总之，MAGIC 标记器的电冲控提供了一种方便的方法，可以单独标记许多星形细胞，并在不同的发育阶段访问其解剖特征。这项技术将可用于分析各种小鼠模型中的皮质星形体形态特性，而无需使用具有转基因记者线的复杂十字架。

胚胎皮质祖细胞中魔法标记的电化允许从大脑皮层发育的早期到后期标记星形细胞（图1）。这些星形细胞在产后各阶段（P4、P7、P21）的所有皮质层中被发现，因为它们广泛分散到整个大脑皮层中。他们用以 20x 0.8 NA（或更高 NA）目标获得的瓷砖组合图像进行评估，并组装为 Z 堆栈重建（图 2）。MAGIC 标记组合标记启用皮质星形细胞的个性化，并提取有关其体积和形态的信息。使用商业图像分析软件，在各星体图像堆栈的每个光学部分上划定了单个星形细胞的轮廓，以分割和重建每个星形细胞所占据的领土域（图3）。从相同的 Z 图像堆栈中，使用允许提取主星形细胞过程骨架的开放访问软件（图 4）分割了单向皮质星形细胞的分支形态。这些分割和追踪工具提供了对产后14早期至晚期个别皮质星形细胞的领土体积增加（图3）和形态复杂性（图4）的半定量评估。这些方法还揭示了不同皮质星形细胞在同一发展阶段所显示的体积和形态的异质性，如图2、图3和图4所示。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61110/61110fig1v2.jpg"> 图1:在子宫电涌（IUE）中，MAGIC标记（MM）的示意图表示，以标记皮质祖先及其在大脑发育过程中的后裔。（A） MM工具包包括几个编码PB-Cytbow、托尔2-努布、PB和托尔2转置酶的质粒，以及可自我切除的Cre重组酶（seCre）。在MM构造中，三对不兼容的 lox站点（loxN、lox2272 和loxP）侧翼四个不同的 FP 编码序列（EBFP2、m 绿松石2/mCerulean、EYFP、tdTomato/mCherry），并在Cre重组后创造相互排斥的切除可能性。 在Cre行动之前，只表达第一个基因（EBFP2）。在 SeCre 诱导的克里介导切除术后，表示 tdTomato/mCherry（红色 FP）、EYFP（绿色 FP）或 m 绿松石2/mCerulean（青色 FP）。来自多个 MAGIC 标记副本的 FP 共同表达在标记细胞的细胞质（PB-CAG-细胞弓） 或细胞核（Tol2-CAG-Nucbow）中产生颜色组合。PB 和 Tol2 转换端脚框架 MM 盒式磁带允许它们集成到皮质祖先的基因组中，当 MM 构造与 PB 和 Tol2 转位酶编码质粒一起进行共电时。（B-G）IUE连续步骤的图形图示，包括麻醉怀孕小鼠的腹腔切除术（B），在胚胎（D）的横向心室注射MM质粒混合（C），通过精心定位的钳子（E）输送电脉冲，以瞄准两个脑半球之一的皮质祖细胞（F），以及怀孕小鼠腹部（G）的缝合。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61110/61110fig1v2large.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/61110/61110fig2v2.jpg"> 图2:继魔法标记工具包的子宫电镀后，在产后阶段发现五颜六色的星形细胞散落在整个大脑皮层中。E15.5 小鼠皮质祖子中驱动 MM、seCre、PB 和 Tol2 转置酶表达的质粒 IUE 导致在 P4、P7 和 P21 上标记了 2-3 层神经元和星形细胞。表达水平和调色板取决于整合在皮质祖先基因组中的MM转基因的数量。从在 80μm 下垂大脑部分获得的瓷砖共存图像堆栈的最大强度投影的蒙太奇。缩放栏:200μm。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61110/61110fig2v2large.jpg" target="_blank">请单击此处查看此数字的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61110/61110fig3v2.jpg"> 图3:在不同的发展阶段，皮质星形细胞属域的分割。在三个不同的发展阶段，分别对裁剪的共星体 Z 堆栈构图单个星形细胞（A-F）及其关联的领土域进行最大强度投影:P4（A-B、A'-B）、P7（C-D、C-D）和P21（E-F、E'F'）。缩放栏:20μm。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61110/61110fig3v2large.jpg" target="_blank">请单击此处查看此数字的较大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61110/61110fig4v2.jpg"> 图4:追踪皮质星形细胞植树。两个不同的星形细胞从共晶Z堆（A-D）中裁剪，其树干重建与Vaa3D（A'-D'）在两个不同的发展阶段收集，分别:P7（A-B，A'-B'）和P21（C-D，C'-D'）。缩放栏:20μm。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61110/61110fig4v2large.jpg" target="_blank">请单击此处查看此数字的较大版本。</a>

Watch this Scientific Journal Video about In Utero Electroporation of Multiaddressable Genome-Integrating Color MAGIC Markers to Individualize Cortical Mouse Astrocytes at JoVE.com

In Utero Electroporation of Multiaddressable Genome-Integrating Color MAGIC Markers to Individualize Cortical Mouse Astrocytes

星形细胞均匀地平铺大脑皮层，使得对其复杂形态的分析在细胞水平上具有挑战性。此处提供的协议使用基于子宫电镀的多色标签，挑出皮质星形细胞，并使用用户友好的图像分析管道分析其体积和形态。

在多地址基因组集成颜色 （MAGIC） 标记的 Utero 电冲中， 以个性化皮质小鼠星形细胞

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at ...

in-utero-electroporation-multiaddressable-genome-integrating-color

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

In Utero Electroporation of Multiaddressable Genome-Integrating Color (MAGIC) Markers to Individualize Cortical Mouse Astrocytes

5.4K 次观看.  Institut des Neurosciences de Montpellier. 星形细胞均匀地平铺大脑皮层，使得对其复杂形态的分析在细胞水平上具有挑战性。此处提供的协议使用基于子宫电镀的多色标签，挑出皮质星形细胞，并使用用户友好的图像分析管道分析其体积和形态。

视频: In Utero Electroporation of Multiaddressable Genome-Integrating Color MAGIC Markers to Individualize Cortical Mouse Astrocytes

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

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

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

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

在子宫内的电穿孔其次原发性神经文化为研究基因的功能在皮层神经元的一个子集

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

科研

神经科学

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos1,2 that we have successfully adapted3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick5. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described6. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.
To date, in utero electroporation has been most widely used for the analysis of neocortical development1,2,3,4. More recent studies have targeted the embryonic retina7,8,9 and thalamus10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.

高效的基因传递到多个中枢神经系统使用新界在子宫内电穿孔

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

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

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

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

在子宫内电穿孔：鼠标控制时空基因Transefection

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

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘<em&gt;在子宫内</em&gt;电穿孔

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Neonatal Pial Surface Electroporation

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and gliogenesis, including after injury. However, methods for genetically interrogating these progenitor populations and tracking their lineages had been limited owing to a lack of specificity or time consuming production of viruses. Thus, progress in this region has been relatively slow with only a handful of investigations of this location. Electroporation has been used for over a decade to study neural stem cell properties in the embryo, and more recently in the postnatal brain. Here we describe an efficient, rapid, and simple technique for the genetic manipulation of pial surface progenitors based on an adapted electroporation approach. Pial surface electroporation allows for facile genetic labeling and manipulation of these progenitors, thus representing a time-saving and economical approach for studying these cells.

The pial surface is a unique progenitor zone in the CNS that is receiving increasing attention. Herein, we detail a method for rapid genetic manipulation of this progenitor zone using a modified electroporation method. This procedure can be used for cellular and molecular investigations of cell lineages and signaling pathways involved in cell differentiation and to elucidate the fate and properties of daughter cells.

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 ...

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

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

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

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

一种新的结合阵列全息、全外测序和宫内电穿孔的方法来识别脑畸形的致病基因

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

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

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

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

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

在子宫内电途径研究神经元亚群的兴奋性和单细胞连接

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

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

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

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

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

宫内电穿孔诱导小鼠大脑皮质中氧合酶的重组

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

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.

双电穿孔，以定时和空间分离的细胞群

In utero electroporation is an in vivo DNA transfer technique extensively used to study the molecular and cellular mechanisms underlying mammalian ...

可视化发育中的小鼠大脑中的神经元和神经胶质细胞动力学

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. This process is crucial for proper brain function, and its dysregulation can result in neurodevelopmental and psychiatric disorders after birth. In fact, many genes responsible for these diseases have been found to be involved in this process, and therefore, revealing how these mutations affect cellular dynamics is important for understanding the pathogenesis of these diseases. This protocol introduces a technique for time-lapse imaging of migrating neurons and glial progenitors in brain slices obtained from mouse embryos. Cells are labeled with fluorescent proteins using in utero electroporation, which visualizes individual cells migrating from the ventricular zone with a high signal-to-noise ratio. Moreover, this in vivo gene transfer system enables us to easily perform gain-of-function or loss-of-function experiments on the given genes by co-electroporation of their expression or knockdown/knockout vectors. Using this protocol, the migratory behavior and migration speed of individual cells, information that is never obtained from fixed brains, can be analyzed.

作者聚焦：探索发育中的大脑中的细胞迁移和基因作用

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. Many genes are involved in this process. This protocol introduces the technique for the time-lapse imaging of migrating neurons and glial progenitors.

胚胎小鼠脑切片中迁移神经元和神经胶质祖细胞的延时成像

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain ...

在多地址基因组集成颜色（MAGIC）标记的 Utero 电冲中，以个性化皮质小鼠星形细胞

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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

高效的基因传递到多个中枢神经系统使用新界在子宫内电穿孔

在子宫内电穿孔：鼠标控制时空基因Transefection

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘

新生儿软脑膜表面电穿孔

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

在子宫内电途径研究神经元亚群的兴奋性和单细胞连接

宫内电穿孔诱导小鼠大脑皮质中氧合酶的重组

双电穿孔，以定时和空间分离的细胞群

胚胎小鼠脑切片中迁移神经元和神经胶质祖细胞的延时成像

在多地址基因组集成颜色 （MAGIC） 标记的 Utero 电冲中， 以个性化皮质小鼠星形细胞

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

高效的基因传递到多个中枢神经系统使用新界在子宫内电穿孔

在子宫内电穿孔：鼠标控制时空基因Transefection

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘

新生儿软脑膜表面电穿孔

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

在子宫内电途径研究神经元亚群的兴奋性和单细胞连接

宫内电穿孔诱导小鼠大脑皮质中氧合酶的重组

双电穿孔，以定时和空间分离的细胞群

胚胎小鼠脑切片中迁移神经元和神经胶质祖细胞的延时成像

在多地址基因组集成颜色（MAGIC）标记的 Utero 电冲中，以个性化皮质小鼠星形细胞