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>

<ol>
	<li>Clarke, L. E., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+roles+of+astrocytes+in+neural+circuit+development.">Emerging roles of astrocytes in neural circuit development.</a> Nature Reviews Neuroscience. 14 (5), 311-321 (2013).</li><li>Ma, Z., Stork, T., Bergles, D. E., Freeman, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuromodulators+signal+through+astrocytes+to+alter+neural+circuit+activity+and+behaviour.">Neuromodulators signal through astrocytes to alter neural circuit activity and behaviour.</a> Nature. 539 (7629), 428-432 (2016).</li><li>Blanco-Su&#225;rez, E., Caldwell, A. L. M., Allen, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+astrocyte-synapse+interactions+in+CNS+disorders:+Astrocyte-synapse+disease.">Role of astrocyte-synapse interactions in CNS disorders: Astrocyte-synapse disease.</a> The Journal of Physiology. 595 (6), 1903-1916 (2017).</li><li>Stogsdill, J. 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=Astrocytic+neuroligins+control+astrocyte+morphogenesis+and+synaptogenesis.">Astrocytic neuroligins control astrocyte morphogenesis and synaptogenesis.</a> Nature. 551 (7679), 192-197 (2017).</li><li>Srinivasan, 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=New+Transgenic+Mouse+Lines+for+Selectively+Targeting+Astrocytes+and+Studying+Calcium+Signals+in+Astrocyte+Processes+in+Situ+and+In+Vivo.">New Transgenic Mouse Lines for Selectively Targeting Astrocytes and Studying Calcium Signals in Astrocyte Processes in Situ and In Vivo.</a> Neuron. 92 (6), 1181-1195 (2016).</li><li>Tabata, H., Nakajima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+in+utero+gene+transfer+system+to+the+developing+mouse+brain+using+electroporation:+visualization+of+neuronal+migration+in+the+developing+cortex.">Efficient in utero gene transfer system to the developing mouse brain using electroporation: visualization of neuronal migration in the developing cortex.</a> Neuroscience. 103 (4), 865-872 (2001).</li><li>Shimogori, T., Ogawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+application+with+in+utero+electroporation+in+mouse+embryonic+brain.">Gene application with in utero electroporation in mouse embryonic brain.</a> Development, Growth &amp; Differentiation. 50 (6), 499-506 (2008).</li><li>Garc&#237;a-Marqu&#233;s, J., L&#243;pez-Mascaraque, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+Identity+Determines+Astrocyte+Cortical+Heterogeneity.">Clonal Identity Determines Astrocyte Cortical Heterogeneity.</a> Cerebral Cortex. 23 (6), 1463-1472 (2013).</li><li>Mart&#237;n-L&#243;pez, E., Garc&#237;a-Marques, J., N&#250;&#241;ez-Llaves, R., L&#243;pez-Mascaraque, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+Astrocytic+Response+to+Cortical+Injury.">Clonal Astrocytic Response to Cortical Injury.</a> PLoS ONE. 8 (9), 74039 (2013).</li><li>Guti&#233;rrez, 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=Sibling+astrocytes+share+preferential+coupling+via+gap+junctions.">Sibling astrocytes share preferential coupling via gap junctions.</a> Glia. 67 (10), 1852-1858 (2019).</li><li>Lee, Y., Messing, A., Su, M., Brenner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GFAP+promoter+elements+required+for+region-specific+and+astrocyte-specific+expression.">GFAP promoter elements required for region-specific and astrocyte-specific expression.</a> Glia. 56 (5), 481-493 (2008).</li><li>Yoon, H., Walters, G., Paulsen, A. R., Scarisbrick, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte+heterogeneity+across+the+brain+and+spinal+cord+occurs+developmentally,+in+adulthood+and+in+response+to+demyelination.">Astrocyte heterogeneity across the brain and spinal cord occurs developmentally, in adulthood and in response to demyelination.</a> PloS One. 12 (7), 0180697 (2017).</li><li>Ge, W. P., Miyawaki, A., Gage, F. H., Jan, Y. N., Jan, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+generation+of+glia+is+a+major+astrocyte+source+in+postnatal+cortex.">Local generation of glia is a major astrocyte source in postnatal cortex.</a> Nature. 484 (7394), 376-380 (2012).</li><li>Clavreul, 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=Cortical+astrocytes+develop+in+a+plastic+manner+at+both+clonal+and+cellular+levels.">Cortical astrocytes develop in a plastic manner at both clonal and cellular levels.</a> Nature Communications. 10 (1), 4884 (2019).</li><li>Figueres-O&#241;ate, M., Garc&#237;a-Marqu&#233;s, J., L&#243;pez-Mascaraque, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UbC-StarTrack,+a+clonal+method+to+target+the+entire+progeny+of+individual+progenitors.">UbC-StarTrack, a clonal method to target the entire progeny of individual progenitors.</a> Scientific Reports. 6 (1), 33896 (2016).</li><li>Figueres-O&#241;ate, M., Garc&#237;a-Marqu&#233;s, J., Pedraza, M., De Carlos, J. A., L&#243;pez-Mascaraque, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+analyses+of+neural+lineages+after+embryonic+and+postnatal+progenitor+targeting+combining+different+reporters.">Spatiotemporal analyses of neural lineages after embryonic and postnatal progenitor targeting combining different reporters.</a> Frontiers in Neuroscience. 9, 87 (2015).</li><li>Figueres-O&#241;ate, M., S&#225;nchez-Villal&#243;n, M., S&#225;nchez-Gonz&#225;lez, R., L&#243;pez-Mascaraque, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+Tracing+and+Cell+Potential+of+Postnatal+Single+Progenitor+Cells+In+Vivo.">Lineage Tracing and Cell Potential of Postnatal Single Progenitor Cells In Vivo.</a> Stem Cell Reports. 13 (4), 700-712 (2019).</li><li>Peng, H., Ruan, Z., Long, F., Simpson, J. H., Myers, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=V3D+enables+real-time+3D+visualization+and+quantitative+analysis+of+large-scale+biological+image+data+sets.">V3D enables real-time 3D visualization and quantitative analysis of large-scale biological image data sets.</a> Nature Biotechnology. 28 (4), 348-353 (2010).</li><li>Peng, H., Bria, A., Zhou, Z., Iannello, G., Long, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensible+visualization+and+analysis+for+multidimensional+images+using+Vaa3D.">Extensible visualization and analysis for multidimensional images using Vaa3D.</a> Nature Protocols. 9 (1), 193-208 (2014).</li><li>Peng, H., 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=Virtual+finger+boosts+three-dimensional+imaging+and+microsurgery+as+well+as+terabyte+volume+image+visualization+and+analysis.">Virtual finger boosts three-dimensional imaging and microsurgery as well as terabyte volume image visualization and analysis.</a> Nature Communications. 5 (1), 4342 (2014).</li><li>Loulier, K., 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=Multiplex+Cell+and+Lineage+Tracking+with+Combinatorial+Labels.">Multiplex Cell and Lineage Tracking with Combinatorial Labels.</a> Neuron. 81 (3), 505-520 (2014).</li><li>Niwa, H., Yamamura, K., Miyazaki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+selection+for+high-expression+transfectants+with+a+novel+eukaryotic+vector.">Efficient selection for high-expression transfectants with a novel eukaryotic vector.</a> Gene. 108 (2), 193-199 (1991).</li><li>Abdeladim, 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=Multicolor+multiscale+brain+imaging+with+chromatic+multiphoton+serial+microscopy.">Multicolor multiscale brain imaging with chromatic multiphoton serial microscopy.</a> Nature Communications. 10 (1), 1662 (2019).</li><li>Chen, F., LoTurco, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+stable+transgenesis+of+radial+glia+lineage+in+rat+neocortex+by+piggyBac+mediated+transposition.">A method for stable transgenesis of radial glia lineage in rat neocortex by piggyBac mediated transposition.</a> Journal of Neuroscience Methods. 207 (2), 172-180 (2012).</li><li>Siddiqi, F., 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=Fate+Mapping+by+PiggyBac+Transposase+Reveals+That+Neocortical+GLAST++Progenitors+Generate+More+Astrocytes+Than+Nestin++Progenitors+in+Rat+Neocortex.">Fate Mapping by PiggyBac Transposase Reveals That Neocortical GLAST+ Progenitors Generate More Astrocytes Than Nestin+ Progenitors in Rat Neocortex.</a> Cerebral Cortex. 24 (2), 508-520 (2014).</li><li>Yoshida, 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=Simultaneous+expression+of+different+transgenes+in+neurons+and+glia+by+combining+in+utero+electroporation+with+the+Tol2+transposon-mediated+gene+transfer+system.">Simultaneous expression of different transgenes in neurons and glia by combining in utero electroporation with the Tol2 transposon-mediated gene transfer system.</a> Genes to Cells. 15 (5), 501-502 (2010).</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>Pacary, 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=Visualization+and+Genetic+Manipulation+of+Dendrites+and+Spines+in+the+Mouse+Cerebral+Cortex+and+Hippocampus+using+in+utero+Electroporation.">Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using in utero Electroporation.</a> Journal of Visualized Experiments. (65), e4163 (2012).</li></ol>

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...

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>1.1 Bacteria transformation</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>1.2 Plasmid culture</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>1.3 Plasmid DNA preparation</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>2.1 Preparation of the solutions</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>2.2 Preparation of the surgery material</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>2.3 Preparation of the pregnant female mouse</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>3.2 Electroporation</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>4.1 Tissue harvesting and sectioning</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>5. Multichannel confocal imaging</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>6. Astrocyte territorial volume segmentation</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>7. Astrocyte arborization tracing</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?...

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

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

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

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 ...

Isolation and Culture of Mouse Cortical Astrocytes

Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics. In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail. This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups. The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture. Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes. Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers. This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology.

Astrocytes have been recognized to be versatile cells participating in fundamental biological processes that are essential for normal brain development and function, and central nervous system repair. Here we present a rapid procedure to obtain pure mouse astrocyte cultures to study the biology of this major class of central nervous system cells.

小鼠脑皮层星形胶质细胞的分离和培养

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

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 ...

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and associated extracellular matrix. Because they provide a suitable organotypic environment, cortical slice explants are often used to investigate those interactions that control neuronal differentiation and development. Although beneficial, the slice explant model can suffer from drawbacks including aberrant cellular lamination and migration. Here we report a whole cerebral hemisphere explant system for studies of early cortical development that is easier to prepare than cortical slices and shows consistent organotypic migration and lamination. In this model system, early lamination and migration patterns proceed normally for a period of two days in vitro, including the period of preplate splitting, during which prospective cortical layer six forms. We then developed an ex utero electroporation (EUEP) approach that achieves ~80% success in targeting GFP expression to neurons developing in the dorsal medial cortex.
The whole hemisphere explant model makes early cortical development accessible for electroporation, pharmacological intervention and live imaging approaches. This method avoids the survival surgery required of in utero electroporation (IUEP) approaches while improving both transfection and areal targeting consistency. This method will facilitate experimental studies of neuronal proliferation, migration and differentiation.

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

This protocol describes an improved explant procedure that involves ex utero electroporation, dissection and culture of entire cerebral hemispheres from the embryonic mouse. The preparation facilitates pharmacological studies and assays of gene function during early cortical development.

前子宫穿孔和整个半球的外植体早期皮质发育的研究:一个简单的实验方法

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and ...

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 ...

Subtype-selective Electroporation of Cortical Interneurons

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

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

皮质的interneurons的亚型选择性电穿孔

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

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

小鼠视网膜神经节细胞的转染在体内电穿孔

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

小鼠脑皮层星形胶质细胞的分离和培养

外用转基因大鼠通过代

前子宫穿孔和整个半球的外植体早期皮质发育的研究:一个简单的实验方法

遗传操纵鼠标的发展下丘脑通过

皮质的interneurons的亚型选择性电穿孔

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

探索更多视频

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

小鼠视网膜神经节细胞的转染在体内电穿孔

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

小鼠脑皮层星形胶质细胞的分离和培养

外用转基因大鼠通过代

前子宫穿孔和整个半球的外植体早期皮质发育的研究:一个简单的实验方法

遗传操纵鼠标的发展下丘脑通过

皮质的interneurons的亚型选择性电穿孔

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