我们要感谢他们的支持和有益的意见，在此手稿的巴雷西实验室成员。我们感谢亚历山大工人为他不断的技术援助以及照顾动物的工作人员为他们在帮助维护史密斯学院斑马鱼殖民地。我们也感谢本议定书filmin一些贷款显微镜设备迈克Hallacy，Rottenfusser鲁迪和卡尔蔡司显微成像。这项工作是支持由美国国家科学基金会资助的研究资助，0615594。

第1部分：Alexa的594标记的胚胎 1.1显微注射板和针的制备<ol><li> 进行显微注射板 <ol style="list-style-type:lower-alpha;"><li>准备注射液在琼脂糖模具使用的技术，韦斯特，2007年1至5个1毫米的槽由三个板块。这些槽，将snuggly举行一个高效和系统的microinjections的胚胎。板之前，采取3个1毫米，4英寸长的无灯丝毛细血管和小心打破了一半。使用这些2英寸长的毛细血管强力胶，胶水两个侧端在一个平面上。胶第三毛细管掩映在树林，在上面这两个，建立一个与三个毛细血管金字塔形状。晾干和重复，直到足够的“低谷模具”构造。 </li><li>熔化的1.5％琼脂糖倒入20 - 25ML（5毫米深度）在胚胎中期（EM）[5mm的氯化钠，氯化钾0.17mM，0.33mM 氯化钙 ， 硫酸镁 0.33mM 0.00003％亚甲蓝到一个100mm的培养皿。立即将先前作出的金字塔形槽模具到五年，确保模具接触的培养皿底部的侧平盘底部，并完全覆盖，经琼脂糖。 </li><li>当琼脂糖凝固，玻璃毛细管模具必须采取。要删除，切出一个大的琼脂糖平方米，包含三个毛细管模具。轻轻地打跑的琼脂上削减广场和丢弃周边。翻转平方米琼脂件以上，并用细镊子，彻底清除槽模具。广场周围倒入熔化的琼脂糖举行到位的新成型的井，到培养皿。让设置。板可几个星期密封存放在4封口膜° C。在琼脂中的任何增长的迹象丢弃。 </li></ol></li><li> 针microinjections。 <ol style="list-style-type:lower-alpha;"><li>使用一个Micropipet毛细管拉马内部长丝1毫米，4英寸的玻璃毛细管必要的注射针头。 Flamming /布朗Micropipet拖轮使用热量为550针拉，拉力为120，速度为200，和200次/延迟。 </li></ol></li></ol> 1.2胚胎收集和显微注射仪设置<ol><li>受精卵立即收集后他们奠定和维持用EM填补了培养皿中。荧光右旋糖酐注射液是在单细胞阶段的理想，应该由16单元的第2阶段完成。 </li><li>使用宽头的玻璃吸管，驱逐到室温注射填充板与EM井的胚胎。胚胎紧握钝钳轻轻楔形槽的底部。胚胎的绒毛或蛋黄已经失密应该被丢弃。 </li><li>装入稀释荧光Alexa的594解决方案的1 -2μL（重量在0.2 mol氯化钾的5％）的注射针头。允许葡聚糖前往注射针头通过毛细作用，由于嵌入式长丝运行的注射针头的长度提示。 </li><li>将注射针头的立体定向显微毛细管持有人。 </li><li>微量仪器调整的压力，以开始与40-50 PSI和脉冲持续时间为500毫秒。 </li><li>注射针的针尖轻轻地吃草，打破封印钳（理想的直径为0.05 -0.15毫米）。使用steromicroscope，驱逐到一微米葡聚糖之上校准针与矿物油。修改通过调节脉冲的空气压力，持续时间和针尖大小的丸大小。阿丸的大小相当于一个0.8 - 1nl量应保持在整个注射 3 。通常的提示将成为与蛋黄颗粒堵塞;调整压力或打破针尖可能会缓解这一问题，否则一个新的注射针必须加载。 </li></ol> 1.3荧光Alexa的594 Microinjections <ol><li>在高放大倍率的情况下，从槽的一端开始，顺利皮尔斯的蛋壳和胚胎的细胞膜，进入蛋黄。刚下细胞注入葡聚糖的解决方案。请务必取出注射针头与使用相同的顺利进入胚胎运动。将培养皿位置的胚胎内联，并继续注射槽的长度。定期检查的压力和丸大小。要小心，不要移动或注射针头弯曲，当它在胚胎，因为这很可能是致命的损害胚胎和/或折断的注射针头。 </li><li>轻轻推胚胎钳的低谷。他们转移到培养皿中充满了抗生素笔/步的EM Nusslein Volhard和达姆（2002）（1毫升6.5M氯化钙2，1毫升3.5M碳酸氢钠3，25毫升20X 60mg/ml青霉素，100毫克/ EM和10ml ml链霉素ST玉珠），后来移植的准备。孵育28.5 ° C和监测未受精卵或胚胎死亡胚胎。 </li></ol>第2部分：原肠期移植 2.1 Dechorionating和移植板的制备<ol><li> Dechorionating板。Dechorionated胚胎尚未完成的外包需要软底板，不坚持他们的蛋黄和撕裂的plastic.The日至移植前，使用先前所述的1.5％的琼脂糖溶液，使浇筑5 dechorionating板-10毫升熔化琼脂糖到一个100mm的培养皿。只有一层薄薄的琼脂糖（2 - 3mm）的需要，以减轻在dechorination胚胎。放凉。 </li><li> 移植板分隔井专业板需要4。 30毫升到1.5％的琼脂糖溶液倒入一个100mm的培养皿中，并插入一个移植以及模具。模具应包含104三个90度双方组成的三角草皮和一个45度的直角方。该井的设计方方（图1A，紫色框）持有两个胚胎。小心陷阱下模气泡。让琼脂糖硬化。取出模具，揭示了一个凹陷区，含倾斜边缘的方形井。 </li></ol> 2.2胚胎移植器械安装<ol><li> 捐助者和东道国胚胎的定位<ol style="list-style-type:lower-alpha;"><li>主机和捐助者的发展需要进行监测，以维持不久的年龄相匹配的配对。要做到这一点，胚胎可以提出在不同的温度（25-31℃）放慢或加快发展的分别。 </li><li>主机和捐助胚胎应手dechorionated琼脂涂dechorionating板前1小时到所需的原肠胚阶段移植的情况下这将需要5hpf完成年龄。 </li><li>屏蔽前在5.5hpf东道国和捐助国胚胎阶段，装入充满抗生素的EM移植板。胚胎装入个人1毫米，使用玻璃吸管井。一个例子如下：第一行是充满了捐助胚胎（共18个，每口井的两个胚胎）和主机胚胎充满以下两行（一个胚胎每口井，每列9个胚胎）。 </li></ol></li><li> 成立移植仪器 <ol style="list-style-type:lower-alpha;"><li>移植手术进行蔡司Lumar荧光体视显微镜，已完全自动化，操纵杆控制变焦和对焦操作。虽然移植可没有这种显微镜的自动化，并显着增加，特别是当移植的高需要的易用性和效率。 </li><li>将Eppendorf公司AirTram规模的中间位置。 AirTram（图1A，红色框）管连接毛细管的持有人，并保护持有人的Eppendorf自动化显微（图1A） 。这种显微操作是完全自动化和操纵杆控制。同样，这种自动化是没有完全必要的，但显着改善的导航能力周围的胚胎和成针。另外，许多研究者喜欢的，是说，提供更流畅的细胞采集的控制，但是，我们有没有困难与AirTram OilTram。 </li><li>毛细血管使用无菌的Eppendorf TransferTips（ES），有一个斜角15米，从尖端（图1A绿箱）的提示和一个20度的弯曲角度1毫米的开放。 </li></ol></li></ol> 2.3原肠期细胞移植<ol><li>在6hpf，使用显微轻轻地吃草与毛细管的尖端胚胎胚胎细胞提取物等，中线和盾牌是可见的和反对的毛细管尖端的位置旋转。吸进入毛细血管的EM少量，以防止任何机会接触空气 - 水界面，它会损害细胞的细胞。 </li><li>对斑马鱼的命运地图的基础上，针对前脑细胞需要移植细胞在中线正好之间的动物极和盾牌5。轻轻捅破捐助胚胎的外胚层（罗丹明注射或GFP转基因胚胎）在这个位置。细胞在原肠胚外胚层和基本蛋黄之间的厚度较薄，因此，请小心不要刺穿蛋黄，因为这往往会导致在几个小时内死亡。使用AirTram吸细胞慢慢地进入毛细血管。始终保持水的能见度。而在捐赠的一角轻微搅拌，将有助于松散的细胞 - 细胞接触。它是从胚胎中删除之前，应提取约10-50从捐赠者的胚胎细胞。 </li><li>删除从捐助者，同样东方的主机，和皮尔斯的毛细血管和驱逐到确切的SA细胞我的位置之间的动物极和屏蔽主机胚胎。 </li><li>捐助者和东道国的胚胎移植后，从水井中删除，分离并轻轻放在涂琼脂培养皿充满抗生素的EM菜。胚胎移植井增加死亡率。孵育胚胎在28.5 ° C。 </li></ol> 2.4可视化和成像胚胎<ol><li> 安装固定和活胚胎。 <ol style="list-style-type:lower-alpha;"><li>从转基因绿色荧光蛋白或若丹明注入胚胎衍生的主机胚胎可以可视化和成像活着的或固定的胚胎。我们在座的固定和实时成像选项的例子。在30hpf 0.1M磷酸盐缓冲液（PB）的4％，在4℃1 paraformadehyde过夜修复胚胎处死。 </li><li>冲洗胚胎的2倍​​，然后在0.1M PB洗3 × 5分钟。 </li><li>我们所提供的例子，固定主机immunolabeled针对以前 6所述的乙酰化微管蛋白（α- AT的）与抗体的所有轴突。 </li><li>广场胚胎在75％甘油，在室温下沉，然后存放在4 ° C。 </li><li>准备幻灯片装入两个胚胎。创建两个凡士林广场幻灯片相匹配的内部形状和一个方形盖玻片直径的轮廓。 </li><li> Deyolk胚胎的前脑额叶，剖析了整个脑垂直切割的前脑。广场这个组织内的石油以及少量的甘油。东方的组织到相应的位置，并放置在试样的盖玻片。 </li><li>安装在0.75％的琼脂糖的Tricaine 4％溶液（在EM）解决方案，到玻璃底培养皿中的活体标本。琼脂糖凝之前，胚胎是操纵成像的钨针，以正确方向。对于我们的分析中，我们面向的前脑对直接盖玻片。 </li></ol></li><li> 成像 <ol style="list-style-type:lower-alpha;"><li>固定和生活主机使用徕卡SP5激光扫描共聚焦显微镜成像。 Z轴堆叠收购，3 -或4维图像处理完成Volocity软件的使用。 </li></ol></li></ol>结果： 在努力解决前脑星形胶质细胞的作用是必要的，同时这些细胞，并针对不同的基因操作，以小群间脑和telencephalic细胞可视化。为了产生这些类型的嵌合胚胎，其中星形胶质细胞的胚胎内人口的某些部分是不同的基因型或表现型，我们是否使用了一个多层面的方法，结合GFP转基因胚胎，使用标签星形胶质细胞与胚囊使用， - 细胞移植上演。专门针对我们的克隆间脑，我们利用斑马鱼原肠胚的命运之图，有选择性地从动物极和盾牌5（图1B）等距离的中线提取细胞。这些提取出来的TG [GFAP：GFP细胞，然后移植到野生型主机胚囊，当时 ​​提出来30hpf，脑解剖参考地标轴突（乙酰化α-微管蛋白）immunolabeled的，在相同的位置。作为一个例子所示，一台主机的胚胎共聚焦成像显示孤立的绿色荧光蛋白在整个端脑和间脑（图2A）+细胞。的这些GFP +细胞的完整形态可以被观察到这个克隆实验，揭示，这些细胞大多采取的特征径向胶质细胞形态，胞体所在相邻心室区和一个大型年底脚终止在一个径向过程软脑膜表面（图2A，插图）。这些收集到的光片的Z - Stack的三维渲染标记的轴突（电影1）清楚地表明了这些细胞的位置。 可视化细胞移植常用的另一种方法是最初microinject成一个细胞的卵黄细胞谱系一种荧光染料，如Alexa的594，上演野生型GFP转基因胚胎。作为一个例子，我们注入的野生型胚胎Alexa的594在单细胞阶段。我们允许他们开发盾构阶段，原肠胚，然后进行上述脑针对性移植，而是我们移植到TG [GFAP：NUC - GFP转基因主机胚囊。激光扫描共聚焦显微镜的背端脑成像显示，供体细胞的荧光红色集群之间GFP +细胞核内的前脑（图2B）。我们进一步分析了超过两个小时的激光扫描共聚焦的过程中收集的Z -栈每三分钟，这台主机。 4这种timelapse三维渲染使用Volocity软件（即兴）显示罗丹明细胞膜的动态蜂窝架次ND的GFP +的核（电影2）。 <img border="0" src="/files/ftp_upload/1422/1422fig1tmb.jpg" alt="图1" /> <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/1422/1422fig1.jpg">点击她看到图1的放大版本。</a> 图1：移植仪器和实验方法的原理。一）仪器使用，进行原肠胚期移植手术。蔡司Lumar荧光体视显微镜是用来进行这些移植。它的操纵杆控制对焦和变焦（左红圈）。 Transferman NK细胞被用来操纵的毛细血管的位置，这也是操纵杆控制（右红圈）。在安装过程中，60-80胚胎放入个人琼脂井以前在一个100mm的培养皿用塑料模具（紫色概述中）。移植毛细管（TransferTip）由Eppendorf公司生产的具有15μm的内径开幕式和20度的直角头（绿色概述盒）。控制细胞的愿望是与Eppendorf公司的 AirTram（红色概述框） 。二）进入宿主胚胎原肠胚阶段，从供体移植过程的插图。 TG GFAP：GFP的]胚胎（如图所示）或Alexa的594注射（如在协议中所述）捐助胚胎中提取细胞。从中间的盾和动物极中线细胞，并移植到同一地区的主机胚胎。主机是固定的，使用激光扫描共聚焦显微镜成像。 <img border="0" src="/files/ftp_upload/1422/1422fig2tmb.jpg" alt="图2" /> <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/1422/1422fig2.jpg">请点击这里看大图图2。</a> 图2。在斑马鱼的前脑的原肠胚阶段移植例A）野生型胚胎移植TG GFAP：GFP]主机的正面看法。diencephalons和斑马鱼的前脑的端脑细胞。轴突中的红色标记（α-乙酰微管蛋白抗体）和绿色（内源性表达GFP）标记细胞移植。胚胎30 HPF。插图显示GFP +细胞的放射状胶质形态。二）现场[GFAP：NUC - GFP] TG端脑与胚胎移植的细胞荧光（红色）若丹明主机背面观。主机胚胎细胞核标记为绿色（GFP）。虚线勾勒出前脑的前表面。实线表示脑室区。比例尺为10μm。 电影1。除前脑commissures异位标记径向胶质细胞的关系 。胚囊细胞上演TG [GFAP：绿色荧光蛋白 ]的胚胎移植到非GFP转基因野生型线。激光扫描共聚焦的Z - Stack的收集和使用 Volocity软件的3 - D渲染，然后进行处理。最初，图像的30hpf斑马鱼的前脑额叶，和它水平翻转相比，图2B。轴突内的前联合（AC）和光纤后合缝（POC，底部）（A -乙酰化微管蛋白，红）看到。绿色细胞的GFP +细胞移植了前脑和产生明显的放射状胶质细胞形态的根。随着电影的发展，它集中在两个放射状胶质细胞拥有最终费POC的<a href="/files/ftp_upload/1422/Deschene&amp;BarresiMovie1.mov">联系。点击这里下载电影</a> 1。 电影2。从以前与Alexa的594注入一个原肠胚的细胞移植到 TG Timelapse移植Alexa的594标记的前脑细胞成像 。GFAP：NUC - GFP]转基因胚胎。这部电影代表了一个2小时的时间序列，其中Z轴堆叠采取每5分钟的3 - D投影。 Z轴堆叠上的激光扫描共聚焦显微镜，收集和2,4 - D渲染完成使用Volocity软件。移植细胞的标记与Alexa的594（红色），和GFP +原子核是绿色的。这timelapse运行的4D图像开始一个的在30hpf前脑的背视图将旋转关于X轴，作为明确的运动是所有移植细胞的细胞膜的GFP检测+细胞核以及<a href="/files/ftp_upload/1422/Deschene&amp;BarresiMovie2.mov">。点击</a>这里<a href="/files/ftp_upload/1422/Deschene&amp;BarresiMovie2.mov">下载电影2。</a>

<ol>
	<li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+for+the+laboratory+use+of+zebrafish+(Danio.">A guide for the laboratory use of zebrafish (Danio.</a> The Zebrafish Book. , (2007).</li><li>N&#252;sslein-Volhard, C., Dahm, R. e. d. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+A+Practical+Approach.">Zebrafish A Practical Approach.</a> , (2002).</li><li>Graeden, E., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+the+zebrafish+embryonic+brain+by+confocal+microscopy.">Live imaging of the zebrafish embryonic brain by confocal microscopy.</a> J Vis Exp. (26), (2009).</li><li>Gilmour, D. T., Jessen, J. R., Lin, S., N&#252;sslein-Volhard, C., Dahm, R. e. d. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish,+a+practical+approach.">Zebrafish, a practical approach.</a> 1, 121-121 (2002).</li><li>Woo, K., Shih, J., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+maps+of+the+zebrafish+embryo.">Fate maps of the zebrafish embryo.</a> Curr Opin Genet Dev. 5 (4), 439-439 (1995).</li><li>Barresi, M. J., Hutson, L. D., Chien, C. B., Karlstrom, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+regulated+Slit+expression+determines+commissure+and+glial+cell+position+in+the+zebrafish+forebrain.">Hedgehog regulated Slit expression determines commissure and glial cell position in the zebrafish forebrain.</a> Development. 132 (16), 3643-3643 (2005).</li><li>Kozlowski, D. J., Murakami, T., Ho, R. K., Weinberg, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+cell+movement+and+tissue+patterning+in+the+zebrafish+embryo+revealed+by+fate+mapping+with+caged+fluorescein.">Regional cell movement and tissue patterning in the zebrafish embryo revealed by fate mapping with caged fluorescein.</a> Biochem Cell Biol. 75 (5), 551-551 (1997).</li><li>Halloran, M. C., Murakami, T., Ho, R. K., Weinberg, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-induced+gene+expression+in+specific+cells+of+transgenic+zebrafish.">Laser-induced gene expression in specific cells of transgenic zebrafish.</a> Development. 127 (9), 1953-1953 (2000).</li><li>Hardy, M. E., Ross, L. V., Chien, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+gene+misexpression+in+zebrafish+embryos+induced+by+local+heat+shock+using+a+modified+soldering+iron.">Focal gene misexpression in zebrafish embryos induced by local heat shock using a modified soldering iron.</a> Dev Dyn. 236 (11), 3071-3071 (2007).</li></ol>

The authors have nothing to disclose.

生成嵌合胚胎是一个功能强大的工具，地址在几个模型系统，即果蝇（D.melanogaster），蠕虫（线虫），斑马鱼（D.rerio）和鼠标（M.musculus）许多研究问题。我们认为，斑马鱼模型系统是唯一适合在一个相对容易的，快速和灵活的方式产生嵌合体。斑马鱼是脊椎动物发展以外的小鼠模型相比，更容易使母亲。斑马鱼也明显比苍蝇或蠕虫，从而简化了，如细胞移植的胚胎操作。此外，与转基因技术相结合细胞移植程序的能力，使一个大阵可能在基因功能，可以在局部组织特定的方式操纵的实验。例如，小克隆的GFP +或罗丹明标记的细胞，使往往失去了在一个纯合的转基因捐助者，或者是不可能想象差与普通抗体的细胞内标签由于的全细胞形态的表征。此外，通过使用transgenically标记或荧光充满细胞，我们可以随着时间的推移跟踪现场斑马鱼的胚胎供体细胞。 Timelapse单个细胞的成像提供了一个新的分析，在整个有机体中的细胞行为。 我们的实验室还结合使用热休克诱导某些轴突的指导线索，可以在海拔孵化温度（数据未显示）以下供体细胞misexpressed细胞移植的转基因株系。这项技术是一个非常强大的和直接的方式，在本地misexpress蛋白质在空间和时间的具体的方式，这让我们看到了特定的蛋白质如何影响到发展的利益。在我们的例子中，这种技术可以扩展我们的知识背后的狭缝- ROBO的指导线索，在中线 5 commissures和神经胶质细胞的定位功能。 本地misexpressing基因等为重点的紫外线uncaging和本地化heatshock工具（激光或烙铁）的其他方法，请提供替代的方式来操纵基因的表达，并在细胞7-9小簇标记;然而，细胞移植建立一个最终环境的分析，是由紫外线，激光或热细胞移植更容易控制的实验方法诱导的任何创伤。总之，我们已经发现了细胞移植的程序，我们的神经发育生物学研究的一个重要组成部分。生成具有不同特性的细胞克隆是一个发育生物学家在细胞的行为，基因功能和细胞自治的根本问题解决的关键工具。

<table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 名称 </td><td> 类型 </td><td> 公司 </td><td> 产品编号 </td><td> 具体细节 </td></tr><tr><td>培养皿</td><td>工具</td><td> Fisherbrand </td><td> 08-757-13 </td><td> 100 × 15毫米</td></tr><tr><td>玻璃底培养皿</td><td>工具</td><td> MatTek </td><td> P35G - 1.5 - 2.0 - C </td><td> 35 × 15MM，1.5，胶版纸，γ-辐照</td></tr><tr><td>大口径玻璃巴斯德吸管</td><td>工具</td><td> Fisherbrand </td><td> 63A - 53 - WT </td><td></td></tr><tr><td>玻璃纤维毛细管槽模具（无灯丝）和注射针（与长丝） </td><td>工具</td><td>世界精密仪器</td><td> 1B150F - 4 </td><td> 4英寸（100毫米），外径1.5 / 0.84 /内径（mm） </td></tr><tr><td>移植毛细管（TransferTip） </td><td>工具</td><td> Eppendorf公司</td><td> 83000122-8 </td><td> IV型，无菌，诠释。 15μm的; 20度弯曲</td></tr><tr><td>移植模具</td><td>工具</td><td>自适应科学工具</td><td> PT - 1 </td><td> 150三角形的草皮举行个别胚胎</td></tr><tr><td>琼脂糖，I型</td><td>试剂</td><td>西格玛爱秩序</td><td> A6013 - 250G </td><td> 1.5％的琼脂糖在EM </td></tr><tr><td> （抗生素）胚胎中等</td><td>试剂</td><td></td><td></td><td>韦斯特2007年</td></tr><tr><td>磷酸盐缓冲液</td><td>试剂</td><td></td><td></td><td>韦斯特2007年</td></tr><tr><td>看守员钳</td><td>工具</td><td>精细科学的工具</td><td></td><td> 100毫米尖端（沉闷），50毫米尖</td></tr><tr><td>若丹明B葡聚糖</td><td>天堂示踪染料</td><td>分子探针，Invitrogen公司</td><td> D1824 </td><td>兆瓦万大，赖氨酸可以解决的，红色（590nm），绿色（570nm处）光激发的荧光染料</td></tr><tr><td>解剖显微镜</td><td>显微镜</td><td>奥林巴斯</td><td> SZX7 </td><td></td></tr><tr><td>荧光显微镜</td><td>显微镜</td><td>蔡司</td><td> Lumar </td><td>全自动操纵杆控制（Sycop） </td></tr><tr><td>移植器械</td><td>工具</td><td>的Eppendorf公司</td><td> AirTram </td><td></td></tr><tr><td>自动微操作机器人</td><td>工具</td><td>的Eppendorf公司</td><td> TransferMan NK2 </td><td>操纵杆控制</td></tr><tr><td>微操作机器人</td><td>工具</td><td>应用科学仪器公司</td><td> 00-42-101-0000 </td><td> MM33右键</td></tr><tr><td>微量与背压单位</td><td>工具</td><td>应用科学仪器公司</td><td>移动电话伙伴关系倡议- 2 BPU </td><td></td></tr><tr><td> Flamming /布朗Micropipet拖轮</td><td>工具</td><td>萨特仪器公司</td><td>型号P97 </td><td>计划：热火550; 速度200; 时间/延时200; 拉力量120。 </td></tr></tbody></table>

tissue targeted embryonic chimeras: zebrafish gastrula cell transplantation

在发育生物学领域的某些基本问题只能回答当细胞被放置在新颖的环境，或改变细胞的小群体，在更大的范围内时。看一个细胞如何相互作用，并在一个独特的环境行为，描述细胞的功能是必不可少的。确定如何本地化错误表达一个特定的蛋白质影响周围的细胞提供各种发育过程中，蛋白质扮演的角色有见地的信息。我们的实验室使用的斑马鱼模型系统，以独特的古典移植技术相结合产生的基因型或表型嵌合体的遗传方法。我们的研究过程中形成的斑马鱼的前脑commissures神经元的神经胶质细胞间的相互作用。本视频介绍了一个方法，使我们的实验室调查种群间脑星形胶质细胞和具体的指导线索，影响轴突投射，因为他们越过中线的角色的作用。由于其透明度斑马鱼的胚胎是这种类型的异位细胞放置或局部基因的错误表达的理想模型。可以通过使用一个重要的染料或表达一种荧光蛋白的转基因鱼线追踪移植细胞。这里我们展示如何准备用于移植的一个重要的染料示踪的捐赠胚胎，以及如何提取和从原肠期胚胎上演到另一个细胞移植。我们目前的数据显示，斑马鱼胚胎前脑内异位的GFP +转基因细胞，这些细胞的位置和表征方面脑commissures。此外，我们Alexa的594标记的细胞移植到转基因的GFP +主机胚胎的激光扫描共聚焦timelapse显微镜。这些数据提供的证据表明，原肠胚上演移植使异位细胞有针对性的定位，以解决发育生物学中的各种问题。

Watch this Scientific Journal Video about Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation at JoVE.com

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

斑马鱼细胞移植使遗传学和胚胎学的结合，产生组织特异性嵌合体。这个视频演示胚囊上演细胞移植，允许我们的实验室，以调查在合缝形成前脑星形胶质细胞的人口和具体的指导线索的角色。

组织有针对性的胚胎嵌合体：斑马鱼原肠胚细胞移植

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

tissue-targeted-embryonic-chimeras-zebrafish-gastrula-cell

Nanyang Technological University

Research

JoVE Journal

Biology

19.2K 次观看.  Smith College. 斑马鱼细胞移植使遗传学和胚胎学的结合，产生组织特异性嵌合体。这个视频演示胚囊上演细胞移植，允许我们的实验室，以调查在合缝形成前脑星形胶质细胞的人口和具体的指导线索的角色。

视频: Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

在成年斑马鱼的全肾骨髓移植

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

科研

生物学

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

肌动蛋白共沉淀测定;​​ F -肌动蛋白结合蛋白分析

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

人CD40激活的B细胞的生成

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Generating Chimeric Zebrafish Embryos by Transplantation

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

移植嵌合斑马鱼胚胎生成

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

Microdissection of Zebrafish Embryonic Eye Tissues

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post fertilization (dpf), the larvae will show the first visual response. Many genes have been identified to control a proper eye development, but we are far from a complete understanding of the underlying genetic architecture. Whole genome gene expression profiling is a useful tool to elucidate genetic regulatory network for eye development. However, the small size of the embryonic eye in zebrafish makes it challenging to obtain intact and pure eye tissues for expression analysis. For example, the anterior-posterior length of the eye between day 2 and 3 is only approximately 200-300 &mu;m, while the diameter of the lens is less 100 &mu;m. Also, the retinal pigment epithelium (RPE) underlying the retina is just a single-layer epithelium. While gene expression profiles can be obtained from the whole embryo, they do not accurately represent the expression of these tissues. Therefore pure tissue must be obtained for a successful gene expression profiling of eye development. To address this issue, we have developed an approach to microdissect intact retina and retina with RPE attached from 1-3 dpf, which cover major stages of eye morphogenesis. All procedures can be done with fine forceps and general laboratory supplies under standard stereomicroscopes. For retinal dissection, the single-layer RPE is removed and peeled off by brushing action and the preferential adherence of the RPE remnants to the surface of the culture plate for dissection. For RPE-attached retinal dissection, the adherence of RPE to the dissection plate is removed before the dissection so that the RPE can be completely preserved with the retina. A careful lifting action of this tissue can efficiently separate the presumptive choroid and sclera. The lens can be removed in both cases by a chemically etched tungsten needle. In short, our approach can obtain intact eye tissues and has been successfully utilized to study tissue-specific expression profiles of zebrafish retina1, 2 and retinal pigment epithelium3.

This article describes an approach to microdissect zebrafish retinas with and without retinal pigment epithelium attached, from one to three days postfertilization embryos.

斑马鱼胚胎眼组织显微切割的

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post ...

Dechorionation of Medaka Embryos and Cell Transplantation for the Generation of Chimeras

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of the vertebrate genome functions. Manipulation of medaka embryos, such as dechorionation, mounting embryos for imaging and cell transplantation, are key procedures to work on both medaka and zebrafish in a laboratory. Cell transplantation examines cell autonomy of medaka mutations. Chimeras are generated by transplanting labeled cells from donor embryos into unlabeled recipient embryos. Donor cells can be transplanted to specific areas of the recipient embryos based on the fate maps 3 so that clones from transplanted cells can be integrated in the tissue of interest during development. Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. In this video, we show detailed procedures to manipulate medaka embryos.

Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. This video shows step-by-step procedures for how to manipulate medaka embryos, including dechorionation, mounting in agarose for imaging and cell transplantation for the production of chimeras. These procedures are essential to use medaka and zebrafish in a laboratory to take full advantage of their complementary features for the genetic dissection of vertebrate genome functions.

鳉胚胎和细胞移植嵌合体生成Dechorionation

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in ...

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound transgenic fish which express different fluorescent reporters in neighboring cell types provide a means of following cellular interactions 2 and/or tissue-level responses to experimental manipulations over time. In this video, we demonstrate methods that can be used for imaging multiple transgenically labeled cell types serially in individual fish over time courses that can span from minutes to several days. The techniques described are applicable to any study seeking to correlate the "behavior" of neighboring cells types over time, including: 1) serial 'catch and release' methods for imaging a large number of fish over successive days, 2) simplified approaches for separating fluorophores with overlapping excitation/emission profiles (e.g., GFP and YFP), 3) use of hypopigmented mutant lines to extend the time window available for high-resolution imaging into late larval stages of development, 4) use of membrane targeted fluorescent reporters to reveal fine morphological detail of individual cells as well as cellular details in larger populations of cells, and 5) a previously described method for chemically-induced ablation of transgenically targeted cell types; i.e., nitroreductase (NTR) mediated conversion of prodrug substrates, such as metronidazole (MTZ), to cytotoxic derivatives 3,5.
As an example of these approaches, we will visualize the ablation and regeneration of a subtype of retinal bipolar neuron within individual fish over several days. Simultaneously we will monitor several other retinal cell types, including neighboring non-targeted bipolar cells and potential degeneration-stimulated retinal stem cells (i.e., M&#971;ller glia). This strategy is being applied in our lab to characterize cell- and tissue-level (e.g., stem cell niche) responses to the selective loss and regeneration of targeted neuronal cell types.

In this video, techniques for multicolor confocal time-lapse imaging and targeted cell ablation are provided. Time-lapse imaging is used to monitor the behavior of multiple cell types of interest in vivo. Targeted cell ablation facilitates the study neural circuit function and cell-specific neuronal regeneration paradigms.

多色成像定时转基因斑马鱼:可视化由针对性的神经细胞消融激活视网膜干细胞

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

原代细胞培养从果蝇原肠胚

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as tumor-propagating cells, or tumor-initiating cells. It is thought that targeting these self-renewing cells for destruction will block tumor progression and stop relapse, greatly improving patient prognosis1. The most common way to determine the frequency of self-renewing cells within a tumor is a limiting dilution cell transplantation assay, in which tumor cells are transplanted into recipient animals at increasing doses; the proportion of animals that develop tumors is used the calculate the number of self-renewing cells within the original tumor sample2, 3. Ideally, a large number of animals would be used in each limiting dilution experiment to accurately determine the frequency of tumor-propagating cells. However, large scale experiments involving mice are costly, and most limiting dilution assays use only 10-15 mice per experiment. 
Zebrafish have gained prominence as a cancer model, in large part due to their ease of genetic manipulation and the economy by which large scale experiments can be performed. Additionally, the cancer types modeled in zebrafish have been found to closely mimic their counterpart human disease4. While it is possible to transplant tumor cells from one fish to another by sub-lethal irradiation of recipient animals, the regeneration of the immune system after 21 days often causes tumor regression5. The recent creation of syngeneic zebrafish has greatly facilitated tumor transplantation studies 6-8. Because these animals are genetically identical, transplanted tumor cells engraft robustly into recipient fish, and tumor growth can be monitored over long periods of time. Syngeneic zebrafish are ideal for limiting dilution transplantation assays in that tumor cells do not have to adapt to growth in a foreign microenvironment, which may underestimate self-renewing cell frequency9, 10. Additionally, one-cell transplants have been successfully completed using syngeneic zebrafish8 and several hundred animals can be easily and economically transplanted at one time, both of which serve to provide a more accurate estimate of self-renewing cell frequency.
Here, a method is presented for creating primary, fluorescently-labeled T-cell acute lymphoblastic leukemia (T-ALL) in syngeneic zebrafish, and transplanting these tumors at limiting dilution into adult fish to determine self-renewing cell frequency. While leukemia is provided as an example, this protocol is suitable to determine the frequency of tumor-propagating cells using any cancer model in the zebrafish.

Limiting dilution cell transplantation assays are used to determine the frequency of tumor-propagating cells. This protocol describes a method for generating syngeneic zebrafish that develop fluorescently-labeled leukemia and details how to isolate and transplant these leukemia cells at limiting dilution into the peritoneal cavity of adult zebrafish.

量化传播肿瘤细胞的频率使用斑马鱼的同源有限稀释细胞移植

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as ...

Immunostaining of Dissected Zebrafish Embryonic Heart

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics 1,2. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects 3. The expression of any gene can be manipulated via morpholino technology or RNA injection 4. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis 5. 
Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue 6. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. 
 Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. 
Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals.

A rapid way to conduct immunostaining of zebrafish embryonic heart is described. Compared to the whole mount immunostaining approach, this method dramatically increases the penetration of the antibodies, which allows obtaining high resolution images that reveal cellular/subcellular structures in the heart within a much reduced processing time.

免疫染色的解剖斑马鱼胚胎心脏

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...

组织有针对性的胚胎嵌合体：斑马鱼原肠胚细胞移植

摘要

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此视频中的章节

在成年斑马鱼的全肾骨髓移植

肌动蛋白共沉淀测定; F -肌动蛋白结合蛋白分析

人CD40激活的B细胞的生成

移植嵌合斑马鱼胚胎生成

斑马鱼胚胎眼组织显微切割的

鳉胚胎和细胞移植嵌合体生成Dechorionation

多色成像定时转基因斑马鱼:可视化由针对性的神经细胞消融激活视网膜干细胞

原代细胞培养从果蝇原肠胚

量化传播肿瘤细胞的频率使用斑马鱼的同源有限稀释细胞移植

免疫染色的解剖斑马鱼胚胎心脏

组织有针对性的胚胎嵌合体：斑马鱼原肠胚细胞移植

摘要

探索更多视频

此视频中的章节

在成年斑马鱼的全肾骨髓移植

肌动蛋白共沉淀测定;​​ F -肌动蛋白结合蛋白分析

人CD40激活的B细胞的生成

移植嵌合斑马鱼胚胎生成

斑马鱼胚胎眼组织显微切割的

鳉胚胎和细胞移植嵌合体生成Dechorionation

多色成像定时转基因斑马鱼:可视化由针对性的神经细胞消融激活视网膜干细胞

原代细胞培养从果蝇原肠胚

量化传播肿瘤细胞的频率使用斑马鱼的同源有限稀释细胞移植

免疫染色的解剖斑马鱼胚胎心脏

肌动蛋白共沉淀测定; F -肌动蛋白结合蛋白分析