在写这篇文章很有帮助输入Moens实验室的成员。香港是由美国国立卫生研究院/ NICHD的授予＃5R01HD037909 - 08支持的博士后研究员。煤层气是与霍华德休斯医学研究所研究员。

在囊胚或原肠胚阶段有针对性的嵌合体斑马鱼的胚胎移植产生由的一步一步的指导。 使用到复杂的发育过程中获得的洞察力最有力的工具之一是嵌合体胚胎的分析。一种幻想被定义为一个有机体，它包含从一个以上的动物细胞;马赛克是一个从多个基因型的细胞混合，通常是野生型和突变型嵌合型。在斑马鱼中，嵌合体可以随时到主机胚胎细胞在适当的萌芽阶段，从捐赠者胚胎移植。注射到一个细胞阶段的胚胎，一个宗族标记，如荧光染料标记的供体细胞的生成。删除标记的供体细胞胚胎从供体和引入到未标记的主机使用胚胎石油控制的玻璃吸管，装在一个化合物或解剖显微镜。供体细胞可以在某些情况下，一个特定的地区或发展囊胚或原肠胚阶段主机胚胎组织有针对性地选择根据既定的命运地图的主机胚胎移植部位。 第1部分：1 - 细胞期的斑马鱼胚胎注射。 <ol><li> 1 -细胞期胚胎的酶法dechorionation 。 以蛋白质dechorionate胚胎，在1 - 细胞阶段孵育〜1-5在0.5毫克/毫升的链霉蛋白酶的解决方案，如在斑马鱼的图书1中所述“。 Moniter dechorionation紧密合作，并淹没胚胎在胚胎中期（EM）用钢笔/链球菌1尽快chorions开始明显塌陷。一旦从他们chorions发布的，囊胚和原肠期胚胎是脆弱的，将坚持以塑料或​​瓦解，如果暴露在空气中。因此保持dechorionated胚胎在PEN /链球菌的EM淹没，菜之间的传输，使用大口径的火抛光的玻璃吸管，并保持在任琼脂涂层的塑料餐具（1.2％笔/链球菌的EM琼脂糖）或高压灭菌的玻璃培养皿。 </li><li> 注射dechorionated胚胎谱系标记。 准备注射盘插入一个45度角，整个培养皿四分之三的1.2％琼脂糖笔/链球菌的EM最宽的部分玻片。去除幻灯片琼脂糖凝固后留下一个斜面槽。 dechorionated胚胎转移到了充满笔/链球菌的EM注射菜槽。注入1nl卷宗族标记，使用精确的校准消防拉玻璃注射针和高压喷射钻井，在斑马鱼图书1所述。直接注入​​到dechorionated胚胎之间的1蛋黄染料和低分子量血统示踪剂 - 和4 - 细胞阶段。 3％溶液的荧光葡聚糖是足以让捐助源性细胞在嵌合胚胎通过数天的发展检测。当宗族标记是一个基因编码一种荧光蛋白（例如，GFP或RFP），直接注入到早期的1 - 细胞阶段的胚胎细胞。 </li><li> 注射非dechorionated胚胎谱系标记。 准备围绕井模具大致相同的宽度为绒毛膜直径1.2％琼脂糖巩固在EM多井注入菜肴。非dechorionated胚胎转移到一个半满的，用钢笔/链球菌的EM的多井注入菜。删除多余的液体。因此，固定化，胚胎可以宗族标记1nl卷如上所述注入。 </li><li> 提高移植注射胚胎。 提高低密度（每盘40-50胚胎）的胚胎，新鲜笔/链球菌的EM。第一阶段比赛的捐助者和东道国相当密切用于移植的胚胎。对于盾构阶段的移植手术，捐助者的目的，稍微落后的主机;需要注意的是注射的胚胎往往是稍微推迟。在不同温度下，25 ° C，28 ° C，29 ° C培养的胚胎，以错开它们的发展，最大限度地移植可以执行的时间窗口。 </li></ol>第2部分：移植嵌合体的斑马鱼胚胎。 <ol style="list-style-type:none;"><li> 在囊胚阶段移植的体视显微镜 </li></ol><ol style="list-style-type:upper-alpha;"><li> 囊胚移植钻机介绍 在斑马鱼囊胚细胞移植中使用的设备包括一微米的驱动器控制的汉密尔顿注射器连接三路阀通过软管长度的矿物油的水库和一个微量持有人（10升50升） 。组装后的移植钻机，它是充满了矿物油，照顾，以消除系统中的所有气泡。气泡的存在会产生负面影响的能力来控制次发送吸力和压力。使用体视显微镜具有相当高的放大倍率和良好的光学（最好至少80倍的放大倍率）。微量持有者和针定位可以控制由Narishige手册显微注射;但是，如果基地的立体太宽的显微操纵器，允许方便地访问，然后一个适配器，连接到身体体视显微镜也可以从Narishige购买。的显微操纵器的安装必须非常稳定，以避免振动传输移植针;一个磁性底座以及用于此目的的作品。 </li><li> 准备移植吸管 移植针是由玻璃毛细管移液器（见试剂表中的两个选项）被吸引到一个电极拉马上的一个温柔的锥度。打破针关闭的提示，在解剖显微镜下用针的内径稍大，比细胞移植的地步直边razorblade。保持在一个很小的角度，以创建一个斜面，并尽可能顺利地打破razorblade，无锯齿状边缘的轮廓。对于囊胚阶段移植针的外径应约50-60毫米。 </li><li> 在琼脂模具安装移植胚胎 准备浮排在培养皿是半满的熔融1.2％琼脂糖在EM的楔形突起的塑料模具，注塑菜。一旦琼脂糖凝固后，拆除模板，留下一个包含成三角形每个大到足以容纳一个胚胎形井行的琼脂模具。填写用钢笔/链球菌的EM移植模具和囊胚期胚胎分别加载到每口井用火抛光的玻璃吸管，使捐赠者的胚胎都放在一列主机的胚胎被放在相邻列。 </li><li> 移植细胞 胚胎在其两侧移植井的位置;重新定位，在移植过程中需要移植的吸管，小心不要刺破蛋黄。位置在舞台上的菜，所以，当移植针进入胚胎，针推针对其良好的后墙的胚胎。使用显微操作，降低入菜移植针，在一个相当陡峭的角度。理想情况下，移植针应在大约45度角进入胚胎。一旦针尖下面的胚胎培养基的表面，绘制成针扭微米驱动器控制汉密尔顿注射器少量的胚胎培养基。最精确的控制，矿物油和胚胎介质之间的接口应保持在薄，针锥部分，但不能太接近了尾声。用针轻轻的位置捐赠者的胚胎，然后绘制退针，并在所需的位置进入囊胚期胚胎的第。迅速，重创将渗透而造成它推出的胚胎。进针供体细胞，缓慢而仔细地画。如果细胞是太快了，他们很可能会剪切。同时避免进针蛋黄，因为它会绑定到的矿物油，然后将杀死细胞。后所需的细胞数，反向压力略有停止吸痰，并从胚胎中删除针。由胚胎移植盘移动主机带入位置。小的调整主机胚胎的位置，可轻轻转动它使用的移植针，只要小心是采取不触摸蛋黄。当驱逐到宿主胚胎的供体细胞，避免引进大量的胚胎中或任何矿物油，因为这可能会干扰发展或杀胚胎。沿细胞动物植物轴的位置会影响他们的贡献三个胚层，内胚层，中胚层，外胚层2,3之一。因此，细胞移植接近保证金的原肠开始之前，将给予优先上升到内胚层和中胚层，同时向动物极细胞移植将有助于外胚层命运，最常用的表面外胚层，前脑和眼睛。因此，可以完成组织目标的程度，即使在囊胚阶段。已完成后的细胞转移，转让捐助主机对24孔板琼脂涂井的进一步发展。 </li></ol><ol style="list-style-type:none;"><li> 在原肠胚阶段移植的复式显微镜 </li></ol><ol style="list-style-type:upper-alpha;"><li> 说明移植钻机 从针位置的精确控制提供了一个3轴的油压复合显微镜受益细胞转移显微操纵器。这显微移植吸管控制的精细动作，而在吸管吸微米驱动汉密尔顿注射器相似囊胚移植使用的控制。与固定阶段的直立复合式显微镜是可取的，执行自聚焦显微镜细胞转移不会导致胚胎相对移动吸管。然而，如果必须使用一个可调阶段的复式显微镜，那么显微可以被安装到舞台上，而不是显微镜体具有同等效力。适配器提供的显微操纵器安装的选项，无论是从一个制造商的各种复合显微镜的阶段或身体，也可从Narishige。 </li><li> 准备移植吸管 移植针基本上如上所述;胚囊阶段移植的理想针孔径为30-40毫米。盾形成后，包围层（EVL）变得更加贴壁，使其难以渗透到胚胎移植针。为了方便针条目，拉使用microforge针倒钩。第一，顺利把它关闭灯丝的microforge针尖，然后打开针斜角的领先优势，可以使灯丝接触。一旦针接触的灯丝的领先优势，迅速拉了创建一个倒钩，或鱼叉。为了避免损坏细胞，倒刺应直，针尖不应曲线 </li><li> 安装用于移植的胚胎中的甲基纤维素 复式显微镜移植，安装上在3％的甲基纤维素（Sigma公司）溶解在胚胎中期的解决方案的抑郁症幻灯片的胚胎。首先，涂抹在玻璃抑郁症的幻灯片，然后大方量与胚胎中期的洪水抑郁甲基纤维素的竖条纹。使用火抛光的吸管，转让的EM表面上的甲基端的抑郁症幻灯片下一个捐助者和三个盾级主机。选择比年轻的主机（30-50％外包）的捐赠胚胎。粘合到毛细管的长度短了2磅测试渔线两端的一个小环，轻轻滚动捐助者和东道国的胚胎到甲基地带的顶部，并塞进甲基直到安全。如果移植的细胞是针对一个特定的领域，盾构阶段命运的知识地图3,4和盾靶组织的相对位置，使主机胚胎可以正确定位是至关重要的。作为东道主的胚胎放在甲基，轧辊的位置，以便有针对性的区域是最重要的，因为在移植过程中针将进入胚胎切向提供供体细胞，而不破坏蛋黄。 </li><li> 移植细胞 位置移动到焦平面的胚胎移植针。首先着眼于捐助者使用10倍的目标胚胎。然后将胚胎到一边，并没有调整焦平面，使用的显微操纵器的控制重点，使移植针的尖端。位置在一个很浅的角度的微量持有和针，使针头接近水平，将允许在幻灯片上的凹陷边缘。如果胚胎是正确安装在甲基纤维素，它不会推出移植针进入，除非胚胎是太旧，使针容易渗透。包围层似乎锻炼胚胎的年龄，因此，原肠胚阶段移植是最好的盾形成后执行。圆顶阶段后，蛋黄只是驻留在细胞的胚盘帽，逐步外包的收益变薄。为了避免针头刺破蛋黄，应该进入一个焦平面，只是稍微比的EVL更深的胚胎。删除从捐赠者的胚胎细胞的大量时，左右移动细胞采取了针，为了避免意外地吸吮进针蛋黄。如果从一个捐赠者的胚胎细胞被移植到多台主机，最好是只有一次进入针捐助，以尽量减少损害的可能性。供体细胞，然后转移到所需的位置，在长达三个主机胚胎。当一台主机胚胎转移到两个或两个以上的捐助胚胎细胞，它是最好相同移植针之前，将它们移植到一台主机的胚胎，要考虑到每一个捐赠者的胚胎细胞。这可以最大限度地减少损坏的主机胚胎，并确保这些细胞被转移到同一区域的胚胎，使他们的行为，可以进行比较。很少混合OC内供体细胞移植针之间的小人，因此，细胞应转移到一台主机，当超过一个捐助者。为了提高供体细胞的可能性，将有助于所需的组织，驱逐针通过有针对性的区域，而不是存放在一个丛细胞的供体细胞。一旦细胞转让完成后，放入培养皿和洪水整个幻灯片，仔细地用钢笔/链球菌的EM。在接下来的几个小时，甲基纤维素溶解，释放出的胚胎。 </li></ol><img src="/files/ftp_upload/1394/fig1.jpg" alt="图1" /> 图1：显微镜设置嵌合胚胎。答：在解剖显微镜移植钻机由充油汉密尔顿一微米的驱动器（一）连接3路阀（D）充油水库（b）和移植移液器（C）注射器。移植吸管上安装了一个粗的显微操纵器（E），这是通过磁英尺（F）金属底板（未显示）。移植手术配备底部照明的最佳光学立体显微镜（G）的阶段。乙：一个复式显微镜移植钻机由一个类似的充油的汉密尔顿注射器和微米的驱动器（A，B），但在这种情况下，移植吸管吸管持有人（C），X，Y和Z运动可以控制安装无论是一个粗糙的（d）或罚款（E）显微。在这个例子中的显微操纵器上安装一个固定的阶段显微镜身体，但它也有可能安装阶段的常规显微镜。胚胎，这是抑郁症幻灯片上的甲基固定，可视化，使用10倍的目标（F）。 ：为固定体视显微镜移植的胚胎移植模具。 Dechorionated胚胎单独成井在90毫米培养皿模具铸造成琼脂糖下降。井的尺寸显示。 <img src="/files/ftp_upload/1394/fig2.jpg" alt="图2" /> 图2：安装原肠胚期移植胚胎。答：理想的形状，一个原肠胚移植吸管。一个用锋利的尖斜面艾滋病穿透胚胎。乙：在甲基纤维素固定的捐助者和东道国胚胎。抑郁症幻灯片以及一个甲基地带放下，充斥着一个胚胎培养基中的慷慨。胚胎被添加到胚胎介质，并使用一个小环的甲基纤维素（三）卷上。 DG扩大胚胎图。 1B。 D：捐助胚胎为导向，让最简单的吸管访问，因为在这个阶段的细胞仍然未提交。例如：盾阶段主机胚胎导向，使目标区域是最重要的。将有助于从左边（E）和右（七）双方或向腹侧后脑和脊髓（F）的派生背后脑和颅神经嵴细胞移植到盒装地区。 <img src="/files/ftp_upload/1394/fig3.jpg" alt="图3" /> 图3：嵌合胚胎的代表图像。 （AC）18hpf嵌合胚胎移植产生的背视图显示的屏蔽阶段，左前方。 （A，B）排序分析嵌合体胚胎中暴露出来的细胞表面受体EphA4的功能。左侧面板：罗丹明标记（rhod.）供体细胞（红色）和转基因绿色荧光蛋白表达在特定的后脑部分（绿色）的合并。 （一）野生型细胞有助于控制嵌合胚胎的整个后脑。 （二）EphA4耗尽的供体细胞的特定部分排除在一个WT主机的后脑。 （三）针对不同部位的神经管在盾构阶段移植的供体细胞。供体细胞（红色）有针对性的前脑/脑（左侧面板），或一个WT主机EfnB2a抗体，标志着前脑，中，后脑边界和中段反染色后脑/脊髓（右图）后脑（绿色）。比例尺：50μm的。

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	<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> The Zebrafish Book. , (2000).</li><li>Kimmel, C. B., Warga, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lineage+and+developmental+potential+of+cells+in+the+zebrafish+embryo.">Cell lineage and developmental potential of cells in the zebrafish embryo.</a> Trends Genet. 4, 68-74 (1988).</li><li>Kimmel, C. B., Warga, R. M., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+organization+of+the+zebrafish+fate+map.">Origin and organization of the zebrafish fate map.</a> Development. 108, 581-594 (1990).</li><li>Woo, K., 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=Order+and+coherence+in+the+fate+map+of+the+zebrafish+nervous+system.">Order and coherence in the fate map of the zebrafish nervous system.</a> Development. 121, 2595-2609 (1995).</li><li>Raz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primordial+germ-cell+development:+the+zebrafish+perspective.">Primordial germ-cell development: the zebrafish perspective.</a> Nat Rev Genet. 4, 690-700 (2003).</li><li>Yoon, C., Kawakami, K., Hopkins, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+vasa+homologue+RNA+is+localized+to+the+cleavage+planes+of+2-+and+4-cell-stage+embryos+and+is+expressed+in+the+primordial+germ+cells.">Zebrafish vasa homologue RNA is localized to the cleavage planes of 2- and 4-cell-stage embryos and is expressed in the primordial germ cells.</a> Development. 124, 3157-3165 (1997).</li><li>Koprunner, M., Thisse, C., Thisse, B., Raz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+zebrafish+nanos-related+gene+is+essential+for+the+development+of+primordial+germ+cells.">A zebrafish nanos-related gene is essential for the development of primordial germ cells.</a> Genes Dev. 15 (21), 2877-2885 (2001).</li><li>Weidinger, G., Wolke, U., Koprunner, M., Klinger, M., Raz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+tissues+and+patterning+events+required+for+distinct+steps+in+early+migration+of+zebrafish+primordial+germ+cells.">Identification of tissues and patterning events required for distinct steps in early migration of zebrafish primordial germ cells.</a> Development. 126 (23), 5295-5307 (1999).</li><li>Weidinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+zebrafish+primordial+germ+cell+migration+by+attraction+towards+an+intermediate+target.">Regulation of zebrafish primordial germ cell migration by attraction towards an intermediate target.</a> Development. 129 (1), 25-36 (2002).</li><li>Ciruna, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+maternal-zygotic+mutant+zebrafish+by+germ-line+replacement.">Production of maternal-zygotic mutant zebrafish by germ-line replacement.</a> Proc Natl Acad Sci U S A. 99, 14919-14924 (2002).</li><li>Waskiewicz, A. J., Rikhof, H. A., Moens, 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=Eliminating+zebrafish+pbx+proteins+reveals+a+hindbrain+ground+state.">Eliminating zebrafish pbx proteins reveals a hindbrain ground state.</a> Dev Cell. 3, 723-733 (2002).</li><li>Weidinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dead+end,+a+novel+vertebrate+germ+plasm+component,+is+required+for+zebrafish+primordial+germ+cell+migration+and+survival.">dead end, a novel vertebrate germ plasm component, is required for zebrafish primordial germ cell migration and survival.</a> Curr Biol. 13, 1429-1434 (2003).</li><li>Blaser, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transition+from+non-motile+behaviour+to+directed+migration+during+early+PGC+development+in+zebrafish.">Transition from non-motile behaviour to directed migration during early PGC development in zebrafish.</a> J Cell Sci. 118, 4027-4038 (2005).</li></ol>

与移植可用于生产有针对性的嵌合体的缓解是斑马鱼为脊椎动物模式的大国之一。上面没有说明这个协议的修改，允许母体基因的功能分析与合子发展中的重要作用的基因。由于这些突变的鱼无法生存，直到成年后，有必要转移到其他野生型主机胚胎生殖细胞突变，突变的细胞的“生殖克隆”。生成生殖马赛克涉及移植的原始野生型主机胚胎生殖细胞突变的捐赠。与所有其他在胚胎细胞谱系，生?...

试剂：许多这些程序所用试剂配方，发现在斑马鱼的书，这是在完整<a href="http://zfin.org/zf_info/zfbook/zfbk.html">的</a>在线http://zfin.org/zf_info/zfbook/zfbk.html。食谱提供有准备的鱼水，胚胎中等，不含抗生素，链霉蛋白酶dechorionating，荧光右旋糖酐，甲基纤维素。

generating chimeric zebrafish embryos by transplantation

使用到复杂的发育过程中获得的洞察力最有力的工具之一是嵌合体胚胎的分析。一种幻想被定义为一个有机体，它包含从一个以上的动物细胞;马赛克是一个从多个基因型的细胞混合，通常是野生型和突变型嵌合型。在斑马鱼中，嵌合体可以随时到主机胚胎细胞在适当的萌芽阶段，从捐赠者胚胎移植。注射到一个细胞阶段的胚胎，一个宗族标记，如荧光染料标记的供体细胞的生成。删除标记的供体细胞胚胎从供体和引入到未标记的主机使用胚胎石油控制的玻璃吸管，装在一个化合物或解剖显微镜。供体细胞可以在某些情况下，一个特定的地区或发展囊胚或原肠胚阶段主机胚胎组织有针对性地选择根据既定的命运地图的主机胚胎移植部位。

Watch this Scientific Journal Video about Generating Chimeric Zebrafish Embryos by Transplantation at JoVE.com

Generating Chimeric Zebrafish Embryos by Transplantation

在囊胚或原肠胚阶段有针对性的嵌合体斑马鱼的胚胎移植产生由的一步一步的指导。

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 ...

generating-chimeric-zebrafish-embryos-by-transplantation

Research

JoVE Journal

Biology

21.1K 次观看.  Fred Hutchinson Cancer Research Center - FHCRC. 在囊胚或原肠胚阶段有针对性的嵌合体斑马鱼的胚胎移植产生由的一步一步的指导。

视频: Generating Chimeric Zebrafish Embryos by 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

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.

Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.

显微注射mRNA和斑马鱼的胚胎吗啉反义寡核苷酸。

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression ...

Microinjection of Zebrafish Embryos to Analyze Gene Function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

斑马鱼的胚胎显微注射来分析基因的功能

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

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 ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

在现场的斑马鱼胚胎的基于双光子Photoactivati​​on

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

移植绿色荧光蛋白表达对视网膜和大脑发育的实时成像嵌合斑马鱼胚胎的卵裂球

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

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 ...

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharides1, 2. The modified monosaccharides, processed by the glycan biosynthetic machinery, are incorporated into cell surface glycoconjugates. The bioorthogonal azide or alkyne tags then allow covalent conjugation with fluorescent probes for visualization, or with affinity probes for enrichment and glycoproteomic analysis. This protocol describes the procedures typically used for noninvasive imaging of fucosylated glycans in zebrafish embryos, including: 1) microinjection of one-cell stage embryos with GDP-5-alkynylfucose (GDP-FucAl), 2) labeling fucosylated glycans in the enveloping layer of zebrafish embryos with azide-conjugated fluorophores via biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and 3) imaging by confocal microscopy3. The method described here can be readily extended to visualize other classes of glycans, e.g. glycans containing sialic acid4 and N-acetylgalactosamine5, 6, in developing zebrafish and in other living organisms.

A click-chemistry based method that allows for the rapid, noninvasive, and robust labeling of alkyne-tagged glycans in zebrafish embryos is described. Fucosylated glycans in the enveloping layer of zebrafish embryos in the late gastrulation stage were imaged in this study.

在斑马鱼胚胎的代谢标记和Bioorthogonal点击化学成像聚糖

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or ...

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases1. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease2. Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells3. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.

Cell transplantation is an essential technique for studying tissue regeneration and for developing cell-based therapies of disease. We demonstrate here a microsurgical technique that permits the transplantation of genetically labeled cells directly into the kidney of adult zebrafish fish.

直接进入成年斑马鱼的肾细胞移植

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

斑马鱼胚胎的代谢谱分析

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...