这项工作是由儿科得克萨斯麦戈文医学院的大学卫生署授予NIDDK（K01DK092320）和启动资金的国家机构提供支持。

下面的协议已获得得克萨斯大学健康科学中心在休斯顿的中心实验室动物医学动物福利委员会，作为机构护理和使用委员会大学（协议＃:HSC-AWC-13-135）。 1.识别和选择卵裂球的肾靶向注射<ol><li>之前产生的胚胎，使用爪蟾发展21的正常表理解在胚胎早期的细胞分裂的方向。或者，在Xenbase 11 爪蟾的早期发育阶段的访问的图。 </li><li>访问Xenbase 11交互非洲爪蟾细胞命运的地图来选择卵裂球将针对显微注射。 </li><li>观察到单细胞胚胎拥有一个黑暗色素动物极和植物极，这是白色的，yolky。需要注意的是一层保护膜，称为卵黄ênvelope，涵盖了胚胎。 </li><li>注意，第一次卵裂通常胚胎的左侧和右侧之间发生。这些细胞同样有助于pronephric血统。 </li><li>注意，第二切割划分背侧和胚胎的腹侧半部，导致4细胞胚胎。背细胞体积较小，具有比腹侧细胞（ 图2A和图3A）颜料更少。 <ol><li>识别腹侧卵裂球（Ⅴ;大，暗细胞）上的左，右两侧，从而比背贡献更大显影肾（D;小，重量轻的细胞）分裂球（ 图2A）。 </li><li>如果注入到4细胞胚胎，注入左腹侧卵裂球定位的左肾（ 图3A和第3节）。 </li></ol></li><li>第三切割平分动物和植物性侧，导致8细胞胚胎。在这一点上，有四个动物的卵裂球[左和右腹侧（V1）和背侧（D 1）]和四植物性卵裂球[左和右腹侧（V2）和背侧（D 2）]（ 图2B和图3B）。 <ol><li>找到腹，植物性卵裂球（V2）。这些卵裂球有助于更向显影肾脏在此阶段（ 图2B）的任何其他细胞。要定位一个8细胞胚胎的左肾，注入左V2卵裂球（ 图3B和第3节）。 </li></ol></li><li>请注意，在第四和第五分裂平分动物和植物的卵裂球。两个子代从每个卵裂球生成，从而导致16细胞胚胎。细胞的前身命名。例如，从8细胞期的V2的卵裂球产生至V2.1和V2.2后代在16细胞阶段（ 图2C）。在16细胞期的V2.2细胞提供大部分贡献给显影肾细胞。 </li><li>请注意，第六和第七分裂导致32细胞安莉芳O操作。再次，二子代从每个卵裂球，它被命名为以下其前身生成。例如，从16细胞阶段的V2.2卵裂球引起V2.2.1和V2.2.2在32细胞阶段。有一个在其中细胞在四行中的A，B，C和D（从动物到植物）所确定的32个细胞阶段的替代的命名系统，并且在四列为1，2，3和4（从背部到腹部）。因此，V2.2.2卵裂球，这最有助于显影前肾，是这种替代命名系统（ 图2D）下称为C3。 </li></ol> 2.胚胎的制备<ol><li>制备50毫升Dejelly溶液（2％半胱氨酸，氢氧化钠至pH 8.0）。 </li><li>根据标准方法14从一个单一的男性青蛙隔离两个睾丸。放置在睾丸60毫米培养皿充满10毫升睾丸存储解决方案（1个马克的修改林格氏[MMR; 0.1M的NaCl，2毫米氯化钾，1毫米硫酸镁4，2毫氯化钙 ，5mM的HEPES pH为8，0.1毫摩尔EDTA] 12，1％牛血清白蛋白，50μg/ ml的庆大霉素）。储存在4℃的睾丸。 注意:睾丸可存放约7 - 在4℃下10天，但受精效率将降低睾丸已存储的时间越长。 </li><li>挤雌蛙根据标准方法14获得的蛋。收集鸡蛋100毫米的培养皿。倒出多余的水分。 </li><li>切断¼睾丸，而它在睾丸存储解决方案使用镊子和刀片。这块睾丸转移到含有鸡蛋培养皿。调整用于帐户睾丸，睾丸已多久被存储的大小睾丸部分的尺寸，以及有多少蛋受精。一般采用¼到新鲜解剖睾丸1/3受精鸡蛋一个离合器。 </li><li>切睾丸部分为使用镊子和刀片小块。补充足够的0.3X MMR+ 30毫克/毫升庆大霉素至培养皿以覆盖鸡蛋。摇动MMR在菜混合。 </li><li>等待大约30分钟受精发生在室温下。注意，动物半球（胚胎的着色侧）将坐在后有效受精胚胎的顶部。然后，从使用一个移液管培养皿取出MMR。足够Dejelly解决方案添加到菜覆盖胚胎。 </li><li>在接下来的几分钟里，轻轻间歇漩涡的菜。此时的菜剧烈振荡会引起轴的缺陷。 &#160;在胚胎的透明带会溶解，胚胎会在纷飞的菜的中心聚集。一旦胚胎在培养皿中心密切相互接触，除去Dejelly解决方案与移液管。不离开在Dejelly解胚胎超过5分钟，或胚胎可能被损坏。 </li><li>洗dejellied胚胎3 - 在0.3X 5倍MMR + 30毫克/毫升的庆大霉素通过仔细倾倒或移液关闭MMR和填充新的MMR的菜。不要从菜中删除所有的MMR，或者胚胎可能会被损坏。 </li><li>使用移液管培养皿中删除任何未受精的卵子或睾丸片。 </li><li>孵育14和22℃之间的胚胎。 注意:在较低的温度下生长的胚胎发育比在较高温度下生长的胚胎更慢。发育阶段的时间可在22 Xenbase中找到。 <ol><li>空间他们的发展，代替半从在培养皿中的单个受精胚胎保持在14℃，在培养皿中的胚胎的另一半保持在18℃。这允许两套注射到4单元或8细胞胚胎从单个受精。 </li></ol></li></ol> 3.注射液和胚胎显微注射的制备<ol><li>制备含0.01注射液纳克/ NL膜结合红色荧光蛋白（MEM-RFP）的mRNA 9而将胚显影的4单元或8细胞期。储存在冰注射液，直到准备注入。 </li><li>加载一个7"置换玻璃毛细管成针拔出器，具有与针拔出器壳体的顶部对齐更换管的顶部。热＃2值设置为800，并且上拉值650按下"拉"键拉出针。这将从一个单一的7创建2针"玻璃毛细管。 </li><li>剪掉一个拉动针的前端有一对杜蒙钳的。 注:拉针后，尖端密封关闭，必须切开。到它被切断的针的点越近，直径针尖将是较小的。虽然尖端的直径不会影响注射体积与这里使用的显微注射系统，具有较大直径的前端更容易损坏胚胎。 </li><li>滑并购icropipette夹头到针的背面。接着，滑移大孔O形圈到针的套爪的后面的后面。 </li><li>使用矿物油27号皮下注射针头，小心不要让中针气泡填充针。 </li><li>滑针到微量的柱塞，休息针头插入大孔安装在柱塞上的白色塑料垫片。柱塞应该具有最接近于微量的主体，其次是白间隔物，大孔的O形环，和夹头一个小O形圈。通过拧紧套爪固定针头。轻轻拉动针确保其正确固定。 </li><li>按住了微量控制箱"空"按钮，直到有两声哔声。 </li><li>吸取3微升注射液上一块封口膜。插入针的尖端插入的上封口膜注射溶液的珠。按住"补"上的按钮microinjector控制箱吸取注射液进针。 </li><li>填写60毫米的培养皿中与500微米聚酯内衬5％聚蔗糖网在0.3X孕产妇死亡率+ 30毫克/毫升的庆大霉素。仔细移液器4月20日至30日细胞或8细胞胚胎放入盘中。 </li><li>使用毛发回路14，操纵使要注入朝向针的卵裂球的胚胎。定位的左肾，排队的胚胎，使4-细胞胚或8细胞胚胎的左V2卵裂球左侧腹侧卵裂球面对针。 </li><li>注入的注射溶液10 NL入在盘每个胚的选定卵裂球。 注意:在培养皿底部的网眼稳定的胚胎，并防止它们从轧制，使他们能够在不使用毛发循环的稳定的喷射。 </li><li>转印注射胚胎到培养板的孔已在0.3X MMR + 30毫克/毫升庆大霉素填充有5％聚蔗糖。孵育胚胎注入■在16℃下进行至少一小时，以使注入的卵裂球愈合。 </li><li>转移愈合胚胎成新的培养板的孔已经由阶段9（前原肠胚形成）填充有0.3×MMR + 30毫克/毫升庆大霉素。 </li><li>孵育胚胎在14 - 22°C，直到胚胎达到38级- 40 21。 </li></ol> 4.固定和胚胎的免疫染色<ol><li>制备50毫升硫酸盐MOPS / EGTA /镁/甲醛缓冲液[MEMFA:100毫MOPS（pH 7.4）中，2毫EGTA，1mM的MgSO 4上，3.7％（V / V）的甲醛。 </li><li>使用移液管，把10 - 20台38 - 40胚胎在玻璃小瓶。在100％乙醇加10微升的5％苯佐卡因到小瓶中并翻转小瓶混合。等待10分钟，以麻醉胚胎。 </li><li>使用玻璃吸管的小瓶中取出MMR。如果同时处理多个小瓶，所述小瓶能够直立在24孔细胞培养板保持。 </li><li>用玻璃管TTE，充满MEMFA小瓶。放置在三维摆动平台小瓶在室温下1小时。 </li><li>使用玻璃吸管小瓶取出MEMFA。填用100％甲醇的小瓶中。放置在三维摆动平台小瓶在室温下10分钟。重复此洗涤步骤一次，并在100％甲醇过夜胚胎储存在-20℃。 </li><li>制备1×磷酸盐缓冲盐水，牛血清白蛋白的Triton（PBT）:1×PBS中，2mg / ml的牛血清白蛋白，0.1％的Triton X-100。 </li><li>制备初级抗体溶液:1个树脂与用1 10％山羊血清:5稀释的小鼠单克隆的4A6抗体（标记的中间，远端和连接管20的膜），1:30稀释的小鼠单克隆抗体3G8的（向近端小管20的标签流明），和一个1:兔多克隆RFP抗体250稀释液（以标记MEM-RFP示踪剂）。保存在4℃。 </li><li>准备secondaRY抗体溶液:1个树脂与10％山羊血清，1:500的Alexa 488山羊抗小鼠IgG（原液浓度2毫克/毫升;将标签4A6和3G8），和1:500的Alexa 555的山羊抗 - 兔IgG（股票浓度为2毫克/毫升;以标记MEM-RFP示踪剂）。在4℃储存，覆盖管箔避光。 </li><li>可替代地，收集第一和第二抗体染色后，在4℃下保存用于以后的实验重用。如果抗体是要保存用于重复使用，添加0.01％叠氮化钠。 </li><li>采用免疫荧光染色协议成立18胚胎。 </li></ol> 5.可视化胚胎和针对性Pronephric组织分析<ol><li>筛选免疫染色的胚胎，以验证正确的卵裂球通过在1X荧光立体显微镜下观察示踪剂的荧光注射（以查看整个胚胎）和5X（查看肾）放大率。地方的胚胎与我们的多井玻璃板填充有使用1倍的PBT与尖端移液管LLS切断。操作用吹风循环胚胎。只使用具有共注射示踪剂存在于前肾对胚胎的左侧的胚（ 图3C，F和图4C中，F），用于基因过表达或敲低分析。 </li><li>通过将胚胎在玻璃小瓶中并填充Murray的清除小瓶:此外，清除Murray的清除胚胎（1份苄醇2份苯甲酸苄酯）。可视化用玻璃孔板胚胎。 注:穆雷的清除是一种有机溶剂，并应小心处理。戴上手套，并且只使用玻璃瓶中，并用吸管穆雷的清除。 </li><li>商店的胚胎在4℃在1×PBT为2 - 3周。对胚胎的长期储存，并在室温下洗涤​​在100％甲醇两次10分钟脱水胚胎。在100％甲醇-20℃保存的胚胎。 </li></ol>

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<li>Miller, R. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pronephric+tubulogenesis+requires+Daam1-mediated+planar+cell+polarity+signaling%5BTitle%5D)+AND+%22J.+Am.+Soc.+Nephrol%22%5BJournal%5D)">Pronephric tubulogenesis requires Daam1-mediated planar cell polarity signaling.</a> J. Am. Soc. Nephrol. 22, 1654-1667 (2011).</li>
<li>Vize, P. D., Jones, E. A., Pfister, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+the+Xenopus+pronephric+system%5BTitle%5D)+AND+%22Dev.+Biol%22%5BJournal%5D)">Development of the Xenopus pronephric system.</a> Dev. Biol. 171 (2), 531-540 (1995).</li>
<li>Miller, R. K., Lee, M., McCrea, P. D. Chapter 12: The Xenopus Pronephros: A Kidney Model Making Leaps toward Understanding Tubule Development. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aMiller%2C+RK+%22Xenopus+Development%22">Xenopus Development</a>. Kloc, M., Kubiak, J. Z. , Wiley and Sons. Oxford. (2014).</li>
<li>Taira, M., Otani, H., Jamrich, M., Dawid, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression+of+the+LIM+class+homeobox+gene+Xlim-1+in+pronephros+and+CNS+cell+lineages+of+Xenopus+embryos+is+affected+by+retinoic+acid+and+exogastrulation%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Expression of the LIM class homeobox gene Xlim-1 in pronephros and CNS cell lineages of Xenopus embryos is affected by retinoic acid and exogastrulation.</a> Development. 120, 1525-1536 (1994).</li>
<li>Weber, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+and+function+of+the+tissue-specific+transcription+factor+HNF1+alpha+%28LFB1%29+during+Xenopus+development%5BTitle%5D)+AND+%22Int.+Dev.+Biol%22%5BJournal%5D)">Regulation and function of the tissue-specific transcription factor HNF1 alpha (LFB1) during Xenopus development.</a> Int. Dev. Biol. 40 (1), 297-304 (1996).</li>
<li>Carroll, T. J., Vize, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synergism+between+Pax-8+and+lim-1+in+embryonic+kidney+development%5BTitle%5D)+AND+%22Dev.+Biol%22%5BJournal%5D)">Synergism between Pax-8 and lim-1 in embryonic kidney development.</a> Dev. Biol. 214 (1), 46-59 (1999).</li>
<li>Etkin, L. D., Pearman, B., Ansah-Yiadom, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Replication+of+injected+DNA+templates+in+Xenopus+embryos%5BTitle%5D)+AND+%22Exp.+Cell+Res%22%5BJournal%5D)">Replication of injected DNA templates in Xenopus embryos.</a> Exp. Cell Res. 169 (2), 468-477 (1987).</li>
<li>Vize, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+chloride+conductance+channel+ClC-K+is+a+specific+marker+for+the+Xenopus+pronephric+distal+tubule+and+duct%5BTitle%5D)+AND+%22Gene+Expr.+Patterns%22%5BJournal%5D)">The chloride conductance channel ClC-K is a specific marker for the Xenopus pronephric distal tubule and duct.</a> Gene Expr. Patterns. 3 (3), 347-350 (2003).</li>
<li>Uochi, T. S., Takahashi, S., Ninomiya, H., Fukui, A., Asashima, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Na%2B%2C+K%2B-ATPase+alpha+subunit+requires+gastrulation+in+the+Xenopus+embryo%5BTitle%5D)+AND+%22Dev.+Growth+Differ%22%5BJournal%5D)">The Na+, K+-ATPase alpha subunit requires gastrulation in the Xenopus embryo.</a> Dev. Growth Differ. 39 (5), 571-580 (1997).</li>
<li>Nieuwkoop, P. D., Faber, J. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aNieuwkoop%2C+PD+%22Normal+table+of+Xenopus+laevis+%28Daudin%29%3A+A+systematical+%26+chronological+survey+of+the+development+from+the+fertilized+egg+till+the+end+of+metamorphosis%22">Normal table of Xenopus laevis (Daudin): A systematical & chronological survey of the development from the fertilized egg till the end of metamorphosis</a>. , Garland Publishing, Inc. NY. (1994).</li>
<li>Ubbels, G. A., Hara, K., Koster, C. H., Kirschner, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evidence+for+a+functional+role+of+the+cytoskeleton+in+determination+of+the+dorsoventral+axis+in+Xenopus+laevis+eggs%5BTitle%5D)+AND+%22J.+Embryol.+Exp.+Morphol%22%5BJournal%5D)">Evidence for a functional role of the cytoskeleton in determination of the dorsoventral axis in Xenopus laevis eggs.</a> J. Embryol. Exp. Morphol. 77, 15-37 (1983).</li>
<li>Huang, S., Johnson, K. E., Wang, H. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Blastomeres+show+differential+fate+changes+in+8-cell+Xenopus+laevis+embryos+that+are+rotated+90+degrees+before+the+first+cleavage%5BTitle%5D)+AND+%22Dev.+Growth+Differ%22%5BJournal%5D)">Blastomeres show differential fate changes in 8-cell Xenopus laevis embryos that are rotated 90 degrees before the first cleavage.</a> Dev. Growth Differ. 40 (2), 189-198 (1998).</li>
<li>Colman, A., Drummond, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+stability+and+movement+of+mRNA+in+Xenopus+oocytes+and+embryos%5BTitle%5D)+AND+%22J.+Embryol.+Exp.+Morph%22%5BJournal%5D)">The stability and movement of mRNA in Xenopus oocytes and embryos.</a> J. Embryol. Exp. Morph. 97, 197-209 (1986).</li>
</ol>

The authors declare that they have no competing financial interests.

针对发展中爪蟾胚胎的前肾依赖于识别和注入正确的卵裂球。 8细胞胚胎的卵裂球V2的注射靶向左侧前肾18。这留下对侧右前肾作为内部对照。如果啉拦截或RNA表达用于改变肾发展，对侧右前肾可用于基因敲除或过表达的影响进行比较的左前肾。在这种情况下，如不匹配的控制啉或显性负RNA构建适当的控制应除了对侧内部控制用于分析基因表达的变化的结果。由于爪蟾胚胎的?...

非洲爪蟾胚胎肾中，前肾，是研究肾脏发育和疾病的良好模式。胚胎外部发展，在尺寸是大的，可以大量生产，并通过微注射或外科手术容易操纵。此外，治在哺乳动物和两栖动物肾脏发育的基因是保守的。哺乳动物的肾脏通过三个阶段进行:在头肾，中肾，后肾和1，而两栖类胚胎有前肾和成人两栖动物有后肾。这些肾脏形式的基本滤波单元是肾，和哺乳动物和两栖类所需要的相同的信号级联和电感事件接受nephrogenesis 2，3， 爪蟾前肾包含近端，中间，远端和连接管组成的单肾，和血管球（类似于哺乳动物肾小球）1，4-6（ 图1 </str翁&gt;）。大存在于非洲爪蟾前肾单肾使得它适合作为用于参与肾发育和疾病过程的基因的研究中一个简单的模型。 细胞命运的地图已经建立了早期的爪蟾胚胎，并且是免费提供的Xenbase 7-11在线。在这里，我们描述了用于谱系示踪剂定位的显影爪前肾的显微注射的技术，虽然同样的技术可以适用于针对其它组织，如心脏或眼睛。谱系示踪剂是标签（包括活体染料，荧光标记的葡聚糖，组织化学可检测的酶，和mRNA编码荧光蛋白），可以注射到早期卵裂球，使该细胞的后代的发育过程中的可视化。这个协议利用MEM-RFP的mRNA，编码膜靶向红色荧光蛋白12，作为一个谱系示踪剂。有针对性的显微注射技术在这里描述的4-和8细胞胚胎niques单个卵裂球可以用于注射吗啉击倒的基因表达，或与外源RNA过表达感兴趣的基因。通过注入腹侧，植物卵裂球，主要是胚胎的前肾将有针对性，留下对侧前肾作为发育控制。示踪剂联合注射验证正确的卵裂球注射，并显示该组织中的胚胎从卵裂球注入出现，验证了前肾定位。在头肾的免疫染色允许有多好pronephric肾小管已经有针对性的可视化。过度表达和敲低效果然后可以对胚，其用作发育控制的对侧得分，并且可以用于计算pronephric索引13。细胞命运地图的可用性允许使用这种有针对性的显微注射技术对靶组织OTH尔比前肾，和荧光示踪剂的共注射允许有针对性的显微注射到每个组织分析前进行验证。 期间胚胎显微注射，发育温度应严格调节，因为爪蟾发展的速率在 ​​很大程度上取决于它14。 （ - 16°C 14）为4和8细胞注射，因为开发时间减慢的胚胎应该稍凉培养。在22℃，从第1阶段（1细胞）的开发时间到阶段3（4细胞）是约2小时，同时在16℃下的开发时间到阶段3是约4小时。这需要大约15分钟从4细胞的胚胎去8芯（4级），胚胎在22°C，但在16℃，大约需要30分钟。同样地，在22℃，只需要30分钟的8细胞胚胎进步到16细胞胚胎（阶段5）。这个时间是在16℃增加至45分钟。该refore，是有用的减缓胚胎的发育率，以使有足够的时间在8细胞期注射前胚胎进步到16单元的阶段。此外，生长温度可以调节，以加快或减慢胚胎发育，直至肾脏已发育完全。 蝌蚪级爪蟾胚胎的表皮是相对透明的，允许未经组织15的清扫或清除显影前肾的容易成像。由于爪蟾胚胎的相对透明度，活细胞成像也是可行16,17。整个安装免疫染色以可视化的前肾可能带有该标签的近端，中间，远端和舞台的连接管38建立抗体- 40胚胎允许pronephric发展评价靶基因表达的操作后爪蟾胚胎18-20。

<table><tbody><tr><td>Sodium chloride</td><td>Fisher</td><td>S271-3</td><td></td></tr><tr><td>Potassium chloride</td><td>Fisher</td><td>P217-500</td><td></td></tr><tr><td>Magnesium sulfate&amp;nbsp;</td><td>Fisher</td><td>M63-500</td><td></td></tr><tr><td>Calcium chloride</td><td>Fisher</td><td>C79-500</td><td></td></tr><tr><td>HEPES</td><td>Fisher</td><td>BP310-500</td><td></td></tr><tr><td>EDTA</td><td>Fisher</td><td>S311-500</td><td></td></tr><tr><td>Gentamycin solution, 50 mg/ml</td><td>Amresco</td><td>E737-20ML</td><td></td></tr><tr><td>L-Cysteine, 99%+</td><td>Acros Organics</td><td>52-90-4</td><td></td></tr><tr><td>Ficoll</td><td>Fisher</td><td>BP525-100</td><td></td></tr><tr><td>100 mm x 15 mm Petri dish</td><td>Fisher</td><td>FB0875712</td><td></td></tr><tr><td>Mini Fridge II</td><td>Boekel</td><td>260009</td><td>Benchtop incubator for embryos.</td></tr><tr><td>Gap43-RFP plasmid</td><td></td><td></td><td>For making tracer RNA. Ref: Davidson &lt;em&gt;et al.&lt;/em&gt;, 2006.</td></tr><tr><td>Rhodamine dextran, 10,000 M.W.</td><td>Invitrogen</td><td>D1817</td><td>Tracer.</td></tr><tr><td>Molecular biology grade, USP sterile purified water</td><td>Corning</td><td>46-000-CI</td><td></td></tr><tr><td>7&quot; Drummond replacement tubes</td><td>Drummond</td><td>3-000-203-G/XL</td><td>Microinjection needles.</td></tr><tr><td>Needle puller</td><td>Sutter Instruments</td><td>P-30</td><td></td></tr><tr><td>Fine forceps</td><td>Dumont</td><td>11252-30</td><td>For breaking microinjection needle tip.</td></tr><tr><td>Nanoject II</td><td>Drummond</td><td>3-000-204</td><td>Microinjector.</td></tr><tr><td>Mineral oil, heavy</td><td>Fisher</td><td>CAS 8042-47-5</td><td>Oil for needles.</td></tr><tr><td>27 G Monoject hypodermic needle</td><td>Covidien</td><td>8881200508</td><td>For loading mineral oil into microinjection needle.</td></tr><tr><td>5 ml Luer-Lok syringe</td><td>BD</td><td>309646</td><td>For loading mineral oil into microinjection needle.</td></tr><tr><td>60 mm x 15 mm Petri dish</td><td>Fisher</td><td>FB0875713A</td><td></td></tr><tr><td>800 micron Polyester mesh</td><td>Small Parts</td><td>CMY-0800-C</td><td></td></tr><tr><td>Transfer pipets</td><td>Fisher</td><td>13-711-7M</td><td>For transferring embryos. Cut off the tip so that the embryos are easily taken up by the pipette.</td></tr><tr><td>6-well cell culture plate</td><td>Nest Biotechnology</td><td></td><td></td></tr><tr><td>MOPS</td><td>Fisher</td><td>BP308-500</td><td></td></tr><tr><td>EGTA</td><td>Acros Organics</td><td>67-42-5</td><td></td></tr><tr><td>Formaldehyde</td><td>Fisher</td><td>BP531-500</td><td></td></tr><tr><td>15 mm x 45 mm Screw thread vial</td><td>Fisher</td><td>03-339-25B</td><td>For fixing, staining, and storing embryos.</td></tr><tr><td>24-well cell Culture plate</td><td>Nest Biotechnology</td><td></td><td></td></tr><tr><td>Benzocaine</td><td>Spectrum</td><td>BE130</td><td></td></tr><tr><td>Ethanol</td><td>Fisher</td><td>BP2818-4</td><td></td></tr><tr><td>Methanol</td><td>Fisher</td><td>A412-4</td><td></td></tr><tr><td>Phosphate buffered saline (PBS) 1x powder</td><td>Fisher</td><td>BP661-50</td><td></td></tr><tr><td>Bovine serum albumen</td><td>Fisher</td><td>BP1600-100</td><td></td></tr><tr><td>Triton X-100</td><td>Fisher</td><td>BP151-500</td><td></td></tr><tr><td>3D Mini rocker, model 135</td><td>Denville Scientific</td><td>57281</td><td></td></tr><tr><td>Goat serum, New Zealand origin</td><td>Invitrogen</td><td>16210064</td><td></td></tr><tr><td>Sodium azide</td><td>Fisher</td><td>S227I-25</td><td></td></tr><tr><td>Monoclonal 3G8 antibody</td><td>European Xenopus Resource Centre</td><td></td><td>Primary antibody to label proximal tubules.</td></tr><tr><td>Monoclonal 4A6 antibody</td><td>European Xenopus Resource Centre</td><td></td><td>Primary antibody to label intermediate, distal and connecting tubules.</td></tr><tr><td>anti-RFP pAb, purified IgG/rabbit</td><td>MBL</td><td>PM005</td><td>Primary antibody to label Gap43-RFP tracer.</td></tr><tr><td>Alexa Fluor 488 goat anti-mouse IgG</td><td>Life Technologies</td><td>A11001</td><td>Secondary antibody to label kidney tubules.</td></tr><tr><td>Alexa Fluor 555 goat anti-rabbit IgG</td><td>Life Technologies</td><td>A21428</td><td>Secondary antibody to label Gap43-RFP tracer.</td></tr><tr><td>9 Cavity spot plate</td><td>Corning</td><td>7220-85</td><td></td></tr><tr><td>Benzyl benzoate</td><td>Fisher</td><td>105862500</td><td>Optional - for clearing embryos</td></tr><tr><td>Benzyl alcohol</td><td>Fisher</td><td>A396-500</td><td>Optional - for clearing embryos</td></tr></tbody></table>

technique to target microinjection to the developing xenopus kidney

的非洲爪 蟾 （蛙）中，前肾，胚胎肾由单个肾的，并且可以用作用于肾脏疾病的模型。 爪蟾胚胎是大的，外部发展，并且可以通过微注射或外科手术容易地操纵。此外，命运地图已经建立了早期的爪蟾胚胎。靶向显微注射到单个卵裂球，最终将产生的器官或感兴趣的组织可被用于选择性地过表达或该受限制区域内的击倒的基因表达，从而降低在发育中的胚胎的其余部分次级效应。在这个协议中，我们描述了如何利用建立的爪蟾的命运映射到显影爪蟾肾（该头肾），通过显微注射到目标的4和8细胞胚胎卵裂球专用。沿袭示踪剂的注入使得注入的具体目标的验证。胚胎已经开发到阶段38后 - 40，整个安装免疫染色被用于可视化pronephric发展，通过靶向的细胞的前肾的贡献可以被评估。同样的技术可以适于除了前肾靶向其它组织类型。

的4-和8-细胞爪蟾胚胎用MEM-RFP的mRNA显微注射表现出不同水平的靶向前肾的。 图4示出了级40的胚胎与正确的MEM-RFP的mRNA表达模式。胚胎在左腹侧卵裂球（ 图4A）注射，并分类为MEM-RFP的mRNA的适当的表达模式。除了在近端，中间，远端表达MEM-​​RFP与连接肾脏肾小管，正常注射的胚胎预计将显示荧光的头部，躯干和尾部的表皮。它们也应显示在...

Watch this Scientific Journal Video about Technique to Target Microinjection to the Developing Xenopus Kidney at JoVE.com

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

技术目标显微注射到显影<em&gt;爪</em&gt;肾

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

technique-to-target-microinjection-to-the-developing-xenopus-kidney

Research

JoVE Journal

Developmental Biology

Technique to Target Microinjection to the Developing Xenopus Kidney

11.2K 次观看.  University of Texas McGovern Medical School. Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos. 

视频: Technique to Target Microinjection to the Developing Xenopus Kidney

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

利用共聚焦分析<em&gt;非洲爪蟾</em&gt;调查的Wnt和Shh形态发生素梯度的调节剂

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

科研

发育生物学

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

胚胎血管生成的可视化和定量分析<em&gt;热带非洲爪蟾</em

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

非洲爪蟾卵母细胞的微量注射

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

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 ...

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

的神经植体培养<em&gt;非洲爪蟾</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

神经科学

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the development of neuronal connectivity. During establishment of the retino-tectal projection, optic axons extend from the eye and navigate through distinct regions of the brain to reach their target tissue, the optic tectum. Once optic axons enter the tectum, they elaborate terminal arbors that function to increase the number of synaptic connections they can make with target interneurons in the tectum. Here, we describe a method to express DNA encoding green fluorescent protein (GFP), and gain- and loss-of-function gene constructs, in optic neurons (retinal ganglion cells) in Xenopus embryos. We explain how to microinject a combined DNA/lipofection reagent into eyebuds of one day old embryos such that exogenous genes are expressed in single or small numbers of optic neurons. By tagging genes with GFP or co-injecting with a GFP plasmid, terminal axonal arbors of individual optic neurons with altered molecular signaling can be imaged directly in brains of intact, living Xenopus tadpoles several days later, and their morphology can be quantified. This protocol allows for determination of cell-autonomous molecular mechanisms that underlie the development of optic axon arborization in vivo.

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

This protocol aims to demonstrate how to microinject a DNA/DOTAP mixture into eyebuds of one day old Xenopus laevis embryos, and how to image and reconstruct individual green fluorescent protein (GFP) expressing optic axonal arbors in tectal midbrains of intact, living Xenopus tadpoles.

将DNA微注射到异种胚胎的眼蕾中，以及GFP表达光学轴状轴向轴向的成像，活的Xenopus大白鼠

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the ...

Intravenous Microinjections of Zebrafish Larvae to Study Acute Kidney Injury

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections on 2 dpf zebrafish. This technique represents an efficient and rapid method to deliver soluble substances into the bloodstream of zebrafish larvae, allowing for the injection of 15-20 fish per hour. In addition to AKI studies, this microinjection technique can also be used for other types of experimental studies such as angiography.  We provide a detailed protocol of the technique from equipment required to visual measures of decreased kidney function. In addition, we also demonstrate the process of fixation, whole mount immunohistochemistry with a kidney tubule marker, plastic embedding and sectioning of the larval zebrafish. We demonstrate that zebrafish larvae injected with gentamicin show morphological features consistent with AKI: edema, loss of cell polarity in proximal tubular epithelial cells, and morphological disruption of the tubule.

We describe a technique of microinjecting the aminoglycoside, gentamicin, into 2 days post-fetilization (dpf) zebrafish larvae to induce acute kidney injury (AKI). We also describe a method for whole mount immunohistochemistry, plastic embedding and sectioning of zebrafish larvae to visualize the AKI mediated damage.

斑马鱼幼虫的研究急性肾损伤的静脉Microinjections

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections ...

产生视网膜变性以研究非洲爪蟾的细胞修复策略

Generating Retinal Injury Models in Xenopus Tadpoles

Retinal neurodegenerative diseases are the leading causes of blindness. Among numerous therapeutic strategies being explored, stimulating self-repair recently emerged as particularly appealing. A cellular source of interest for retinal repair is the M&#252;ller glial cell, which harbors stem cell potential and an extraordinary regenerative capacity in anamniotes. This potential is, however, very limited in mammals. Studying the molecular mechanisms underlying retinal regeneration in animal models with regenerative capabilities should provide insights into how to unlock the latent ability of mammalian M&#252;ller cells to regenerate the retina. This is a key step for the development of therapeutic strategies in regenerative medicine. To this aim, we developed several retinal injury paradigms in Xenopus: a mechanical retinal injury, a transgenic line allowing for nitroreductase-mediated photoreceptor conditional ablation, a retinitis pigmentosa model based on CRISPR/Cas9-mediated rhodopsin knockout, and a cytotoxic model driven by intraocular CoCl2 injections. Highlighting their advantages and disadvantages, we describe here this series of protocols that generate various degenerative conditions and allow the study of retinal regeneration in Xenopus.

作者聚焦：探索非洲爪蟾的视网膜再生机制 

We have developed several protocols to induce retinal damage or retinal degeneration in Xenopus laevis tadpoles. These models offer the possibility of studying retinal regeneration mechanisms.

在 非洲爪蟾 蝌蚪中生成视网膜损伤模型

Retinal neurodegenerative diseases are the leading causes of blindness. Among numerous therapeutic strategies being explored, stimulating self-repair ...

技术目标显微注射到显影

摘要

探索更多视频

利用共聚焦分析

胚胎血管生成的可视化和定量分析

非洲爪蟾卵母细胞的微量注射

显微注射mRNA和斑马鱼的胚胎吗啉反义寡核苷酸。

的神经植体培养

将DNA微注射到异种胚胎的眼蕾中，以及GFP表达光学轴状轴向轴向的成像，活的Xenopus大白鼠

斑马鱼幼虫的研究急性肾损伤的静脉Microinjections

在非洲爪蟾蝌蚪中生成视网膜损伤模型

在非洲爪蟾蝌蚪中生成视网膜损伤模型

在非洲爪蟾蝌蚪中生成视网膜损伤模型

技术目标显微注射到显影

摘要

探索更多视频

利用共聚焦分析

胚胎血管生成的可视化和定量分析

非洲爪蟾卵母细胞的微量注射

显微注射mRNA和斑马鱼的胚胎吗啉反义寡核苷酸。

的神经植体培养

将DNA微注射到异种胚胎的眼蕾中，以及GFP表达光学轴状轴向轴向的成像，活的Xenopus大白鼠

斑马鱼幼虫的研究急性肾损伤的静脉Microinjections

在 非洲爪蟾 蝌蚪中生成视网膜损伤模型

在 非洲爪蟾 蝌蚪中生成视网膜损伤模型

在 非洲爪蟾 蝌蚪中生成视网膜损伤模型

在非洲爪蟾蝌蚪中生成视网膜损伤模型

在非洲爪蟾蝌蚪中生成视网膜损伤模型

在非洲爪蟾蝌蚪中生成视网膜损伤模型