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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Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+12:+The+Xenopus+Pronephros:+A+Kidney+Model+Making+Leaps+toward+Understanding+Tubule+Development.">Chapter 12: The Xenopus Pronephros: A Kidney Model Making Leaps toward Understanding Tubule Development.</a> Xenopus Development. , (2014).</li><li>Taira, M., Otani, H., Jamrich, M., Dawid, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+and+function+of+the+tissue-specific+transcription+factor+HNF1+alpha+(LFB1)+during+Xenopus+development.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergism+between+Pax-8+and+lim-1+in+embryonic+kidney+development.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+of+injected+DNA+templates+in+Xenopus+embryos.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chloride+conductance+channel+ClC-K+is+a+specific+marker+for+the+Xenopus+pronephric+distal+tubule+and+duct.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Na+,+K+-ATPase+alpha+subunit+requires+gastrulation+in+the+Xenopus+embryo.">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://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Normal table of Xenopus laevis (Daudin): A systematical &amp; chronological survey of the development from the fertilized egg till the end of metamorphosis. , (1994).</li><li>Ubbels, G. A., Hara, K., Koster, C. H., Kirschner, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+a+functional+role+of+the+cytoskeleton+in+determination+of+the+dorsoventral+axis+in+Xenopus+laevis+eggs.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blastomeres+show+differential+fate+changes+in+8-cell+Xenopus+laevis+embryos+that+are+rotated+90+degrees+before+the+first+cleavage.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stability+and+movement+of+mRNA+in+Xenopus+oocytes+and+embryos.">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。由于爪蟾胚胎的相对透明度，肾脏缺陷可以很容易地使用多种技术进行定量。例如，基因敲除或过度表达，可以简单地通过计算胚的pronephric索引量化免疫染色用抗体3G8 13，该比较对胚胎的注入侧和对照侧的近端小管的发展。除了 ​​研究pronephric发展，该技术可以很容易地适应目标通过选择合适的卵裂球其它组织使用已建立的命运映射7-10注入。除了靶向其它组织类型，该协议也可以被用来研究在不同阶段肾发展。该协议看着阶段40胚胎抗体3G8和4A6染色，其检测分化的肾组织。年轻的胚胎可以与不同肾抗体免疫染色， 或原位杂交5,6 20进行。例如，该抗体LIM1可以检测显影前肾21,22。同样，LIM1，PAX8和hnf1-β转录可以在舞台12.5 22-24 原位杂交开始检测</sup&gt;。其他后原位杂交标记可用于在以后的发展阶段，以检测肾脏的不同区域。例如，探针设计为检测在血管球NPHS1，clckb，slc5a1和ATP1A1的表达，远端和连接管，近端小管，并且在整个肾脏，分别已经描述25，5，26，27。评估肾跨使用不同的抗体或原位分析将允许更好地了解治疗如何改变肾脏发育多个阶段。 该协议的一个关键方面是正确的选择和卵裂球的标识被注入。在这个协议中，我们将描述如何通过注入8细胞胚胎的左侧V2卵裂球或4-细胞胚胎左侧腹侧卵裂球定位的前肾。如果不正确的卵裂球注入时，前肾将不会被正确定位。因此，它关键是要熟悉早期爪蟾胚胎的前显微注射28的发展和细胞解理面29。胚胎裂解并不总是遵循既定发育阶段的图表，因此，它是有帮助的仅注入胚胎，其中早期细胞分裂看"正常"和适当的卵裂球可以容易地识别。这将减少已不当靶向的胚胎的数量。此外，它也是重要的胚胎的卵裂球，以在注射时被完全分割。例如，如果卵裂球V1和8细胞胚胎的V2之间的裂解是不显微注射之前完成，所述示踪剂可扩展到V 1以及V 2。这会降低显微注射的靶向效率，将所得的胚胎可能显示示踪剂分布，看起来更类似于在4-细胞阶段，而不是8细胞期注入的胚胎。 一个本协议的其他重要部分是核实前肾被正确通过显微注射的目标。这之前，必须分析pronephric做开发，因为胚胎还没有正确定位可能会扭曲所产生的数据集中的前肾。要确保正确定位，分析每个胚胎注射示踪分布。胚胎注入（左）侧应显示绝大多数荧光示踪剂的。如果胚胎的控制（右）侧示出了荧光的显着量，那么这个胚胎应被丢弃。如果发生这种情况，要么不正确的卵裂球注入或注入的卵裂球没有注射前完成切割。第二，对胚胎的注射（左）侧的荧光，必须正确地定位的前肾。如果胚胎在8细胞期，背部和腹部的体节，侧板，后肠，proctodeum，注入树干躯干和神经嵴的皮肤，预计将显示流感orescence除了前肾6，29:除了在4-细胞阶段注入的应在皮肤和体节的荧光示踪剂的更宽分布以及的靶向为8细胞注射所列的组织，胚胎水泥腺，听囊，镜头和嗅placode。如果胚胎不显示正确的组织定位，它应该被丢弃之前分析（ 图5）。 在这个协议中描述的靶向注射提供在所需的组织操纵基因表达的一种手段。这种技术的一个限制是，尽管这些注射靶向，只要不影响仅在显影肾脏。在该技术中所描述的4-和8-细胞注射比在单细胞阶段，其中的基因表达将在整个胚胎被改变完成注射更有针对性，并在两细胞阶段，完成注射，其中的半胚胎显示改变基因表达离子。他们不这样做，但是，目标只有一个组织。虽然在4-细胞胚胎和8细胞胚胎的卵裂球V2腹侧卵裂球注射改变在头肾基因的表达，它们还改变在其他组织中的基因表达。其他受影响的组织包括皮肤，体节，肠道和神经嵴。因此，要明白的是，虽然4-和8-细胞注射比一个或两个细胞注射更有针对性的，它们将仍然改变在多种组织的基因表达是重要的。除了在4和8细胞阶段注入的胚胎，注射剂，也可在2-，16-，以及32-细胞阶段进行。在2-细胞阶段注射的胚胎将有更广泛的影响比在稍后阶段注射的胚胎组织。虽然越来越多的技术挑战，在16和32细胞期胚胎注入将有一个窄的范围比的影响在4和8细胞期胚胎注射组织。 该MEM-RFP血统跟踪R在本协议中使用微量注射验证适当的目标。 MEM-RFP的mRNA标记细胞后细胞膜已经从所注入的RNA的翻译的蛋白。根据需要考虑到胚胎死亡和期望的示踪荧光的强度在这个协议（在10 NL注射0.1纳克MEM-RFP的mRNA）中使用的MEM-RFP谱系示踪剂的浓度应被修改。除了修改谱系示踪剂注入的量，不同的谱系示踪剂，如罗丹明 - 葡聚糖，量子点，或组织化学可检测的β半乳糖苷酶，可被用来代替MEM-RFP。谱系示踪剂成为作为细胞分裂更稀，导致荧光的水平减小，胚胎发育。这种技术的另外的应用是共同注入的外源RNA或吗啉代与谱系示踪剂以过表达或击倒感兴趣的基因的表达。谱系示踪剂用来验证前肾的正确定位，但不能以指示共注射外源RNA或吗啉代在胚胎本地化。外源RNA和吗啉可能不会以相同的速率作为共注射示踪剂通过注入卵裂球蔓延，潜在地导致具有示踪剂，但不是外源RNA或吗啉代本30的子细胞。实际上，我们已经观察到，注入单细胞两种不同的RNA构建体并不总是共定位（未公布的数据）。因此，在一个小区中的示踪剂的存在并不一定表示该共注射外源RNA或吗啉代是始终存在于该细胞。 该协议详细介绍了针对显微注射到引起发展中国家爪蟾胚胎的前肾卵裂球的过程。基因表达通常是通过吗啉或外源RNA，这两者都能够防止起因早期发育异常的喷射操作在爪蟾胚胎如果注入一维或两个细胞胚胎。胚胎在所有胚胎发育的细胞的单细胞阶段展览击倒或过度注入。如果基因是必要进行适当的早期发育过程中，胚胎可能不会制定一个头肾。在稍后的阶段，如这里详细描述的8细胞注射，进行注射可导致在经历原肠胚形成和最终pronephric发展18胚。因此，这里所讨论的有针对性的注射可以克服由一些基因的表达的改变的原肠胚形成的缺陷，使胚胎通过pronephric发展进步。在未来，该协议也可以用于靶向CRISPR构建体至所需的卵裂球。

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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 et al., 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&#34; 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>27G 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 distal tubule and duct.</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 distal tubule and duct.</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。它们也应显示在听囊荧光，水泥腺，proctodeum，和体节（ 图4C）。用荧光立体显微镜（ 图4B）来测定的MEM-RFP荧光的在8正确注射的胚胎的肾脏的分布。在表皮高水平MEM-RFP表达胚胎阻塞肾脏区域的检测被评价为"闭塞"。在近端检测MEM-RFP的表达，因为闭塞那些没有拿下所有的胚胎中间，远端和连接管。体视（ 图4D-F）和共焦显微镜（ 图4G-I）被用来验证用3G8和4A6进行免疫染色的MEM-RFP和肾组织的共定位。共焦成像验证MEM-RFP的共定位与近端，肾的中间，远端和连接管，这表明MEM-RFP的显微注射入左腹侧卵裂球正确定位到肾。 在8细胞期用MEM-RFP的mRNA注射的胚胎（ 图5A）示出了较窄的范围中显示荧光组织，表明8-细胞注射减少潜在的次级效应的肾脏。像在4-细胞期，EMBR注射的胚胎YOS在近端处的8细胞期显示荧光注入，肾的中间，远端和连接管，以及在体节和proctodeum（ 图5C-F）。不像在4-细胞阶段注射的胚胎中，水泥腺和听囊未标示用MEM-RFP。出10正确定位的胚胎，一个胚胎表明MEM-RFP在几乎所有的近端小管，和3显示的MEM-RFP在几乎所有的肾脏（ 图5B）的中间和远端小管。 7胚胎的连接管用MEM-RFP被部分标记。此外，在8细胞期注射的胚胎的肾脏有更易于可视化，且只有一个为包藏进行评分。使用立体显微镜（ 图5D-F）测定的MEM-RFP和抗体标记的肾（3G8和4A6）的共定位，并使用共聚焦显微镜（ 图5G-I）中进行验证。近端，中间，远端和连接涂肾脏bules被标记用MEM-RFP在8细胞阶段注射的胚胎，表明靶向V2的卵裂球的注射标签肾脏，而在较少的组织中显示荧光比在4芯的左腹侧卵裂球注射的胚胎阶段。 此法是利用荧光标记的基因作为示踪剂来指示组织中通过显微注射的目标。它分析前筛选胚胎一致示踪剂定位是重要的，并且不显示预期的示踪剂分布图案任何胚胎应被丢弃。 图6示出了级40的胚胎被错误地用MEM-RFP的mRNA定位于的一个例子4-细胞阶段。 MEM-RFP表达位于胚胎代替的左侧（ 图6A）的右侧。此外，大多数的mRNA表达的是在较低的躯干和尾部的皮肤上，用在体节的小表情。没有表达见于近端，中间，远端和连接管（ 图6B），证实了肾脏的不当定位。因此，该胚胎不应该用于分析。 <img alt="图1" src="/files/ftp_upload/53799/53799fig1.jpg" /> 图 1: 非洲爪蟾 胚胎肾一个级35 爪蟾胚胎的肾脏的图，示出在近端，中间，远端，和连接细管。基于Raciti 等 。 （2008年）6。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53799/53799fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53799/53799fig2.jpg" /> 图2:Xenopu小号胚胎命运地图。地图命运识别和命名有助于发展前肾卵裂球。 A）效果图4细胞胚胎。 B）8细胞胚胎的命运映射。 C）效果图16细胞胚胎。 D）效果图32细胞胚胎。 32细胞胚胎卵裂球的使用备用卵裂球的命名系统还标注。根据穆迪（1987）8,9和穆迪和克莱恩（1990）命运映射7。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53799/53799fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53799/53799fig3.jpg" /> 图 3:是面向 非洲爪蟾 头肾 注入方案红色箭头指示细胞注入。 A）动物和4细胞胚胎的腹示出左侧腹部的B注射液lastomere瞄准左侧前肾。红色箭头指示左腹侧卵裂球。 B）动物并显示左V2卵裂球注射的8细胞胚胎腹观点为目标的左侧前肾。红色箭头指示左V2卵裂球。 AB）上放大4倍立视拍摄的胚胎图像。根据非洲爪蟾的命运映射注射穆迪（1987）8,9和穆迪和克莱恩（1990）开发的7。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53799/53799fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53799/53799fig4.jpg" /> 图 4: 在4-细胞期靶向胚的实例免疫染色阶段40 爪蟾胚胎示出在使用MEM-RFP的mRNA作为示踪剂4-细胞阶段靶向注射到左侧前肾。 MEM-RFP标签细胞膜红色。 A）示意图，显示注射MEM-RFP的mRNA成4细胞胚胎的左侧腹卵裂球。在适当的注射胚胎的肾脏MEM-RFP荧光二）组织定位。 C）的阶段40胚胎在4细胞期用MEM-RFP注射在左腹侧卵裂球。 MEM-RFP定位以红色显示，而肾标记为绿色。 1X的放大倍率。 D）的肾脏与3G8染色以标记近端小管的内腔，和4A6标记的细胞的细胞膜中的中间，远端和连接管。放大5倍。 E）该地区在显示MEM-RFP的本地化肾肿大。放大5倍。 F）的合并图像，其显示与肾脏的MEM-RFP示踪剂共定位。放大5倍。 CF）与奥林巴斯DP71相机立视拍摄的胚胎图像。 G）与3G8和4A6染色第二胚胎的肾脏的共聚焦图像。高）肾MEM-RFP的本地化及周围组织。我）合并图像抄MEM-RFP，3G8和4A6肾脏的翼共定位。 GI），使用最大投影在放大20倍共聚焦图像。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53799/53799fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53799/53799fig5.jpg" /> 图 5: 在8细胞阶段靶向胚的实例免疫染色阶段40 爪蟾胚胎示出在使用MEM-RFP的mRNA作为示踪剂的8细胞期靶向注射到左侧前肾。 MEM-RFP标签细胞膜红色。 A）示意图，显示注射MEM-RFP的mRNA为8细胞胚胎的左V2卵裂球。在适当的注射胚胎的肾脏MEM-RFP荧光二）组织定位。 C）的阶段40胚胎在8细胞期用MEM-RFP注射在左V2卵裂球。 MEM-RFP的本地化显示在红色，而肾标记为绿色。 1X的放大倍率。 D）的肾脏与3G8染色以标记近端小管的内腔，和4A6标记的细胞的细胞膜中的中间，远端和连接管。放大5倍。 E）该地区在显示MEM-RFP的本地化肾肿大。放大5倍。 F）的合并图像，其显示与肾脏的MEM-RFP示踪剂共定位。放大5倍。 CF）与奥林巴斯DP71相机立视拍摄的胚胎图像。 G）与3G8和4A6染色第二胚胎的肾脏的共聚焦图像。高）肾MEM-RFP的本地化及周围组织。 Ⅰ）合并图像显示的MEM-RFP，3G8，以及4A6在肾脏的共定位。 GI），使用最大投影在20X放大倍率拍摄共聚焦图像。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53799/53799fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图6:胚胎的实施例不正确地定位在4-细胞阶段免疫染色阶段40爪蟾胚胎示出在使用MEM-RFP的mRNA作为示踪剂4-细胞阶段左侧前肾的不正确的定位。 MEM-RFP标签细胞膜红色。 a）第一阶段40胚胎显示指示注射MEM-RFP到不正确的卵裂球的不当分配。除了具有指示注入不正确卵裂球不正确示踪剂分布，这胚胎注射在右侧。 1X的放大倍率。 B）表现出缺乏MEM-RFP与抗体标记的肾（3G8，4A6）共定位的肾合并后的图像。放大5倍。 AB）在立体镜取胚图像。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53799/53799fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

Technique to Target Microinjection to the Developing Xenopus Kidney

11.1K 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

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

操作和<em&gt;在体外</em中&gt;成熟<em&gt;非洲爪蟾</em&gt;卵母细胞，其次是胞浆内单精子注射，研究胚胎发育

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

科研

发育生物学

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 ...

Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression

Using chicken embryos it is possible to test directly the effects of either growth factors or specific inhibitors of signaling pathways on gene expression and activation of signal transduction pathways. This technique allows the delivery of signaling molecules at precisely defined developmental stages for specific times. After this embryos can be harvested and gene expression examined, for example by in situ hybridization, or activation of signal transduction pathways observed with immunostaining.
In this video heparin beads soaked in FGF18 or AG 1-X2 beads soaked in U0126, a MEK inhibitor, are grafted into the limb bud in ovo. This shows that FGF18 induces expression of MyoD and ERK phosphorylation and both endogenous and FGF18 induced MyoD expression is inhibited by U0126. Beads soaked in a retinoic acid antagonist can potentiate premature MyoD induction by FGF18.
This approach can be used with a wide range of different growth factors and inhibitors and is easily adapted to other tissues in the developing embryo.

By grafting beads soaked in growth factors or specific inhibitors of signaling pathways into developing embryos it is possible to directly test their effects in vivo. In this protocol beads are grafted into the limb bud to determine the effects of these molecules on gene expression and signal transduction.

珠嫁接到发展鸡胚四肢识别信号转导途径影响基因表达

Using chicken embryos it is possible to test directly the effects of either growth factors or specific inhibitors of signaling pathways on gene expression ...

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

干细胞样<em&gt;爪</em&gt;胚胎外植体，研究早期神经发育特点<em&gt;体外</em&gt;和<em&gt;在体内</em

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance to man of conventional invertebrate model systems and the complexity of genomes in vertebrates are critical challenges to modeling human normal and pathological conditions. The chordate Ciona intestinalis is an invertebrate chordate that emerged from a common ancestor with the vertebrates and may represent features at the interface between invertebrates and vertebrates. A common body plan with much simpler cellular and genomic composition should unveil gene regulatory network (GRN) links and functional genomics readouts explaining phenomena in the vertebrate condition. The compact genome of Ciona, a fixed embryonic lineage with few divisions and large cells, combined with versatile community tools foster efficient gene functional analyses in this organism. Here, we present several crucial methods for this promising model organism, which belongs to the closest sister group to vertebrates. We present protocols for transient transgenesis by electroporation, along with microinjection-mediated gene knockdown, which together provide the means to study gene function and genomic regulatory elements. We extend our protocols to provide information on how community databases are utilized for in silico design of gene regulatory or gene functional experiments. An example study demonstrates how novel information can be gained on the interplay, and its quantification, of selected neural factors conserved between Ciona and man. Furthermore, we show examples of differential subcellular localization in embryonic cells, following DNA electroporation in Ciona zygotes. Finally, we discuss the potential of these protocols to be adapted for tissue specific gene interference with emerging gene editing methods. The in vivo approaches in Ciona overcome major shortcomings of classical model organisms in the quest of unraveling conserved mechanisms in the chordate developmental program, relevant to stem cell research, drug discovery, and subsequent clinical application.

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

We present transient transgenesis and gene knockdown in Ciona intestinalis, a chordate sister group to vertebrates, using microinjection and electroporation techniques. Such methods facilitate functional genomics in this simple invertebrate that features rudimentary characteristics of vertebrates, including notochord and head sensory epithelia, and many orthologs of human disease associated genes.

胚胎显微注射和电的脊索动物<em&gt;玻璃海鞘</em

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance ...

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global DNA methylation level of chondrocytes is considered to be a suitable biomarker for the loss of the chondrocyte phenotype. However, results based on different experimental methods can be inconsistent. Therefore, it is important to establish a precise, simple, and rapid method to quantify global DNA methylation levels during chondrocyte dedifferentiation.
Current genome-wide methylation analysis techniques largely rely on bisulfite genomic sequencing. Due to DNA degradation during bisulfite conversion, these methods typically require a large sample volume. Other methods used to quantify global DNA methylation levels include high-performance liquid chromatography (HPLC). However, HPLC requires complete digestion of genomic DNA. Additionally, the prohibitively high cost of HPLC instruments limits HPLC&#39;s wider application.
In this study, genomic DNA (gDNA) was extracted from human chondrocytes cultured with varying number of passages. The gDNA methylation level was detected using a methylation-specific dot blot assay. In this dot blot approach, a gDNA mixture containing the methylated DNA to be detected was spotted directly onto an N+ membrane as a dot inside a previously drawn circular template pattern. Compared with other gel electrophoresis-based blotting approaches and other complex blotting procedures, the dot blot method saves significant time. In addition, dot blots can detect overall DNA methylation level using a commercially available 5-mC antibody. We found that the DNA methylation level differed between the monolayer subcultures, and therefore could play a key role in chondrocyte dedifferentiation. The 5-mC dot blot is a reliable, simple, and rapid method to detect the general DNA methylation level to evaluate chondrocyte phenotype.

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

We present a method to quantify DNA methylation based on the 5-methylcytosine (5-mC) dot blot. We determined the 5-mC levels during chondrocyte dedifferentiation. This simple technique could be used to quickly determine the chondrocyte phenotype in ACI treatment.

5-mC点印迹测定定量软骨细胞去分化的DNA甲基化水平<em&gt;体外</em

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global ...

Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to &#34;dial&#34; the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.

This work presents a new in vivo model of segmental kidney injury using kidney GFP transgenic zebrafish. The model allows for the induction of the targeted ablation of kidney epithelial cells to show the cellular mechanisms of nephron injury and repair.

精确的细胞消融方法建模发展中的斑马鱼急性肾损伤

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore ...

Generation of Genetically Modified Mice through the Microinjection of Oocytes

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear injection of DNA expression constructs into fertilized oocytes remains the most commonly used technique to generate transgenic mice for overexpression. With the introduction of CRISPR technology for gene targeting, pronuclear injection into fertilized oocytes has been extended to the generation of both knockout and knockin mice. This work describes the preparation of DNA for injection and the generation of CRISPR guides for gene targeting, with a particular emphasis on quality control. The genotyping procedures required for the identification of potential founders are critical. Innovative genotyping strategies that take advantage of the "multiplexing" capabilities of CRISPR are presented herein. Surgical procedures are also outlined. Together, the steps of the protocol will allow for the generation of genetically modified mice and for the subsequent establishment of mouse colonies for a plethora of research fields, including immunology, neuroscience, cancer, physiology, development, and others.

The microinjection of mouse oocytes is commonly used for both classic transgenesis (i.e., the random integration of transgenes) and CRISPR-mediated gene targeting. This protocol reviews the latest developments in microinjection, with a particular emphasis on quality control and genotyping strategies.

通过显微注射卵母细胞生成转基因小鼠

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C） 损伤的反应

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

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 ...