We are grateful to Gurdon laboratory members for useful discussion. K.M. is a Research Fellow at Wolfson College and is supported by the Herchel Smith Postdoctoral Fellowship and the Great Britain Sasakawa Foundation. Gurdon laboratory is supported by grants from the Wellcome Trust (RG69899) and MRC to J.B.G.

注:所有实验用青蛙进行了继英国内政部的要求。 1.准备爪蛙卵母细胞<ol><li>大约一个星期的卵母细胞采集前，用1毫升无菌注射器用27G的针头注射雌蛙用孕马血清促性腺激素（PMSG）预吸150ü。注入皮下做成背淋巴囊。 注:这是最佳的使用青蛙未下蛋很长一段时间（至少半年以上），或者从来没有产卵。 </li><li> （:对，晚上7点到上午07点:离上午07时 - 下午7时）在水箱中的光控室设置在18°C储存预先打底的青蛙。 </li><li>对卵母细胞收集的当天，制备1升的1个修饰巴特的解决方案（MBS）从10倍的MBS库存稀释用双蒸水（DDH 2 O）和添加青霉素和链霉素以10微克/毫升的最终浓度每个（MBS + PS）。 MBS 10倍的股票由880毫米氯化钠，10毫米氯化钾，24毫米的NaHCO 3，8.2毫米硫酸镁4 7H 2 O，3.3毫米的Ca（NO 3）2·4H 2 O，4.1毫米氯化钙2·6H 2 O，100毫米的HEPES，pH值7.5。 </li><li>通过皮下注射120毫克（以400微升）三卡因甲磺酸（MS222）麻醉一个预先打底的青蛙。不断注入的青蛙在一个小水箱加水。 </li><li> 10分钟后，用自成功麻醉的青蛙没有对此作出回应翻过来检查麻醉的青蛙。如果麻醉是不完整的，加冰块放入罐，等待另一个10分钟。 </li><li>捡起小坦克，地点在背斜卧青蛙在潮湿擦拭。 </li><li>使用镊子和小手术剪，捏皮肤，使一个小切口（2厘米），在腹部，横向于中线的下部。再拍切口的另一边。 </li><li>切割后的皮肤，解除肌肉层的Wi第镊子，使切口在肌肉层。 </li><li>观察卵巢的肌肉层被切断后，拉出卵巢从使用镊子切口。 </li><li>转印萃取卵巢到50ml管的MBS溶液。 注:尝试从两个切口收集尽可能多的卵巢越好。 </li><li>屠宰雌蛙放血，随后冻结了后续适当处置。 </li><li>与MBS + PS冲洗提取卵巢几次，直到血冲走。然后，将卵巢转移至含有的MBS + PS一个9厘米培养皿 注意:检查卵母细胞的质量解剖显微镜此时下。如果卵母细胞有明显质量差，如卵母细胞色素不均的动物一半（ 图1A），不进一步处理，而是获得一个新的卵巢。理想情况下，卵母细胞应该有同样色素的动物半球和大约同样大小。 </li><li>逗卵巢切成小块（1-2厘米2），并收集在一个新的50ml管中5毫升的卵母细胞。 </li><li>冲洗5毫升撕裂卵巢几次与MBS + PS，并添加MBS + PS到15ml。 </li><li>加入7个单位的酶进行defolliculation到卵巢暂停（见材料清单）。 注:每瓶冻干酶用10毫升DDH 2 O的（28单位/毫升）。将小瓶在冰上与偶尔轻轻旋转30分钟。等份加入250μl并储存于-80℃直至使用。 </li><li>同（在15转/分，相当于约0.014 XG速度）轻轻摇动的摇动孵育卵巢片与酶1小时。 注:对于几乎完全去除毛囊细胞，2小时到2小时15分钟孵育酶通常需要的。潜伏期较长，需要对卵母细胞的核移植实验中16。 </li><li>经过1小时温育后，拿起含有MBS少量处理的卵母细胞（10-20卵母细胞），并转移到一个6cm培养皿中+ PS检查EXTE核苷酸在解剖显微镜下defolliculation的。如果卵母细胞被彼此分离，并且至少部分地defolliculated（ 图1B），立即进行下一站反应（步骤18）。如果卵母细胞仍然深受毛囊细胞包围，孵育一个额外的15分钟最大值。 </li><li>补充充足的MBS + PS的，使反应停止，并丢弃上清液。重复洗涤10次。 注:加入的MBS + PS通过倾倒到50ml管的壁，而不是直接向卵母细胞。悬挂在MBS + PS后，小未成熟卵母细胞往往会漂浮在洗涤液的顶部和丢弃这样的小浮卵母细胞。 </li><li>在洗涤后，转移到含有MBS + PS一9厘米培养皿 注:从这一步开始，卵母细胞保持在16-18℃，尽可能多地。这可以通过保持包含卵母细胞的碟上的温度控制的阶段来完成。 </li><li>根据杜蒙的分类选择阶段VI的卵母细胞用于随后的用途，并转移到含有MBS + PS卵母细胞转移的新9厘米培养皿可以通过使用玻璃吸管与合适的尖端尺寸（大于所述卵母细胞的大小）来进行。 注:良好质量的卵母细胞应该具有均匀的着色动物半球与动物半球和植物性半球之间明显的对比，并进行近似相等的尺寸（ 图1C）。阶段六卵母细胞代表完全成熟卵母细胞，可以孕酮回应（卵母细胞直径约1.2-1.4毫米）。 </li></ol> 2.注射反义寡核苷酸或mRNA的卵母细胞成注:本节中的所有步骤应在16-18°C下进行在显微镜舞台装有温度控制的循环水，或在塑料盒子，里面装满冰块。 <ol><li>传送200-300选的卵母细胞成新9厘米培养皿填充MBS + PS，其中显微注射将被执行。 注:在EACH条件，200-300卵母细胞通常被使用。总计约1000的卵母细胞被用来在一个实验中。 </li><li>对于准备注射反义寡核苷酸或mRNA的，弹出微量的金属柱塞。 </li><li>使用注射器填充有矿物油的玻璃毛细管，并插入玻璃毛细管填充有油，以微量的金属柱塞。 注:玻璃毛细管是由微量拉马拉。这些针都保持在一个盒子里，而不会损坏的提示。 </li><li>通过瞄准作为小直径端部的注射尽可能削减在显微镜下利用小手术剪针的尖端。 注:针尖可以通过使用微锻造或微量倒角进行锐化。到斜面的针以25°的角度提高穿透卵母细胞壁的能力。 </li><li>放置一小条封口膜对解剖显微镜的阶段和分配的反义寡核苷酸或mRNA溶液的3微升下降。 </li><li>移动TI要注入P注射针进入小液滴的溶液中，并填写与溶液针头。 注意:如果该注射针的尖端太小，气泡应该出现在金属柱塞的尖端。然后，使上注射针，直到这不会发生一个更大的小费。 </li><li>马克注射针大约0.5mm远离小费。这个标志被用作注入深度的指标。 </li><li>插入针的尖端插入沿赤道边界的卵母细胞通过旨在对卵母细胞的中心点（生发囊泡下方），同时轻轻地保持卵母细胞的相对侧，以在注入点与钳，用于防止在注射过程中不希望的卵母细胞的运动。 注:查找游离毛囊细胞（ 图1B）的区域，并从该点插入针，因为即使一个微细针不能经常渗入一层卵泡细胞。 </li><li>弹出4.6纳克/ 4.6 NL或9.2纳克/ 9.2 NL ANTIS的恩瑟寡聚或mRNA的适当体积（250皮克至13.8纳克）通过使用脚踏开关。反义寡核苷酸的制备的细节在7说明。 </li><li>传输所注入的卵母细胞成6cm培养皿中填充有MBS + PS补充有0.1％BSA（卵母细胞孵育培养基中;用0.45微米的微孔过滤器灭菌溶液）。 </li><li>孵化的卵母细胞在16 ℃或18℃下进行1-2天。 注:注入的卵母细胞可以经受体外成熟数小时注射后，在这种情况下，注射的4.6毫微克的反义寡核苷酸是优选的。一般的规则是，越短潜伏期，更好的早期胚胎发育。 </li></ol> 3. 在卵母细胞体外成熟（IVM） <ol><li>对卵母细胞成熟的实验日晚，取-80黄体酮原液（30毫米乙醇）℃的冰柜和溶解沉淀在室温统perature偶尔漩涡。 注:孕酮库存通常在室温下放置30分钟到1小时。 </li><li>加入5微升孕酮股票的50ml的MBS + PS的（成熟介质;孕酮在3μM终浓度），并摇动的摇动器进行30分钟，以在溶液中均匀分配孕酮。 </li><li>准备琼脂糖涂层6厘米菜肴体外成熟治疗。 <ol><li>添加1克琼脂糖至50ml 1×MBS的无镁和钙。 </li><li>通过加热，在微波溶解琼脂糖。 </li><li>倒入一个小体积的琼脂糖溶液至6cm菜肴以覆盖培养皿底部。 </li><li>等待至少30分钟，直至凝固。 </li></ol>注:琼脂糖涂层防止卵母细胞粘附到菜肴。一旦在体外成熟卵母细胞紧密附在菜，这是最有可能的卵母细胞会被刺激，他们从DIS支队在收到激活HES。 </li><li>倒入5-8成熟培养基中琼脂糖涂层的菜肴和转移200-300卵母细胞到每个6厘米菜肴。 注:尽量与卵母细胞培养液中少量转移卵母细胞。 </li><li>孵育16小时，在16℃下。 注:例如，开始治疗下午5点，结束上午9点第二天。 </li></ol> 4.内单精子注射（ICSI） <ol><li>冷冻精子备料 注:以下精子的准备应开始卵母细胞成熟的实验之前完成，而冷冻精子库存保持在-80℃下ICSI。 <ol><li>通过以下步骤1.4到1.11，除了拉脂肪收集从雄性青蛙睾丸和从使用镊子和去除来自脂肪睾丸的睾丸切口出来。 </li><li>在1×Marc的改性林格氏（MMR，100 mM氯化钠，2mM的氯化钾，1mM的MgSO 4干燥，2mM的氯化钙 ，5mM的HEPES，pH值7.4）冲洗睾丸。 </li><li>去除脂肪的ð血管通过轧制在干净的纸巾洗过睾丸，然后通过除去使用镊子其余血管。 </li><li>将每个睾丸中含有1ml 1X MMR的3.5公分的培养皿。 </li><li>用细剪刀纵向切开，并撕成小块，用钳，直到没有大的碎片依然存在。 </li><li>移液器上下用1ml塑料尖端的端部被切断，并且负载1毫升粉碎睾丸溶液到玻璃匀浆器。 </li><li>由2-3笔划均质它们，然后将其倾溶液在连接到一个15毫升的离心管的顶部50微米孔径的过滤器。使该溶液通过过滤器。 </li><li>通过重新悬浮1毫升1X MMR的剩余切睾丸重复步骤4.1.7。最后，洗均质几次与1X MMR并通过过滤器传递。 </li><li>重复步骤4.1.5至4.1.8的其它睾丸和池2睾丸到一个15ml离心管中。 </li><li>旋转的细胞在800 XG在46℃持续20分钟。 </li><li>弃上清，重悬沉淀的细胞在2毫升1X MMR的。然后，转移到新的管中。 注意:请确保没有可见的血细胞都存在。 </li><li>在14毫升超清晰的离心管中，准备阶段梯度。底层:4毫升30％碘克沙醇。第二底层:1毫升20％碘克沙醇。第三底层:5毫升12％碘克沙醇。顶层:睾丸在2毫升1X MMR细胞。用1毫升枪头覆盖梯度轻轻地（覆盖慢慢不打扰底部阶段）。 注意:只有一层的30％碘克沙醇也应该只隔离精子。 </li><li>旋在使用超离心机以10,000 xg离心在4℃下进行15分钟（减速不休息）的SW40转子。 </li><li>轻轻地取出从超速离心机管。确认该梯度的每一层，将沉淀在底部之间的接口。 </li><li>收集沉淀的精子部分。 </li><li>悬浮精子像素让1X MMR的洗出碘克沙醇。旋，在800×g离心在4℃下20分钟。 注意:如果大量的精子的自旋，再旋后仍保留在悬浮液中的上清液在3220 xg离心在4℃下20分钟，并凝聚沉淀的精子。 </li><li>弃去上清，重悬精子沉淀在1ml 1X核的制备缓冲液（NPB; 250mM蔗糖，0.5mM的亚精胺三盐酸盐，0.2mM的精胺四盐酸盐，1mM EDTA中，15毫米的HEPES，pH值7.7）13。然后，转移到Eppendorf管中。 </li><li>添加50微升10毫克/毫升毛地黄皂苷溶液在1×NPB（重悬毛地黄皂苷粉在DMSO中，在50毫克/毫升，并冷冻等分试样在-80℃。使用前，稀释50毫克/毫升等分试样的10毫克/毫升，带有1 NPB。未使用10毫克/毫升毛地黄皂苷可以冷冻并储存在-20℃，并重新使用）的精子溶液至1ml。 </li><li>孵育5分钟，在室温下进行。 </li><li>检查通透率DAPI染色（0.3微克/毫升DAPI作为FINA升浓度）。如果小于95％的细胞被透化，孵育长一点。 注:有时，如果太多的精子细胞培养在相同的时间（特别是当精子从多个睾丸收集），则难以透，在这种情况下，可能有必要增加一个额外的少量的毛地黄皂苷;例如，如果精子的20％不透，额外的20微升毛地黄皂苷的溶液。 </li><li>通过加入10％的BSA至3％的最终浓度停止透。 </li><li>旋转细胞在4℃下。 注:尽量少离心速度尽可能通过启动用100×g离心5分钟。如果没有足够的，增加的速度和/或时间。 </li><li>以相同的速度作为步骤4.1.22洗细胞用0.3％BSA的1×NPB和离心机。同时，准备了精子存储缓冲区（SSB;2毫升2X NPB的，120微升10％BSA，1.2毫升蒸压甘油，680微升水中2 O的）。 </li><li>加入500μlSSB到精子颗粒。吹打数次上下重悬带，为了不损伤细胞的切割尖端。 </li><li>允许过夜平衡在4℃。 </li><li>吸取细胞上下数次切割尖端。 </li><li>稀几个微升在1×MMR或在1×PBS中的10倍的计数使用血球细胞。 </li><li>冻结为在30000精子/微升（或更高）直接在-80℃下，并储存在-80℃下在单次使用等份等分试样。 </li></ol></li><li> ICSI 体外成熟卵母细胞 注:所有涉及到的卵母细胞的过程应在16-18°C下进行。 <ol><li>准备一个玻璃吸管转移成熟卵母细胞。 注:玻璃吸管必须足够宽，以适应卵母细胞不受挤压。 </li><li>移动卵母细胞含有孕酮中16小时后，转让的卵母细胞为一块2.4英寸的琼脂糖涂层培养皿充满MBS + PS洗涤。 注:重要的是，米atured卵母细胞必须极其慎重对待。粗化处理的卵母细胞可诱导精子注射前自发激活。 </li><li>通过确认的白色斑点的出现，这意味着胚泡破裂（ 图2）计数成熟卵母细胞。如果成熟率小于80％，则可能无法获得足够的数量幸存的胚胎用于进一步的分析。 注:剩余的毛囊细胞被卵母细胞成熟后剥离（ 图2）。 </li><li>从MBS洗涤到一个新的琼脂糖包被的6cm皿填充有喷射的介质转印的卵母细胞（准备500毫升将30g聚蔗糖（6％），20毫升10×的MMR和500微升的1000倍的PS股票）。 </li><li>开始前的ICSI孵育至少30分钟。 </li><li>设置了微量按照步骤2.2-2.4中所述。 注:注射针的尖端尺寸保持尽可能小由以下步骤2.6中描述的方法。直径针是20-40微米。要斜面针尖按照步骤2.4中所述可能允许有效的注射。 </li><li>取精等分稀释精子悬液精子稀释液（SDB; 250毫米蔗糖，75毫米氯化钾，0.5毫米亚精胺，0.2毫米精胺，200μMHEPES pH值7.5）。 注:稀释可以根据针的大小等进行变化。淡化30,000精子/微升股票120倍的初始尝试，如果需要进一步调整。 </li><li>放置一小条封口膜对解剖显微镜的阶段。混合精注射液吹打和分配精子的解决方案几微升下降。 </li><li>吸稀释精子悬浮液进针，注入4.6 NL入含0.3微克/毫升的DAPI在显微镜载玻片10连续下降，并且迅速地计数精子每次注射递送上的荧光显微镜的数量。 注:瞄准每次注射1-2个精子。如果有必要，稀释精子注射液更并再次检查每次注射精子的数目，直至达到所需的浓度。 </li><li>弹出测试注射液，并填写了正确的浓度一个新的精子注射液。 </li><li>注入4.6 NL精子解决100成熟的卵母细胞。 注意:连续注入该溶液是很重要的。首先确定所需喷出4.6 NL（通常1-2秒）的时间。重复以下的处理;在注入卵母细胞，等待几秒钟，取出针和模拟注射注射液，等待几秒钟，在注入卵母细胞。这种连续注射也防止阻挡针尖。可替代地，利用泵的方法可能被用来13。 </li><li>后约100次注射，喷射剩余的精子溶液和（分配前使用相同的精子解决方案，但吸液管上下）重新填充精子溶液。 注意:如果针不吸精解好了，切需要尖端勒。如果没有改善的情况下，准备一个新的针头。 </li><li>重复注射过程，直到所有的卵母细胞注射。 注意:如果卵母细胞的质量是好的，注入成功后，注入卵母细胞的收缩中的注射时间20分钟观察。 </li><li>将注入菜到16°C或18 &#160; °Ç孵化器。 </li><li> 4-5小时注射后，检查胚胎ICSI的卵裂率。 注意:这些胚胎卵裂沟可能不明确的，因为这些正常的受精胚胎。有些胚胎也显示异常分裂，这可能是由多个单精子注射引起的。 </li><li>转印裂解胚胎包括异常裂解胚胎孵化介质（准备500毫升20克聚蔗糖（4％），5毫升10×的MMR和500微升的1000倍的PS股票）。 </li><li>胚胎孵育无论是在16 &#160; °C或18℃培养箱过夜。 </li><li>第二天早上，转让安莉芳操作系统0.1X MMR和计数存活的胚胎。 </li><li>平均来说，精子注射的卵母细胞约10％可发展到游泳蝌蚪阶段（ 图3A）。 </li></ol></li></ol>

<ol>
	<li>Miyamoto, K., Gurdon, J. B., Trounson, A., Gosden, R., Eichenlaub-Ritter, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+the+amphibian+egg+to+understand+the+mammalian+oocyte.">Insights into the amphibian egg to understand the mammalian oocyte.</a> Biology and Pathology of the Oocyte: Role in Fertility, Medicine, and Nuclear Reprogramming. , 1-11 (2013).</li><li>Heasman, J., Kofron, M., Wylie, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta-catenin+signaling+activity+dissected+in+the+early+Xenopus+embryo:+a+novel+antisense+approach.">Beta-catenin signaling activity dissected in the early Xenopus embryo: a novel antisense approach.</a> Developmental biology. 222 (1), 124-134 (2000).</li><li>Oelgeschlager, M., Kuroda, H., Reversade, B., De Robertis, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chordin+is+required+for+the+Spemann+organizer+transplantation+phenomenon+in+Xenopus+embryos.">Chordin is required for the Spemann organizer transplantation phenomenon in Xenopus embryos.</a> Developmental cell. 4 (2), 219-230 (2003).</li><li>Szenker, E., Lacoste, N., Almouzni, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+developmental+requirement+for+HIRA-dependent+H3.3+deposition+revealed+at+gastrulation+in+Xenopus.">A developmental requirement for HIRA-dependent H3.3 deposition revealed at gastrulation in Xenopus.</a> Cell reports. 1 (6), 730-740 (2012).</li><li>Heasman, J., Holwill, S., Wylie, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fertilization+of+cultured+Xenopus+oocytes+and+use+in+studies+of+maternally+inherited+molecules.">Fertilization of cultured Xenopus oocytes and use in studies of maternally inherited molecules.</a> Methods in cell biology. 36, 213-230 (1991).</li><li>Heasman, J., 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=Overexpression+of+cadherins+and+underexpression+of+beta-catenin+inhibit+dorsal+mesoderm+induction+in+early+Xenopus+embryos.">Overexpression of cadherins and underexpression of beta-catenin inhibit dorsal mesoderm induction in early Xenopus embryos.</a> Cell. 79 (5), 791-803 (1994).</li><li>Hulstrand, A. M., Schneider, P. N., Houston, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+antisense+oligonucleotides+in+Xenopus+oocytes.">The use of antisense oligonucleotides in Xenopus oocytes.</a> Methods. 51 (1), 75-81 (2010).</li><li>Miyamoto, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+Wave1+is+required+for+reprogramming+transcription+in+oocytes+and+for+normal+development.">Nuclear Wave1 is required for reprogramming transcription in oocytes and for normal development.</a> Science. 341 (6149), 1002-1005 (2013).</li><li>Halley-Stott, R. P., 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=Mammalian+nuclear+transplantation+to+Germinal+Vesicle+stage+Xenopus+oocytes+-+a+method+for+quantitative+transcriptional+reprogramming.">Mammalian nuclear transplantation to Germinal Vesicle stage Xenopus oocytes - a method for quantitative transcriptional reprogramming.</a> Methods. 51 (1), 56-65 (2010).</li><li>Astrand, C., Belikov, S., Wrange, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+acetylation+characterizes+chromatin+presetting+by+NF1+and+Oct1+and+enhances+glucocorticoid+receptor+binding+to+the+MMTV+promoter.">Histone acetylation characterizes chromatin presetting by NF1 and Oct1 and enhances glucocorticoid receptor binding to the MMTV promoter.</a> Experimental cell research. 315 (15), 2604-2615 (2009).</li><li>Amaya, E., Kroll, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+generating+transgenic+frog+embryos.">A method for generating transgenic frog embryos.</a> Methods in molecular biology. 97, 393-414 (1999).</li><li>Kroll, K. L., Amaya, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+Xenopus+embryos+from+sperm+nuclear+transplantations+reveal+FGF+signaling+requirements+during+gastrulation.">Transgenic Xenopus embryos from sperm nuclear transplantations reveal FGF signaling requirements during gastrulation.</a> Development. 122 (10), 3173-3183 (1996).</li><li>Smith, S. J., Fairclough, L., Latinkic, B. V., Sparrow, D. B., Mohun, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+laevis+transgenesis+by+sperm+nuclear+injection.">Xenopus laevis transgenesis by sperm nuclear injection.</a> Nature protocols. 1 (5), 2195-2203 (2006).</li><li>Drury, K. C., Schorderet-Slatkine, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+cycloheximide+on+the+'autocatalytic'+nature+of+the+maturation+promoting+factor+(MPF)+in+oocytes+of+Xenopus+laevis.">Effects of cycloheximide on the 'autocatalytic' nature of the maturation promoting factor (MPF) in oocytes of Xenopus laevis.</a> Cell. 4 (3), 269-274 (1975).</li><li>Smith, L. D., Ecker, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+oocyte+nucleus+in+physiological+maturation+in+Rana+pipiens.">Role of the oocyte nucleus in physiological maturation in Rana pipiens.</a> Developmental biology. 19 (3), 281-309 (1969).</li><li>Miyamoto, K., Pasque, V., Jullien, J., Gurdon, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+actin+polymerization+is+required+for+transcriptional+reprogramming+of+Oct4+by+oocytes.">Nuclear actin polymerization is required for transcriptional reprogramming of Oct4 by oocytes.</a> Genes &amp; development. 25 (9), 946-958 (2011).</li><li>Schneider, P. N., Hulstrand, A. M., Houston, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fertilization+of+Xenopus+oocytes+using+the+host+transfer+method.">Fertilization of Xenopus oocytes using the host transfer method.</a> J. Vis. Exp. (45), (2010).</li><li>Ishibashi, S., Cliffe, R., Amaya, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+bi-allelic+mutation+rates+using+TALENs+in+Xenopus+tropicalis).">Highly efficient bi-allelic mutation rates using TALENs in Xenopus tropicalis).</a> Biology open. 1 (12), 1273-1276 (2012).</li><li>Sakane, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+mutagenesis+of+multiple+and+paralogous+genes+in+Xenopus+laevis+using+two+pairs+of+transcription+activator-like+effector+nucleases.">Targeted mutagenesis of multiple and paralogous genes in Xenopus laevis using two pairs of transcription activator-like effector nucleases.</a> Development, growth &amp; differentiation. 56 (1), 108-114 (2014).</li><li>Nakayama, T., 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=Simple+and+efficient+CRISPR/Cas9-mediated+targeted+mutagenesis+in+Xenopus+tropicalis.">Simple and efficient CRISPR/Cas9-mediated targeted mutagenesis in Xenopus tropicalis.</a> Genesis. 51 (12), 835-843 (2013).</li><li>Guo, X., 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=Efficient+RNA/Cas9-mediated+genome+editing+in+Xenopus+tropicalis.">Efficient RNA/Cas9-mediated genome editing in Xenopus tropicalis.</a> Development. 141 (3), 707-714 (2014).</li></ol>

The authors have nothing to disclose.

我们在这里详细的新方法来消耗母体因素或过表达外源性因素受精前。该系统需要两个显微注射，而是跳过青蛙的手术，在主机传送方法7,17使用。它是最佳的以除去蛙手术步骤在动物护理方面。此外，我们也没有考虑到对主机雌蛙的转移实验的质量，这意味着我们可以摆脱影响的实验的成功的一个生物因素。因此，在这个系统中，从PMSG打底的蛙卵母细胞得到的质量是一个重要的生物因子是关键成功实验。卵母细胞的质量可以收集卵巢或之后defolliculation后进行判断。如果有任何异常时，在这些阶段观察到的，这是最佳的，从一个新的蛙收集另一个卵巢。当你可以检查卵母细胞质量的另一点是卵母细胞成熟后。如果小于80％的孕激素电子处理卵母细胞成熟表现的迹象，你可能无法获得足够数量的胚胎进行进一步的分析。使用酶促defolliculation代替手工defolliculation也是使用这种系统，以减少所需的defolliculation劳动的另一优点。然而，它仍然是可能的，手动defolliculation可以给出比酶处理更好的发展。 如图3中 ， 在体外几乎10％的成熟卵母细胞可到达游泳蝌蚪阶段。这些数据是从13个独立实验包括那些报告在8和新注射实验收集。主机传输方法需要在每个治疗获得有意义的数据，因为转移的卵母细胞的30-60％，可以达到神经胚阶段，17 75-150卵母细胞。我们的方法通常与200-300的卵母细胞中各实验组启动自裂解的胚胎的约40-60％可达到肌肉反应的阶段。这些数据表明，这两种方法都支持一个合理的发育率。到目前为止，我们已获得来自对照的mRNA注射的和控制的反义寡 - 注射的卵母细胞用这种技术10活着青蛙，表明该方法支持开发通过变态。 我们的卵母细胞操纵ICSI方法提供了一个机会，很早期胚胎发育过程中，测试的母体因素的作用下，受精后不久。此外，染色质修饰因子的受精前过表达使得可以重塑染色质母体受精前对理解所必需的开发产妇染色质状态。这一战略也可能会与目前的基因编辑系统，如转录激活因子样效应核酸酶（TALEN）18,19和CRISPR / CAS9 20,21因为这些即使受精前可表现正常工作。因此，我们的新方法有势的人要用于在未来的许多应用。

非洲爪蟾是一种广泛使用的，功能强大的模式生物研究开发1。这是因为， 爪蟾卵是非常大（约1.2-1.4毫米，相较于哺乳动物对口为约0.1毫米）和丰富。鸡蛋中含有母体合成和存储组件，这是足以驱动胚胎发育至中期囊胚转换（MBT，在舞台8-8.5发生与4,000-8,000细胞）。 MBT伴随合子基因的激活，然后产生了直接的进一步发展胚胎的基因产物。 许多研究旨在确定为发展的重要母体因素。许多研究依靠注射反义寡核苷酸（寡）包括吗啉代寡核苷酸为受精胚胎，其中产妇蛋白质的降解情况下，可在原肠期2-4被观察到。可替代地，的mRNA注射到受精的青霉bryos干扰基因的功能或跟踪过表达的蛋白质的命运。然而，注射入单细胞阶段的胚胎通常不影响母体蛋白质的表达水平在MBT前非常早期胚胎阶段。 Heasman和怀利建立主机传输方法来克服这个问题，5。在他们的方法中，手动defolliculated卵母细胞被注射以反义寡聚物，并转移到宿主雌性6。蛋白质受精之前下调，使早期胚胎发育下调蛋白的作用可以检查。该产妇消耗的方法导致产妇蛋白质的几个独特的发展角色的身份，在7审查。 在这份报告中，我们详细介绍我们最近开发的方法，其中受精前母体消耗mRNA的表达，或在没有人工defolliculation和主机传输实现8。手动defolliculation需要大量的时间和主机传输往往需要熟练的技术和动物手术特定许可证，从而妨碍了经常使用的产妇消耗的方法。 Defolliculation和主机传输是通过酶defolliculation 9,10和胞浆内单精子注射（ICSI）11-13，分别取代。 ICSI到鸡蛋最初用于产生转基因青蛙12。精子和胚胎细胞核也移植到体外成熟的卵母细胞的两栖11,14,15。这里，我们显示了一步一步方法注入精子在体外成熟的卵母细胞即进行预注射反义寡聚物或mRNA。

<table><tbody><tr><td>Pregnant Mare Serum Gonadotropin (PMSG)&amp;nbsp;</td><td>MSD Animal Health</td><td>Vm 01708/4309</td><td>PMSG-Intervet 5000iu powder and solvent for solution for injection</td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfonate salt (MS222)</td><td>Sigma</td><td>A5040</td><td>For anesthesia</td></tr><tr><td>Liberase TM Research Grade</td><td>Roche</td><td>05 401 127 001</td><td>For oocyte defolliculation. Store at -20&amp;nbsp;&amp;#176;C.</td></tr><tr><td>Drummond Nanoject</td><td>Drummond Scientific Company</td><td>3-000-205/206</td><td>For microinjection</td></tr><tr><td>glass capillary&amp;nbsp;</td><td>Alpha laboratories</td><td>7&amp;rdquo; Drummond #3-000-203-G/XL</td><td>For microinjection</td></tr><tr><td>Micropipette Puller&amp;nbsp;</td><td>SUTTER Instrument</td><td>Model D-97</td><td>For microinjection</td></tr><tr><td>UltraPure Agarose</td><td>Invitrogen</td><td>16500-500</td><td>For &lt;em&gt;in vitro&lt;/em&gt; maturation and ICSI</td></tr><tr><td>14 ml ultra-clear centrifuge tube&amp;nbsp;</td><td>Beckman Coulter United Kingdom Ltd</td><td>344060</td><td>For sperm purification</td></tr><tr><td>OptiPrep Density Gradient Medium (Iodixanol)</td><td>Sigma</td><td>D1556</td><td>For sperm purification</td></tr><tr><td>Digitonin</td><td>Sigma</td><td>D141</td><td>Cell permeabilization reagent</td></tr><tr><td>Shaker</td><td>Hybaid&amp;nbsp;</td><td>HB-SHK-1</td><td>For oocyte defolliculation</td></tr><tr><td>Dissecting microscope (Stereo zoom microscope)</td><td>ZEISS</td><td>Semi SV6</td><td>For oocyte collection and microinjection</td></tr><tr><td>50 &amp;mu;m pore filter</td><td>CellTrics</td><td>04-0042-2317</td><td>For sperm purification</td></tr><tr><td>Ultracentrifuge</td><td>Beckman Coulter</td><td>Optima L-100XP</td><td>For sperm purification</td></tr><tr><td>50 ml centrifuge tube</td><td>Cellstar Greiner Bio-One</td><td>227261 or 210261</td><td>Oocyte collection and defolliculation reaction</td></tr><tr><td>15 ml centrifuge tube</td><td>FALCON</td><td>352097</td><td>For sperm purification</td></tr><tr><td>90 mm Petri dish&amp;nbsp;</td><td>Thermo Scientific</td><td>101VR20/C</td><td></td></tr><tr><td>Easy-Grip Cell Culture Dish, 60&amp;nbsp;x&amp;nbsp;15 mm</td><td>FALCON</td><td>353004</td><td></td></tr><tr><td>Easy-Grip Cell Culture Dish, 35&amp;nbsp;x&amp;nbsp;15 mm</td><td>FALCON</td><td>351008</td><td></td></tr></tbody></table>

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.

使用体外成熟卵母细胞受精胚胎的胚胎发育进行了检查（ 图3A）。 GV卵母细胞的MII阶段的成熟率是可变的，在很大程度上取决于卵子的质量。在良好的实验中，GV卵母细胞几乎100％响应卵母细胞成熟的孕激素和迹象，最终在信息产业部的阶段成为鸡蛋。所有成熟的卵母细胞进行ICSI和大约25％的裂解注入卵的（ 图3A中，n = 13，用于控制扰频的寡注射的卵母细胞和n = 7，用于无寡聚注入卵母细胞）。约60％或80％的胚胎裂解，从控制低聚注射的卵母细胞或没有注射的卵母细胞产生的，分别达到了囊胚/原肠胚阶段。其中囊胚/原肠胚，大约一半寡注入样品在胚胎质量良好（异常裂解和凋亡几乎没有迹象），而囊胚/原肠胚的82％是在正质量好在注入样品（ 图3A）。最后，41％和寡聚注入样品中裂解胚胎的11％达到了肌肉响应和游泳蝌蚪阶段，分别，同时未注射的样品在60％和切割胚胎的29％那样（ 图3A）。这些ICSI胚胎是正常和不正常的胚胎（ 图3B）的混合物中。他们中的一些经历蜕变，发展到成熟的青蛙8。这些结果表明，注射反义寡核苷酸进入GV的卵母细胞，随后IVM和ICSI的，允许高效的早期胚胎发育。虽然注射反义寡核苷酸本身降低胚胎发育，我们依然能够获得足够的胚胎很多实验的目的，如检查发育率，RT-PCR，Western印迹等。 <img alt="图1" src="/files/ftp_upload/52496/52496fig1highres.jpg" /> 无花果<html> URE 1: 非洲爪蟾的典型例子蟾质量好或坏的质量体外成熟卵母细胞的实验。 （一）质量差的卵母细胞的一个例子。例如，不被用于随后的实验中卵母细胞表示片状色素沉着。每个爪蟾卵母细胞是约1.2-1.4毫米的直径。（B）中 ，一种可部分defolliculated卵母细胞。卵母细胞的右半覆盖毛囊细胞，其可以通过血管的存在而辨别。箭头示出了自由囊细胞和成注射针注入的区域。（C）的良好质量的卵母细胞，这是同样大小的，并显示均匀着色的动物半球与动物半球和植物性半球之间鲜明对比的一个例子。 2496 / 52496fig2highres.jpg"/&gt; 图2: 非洲爪蟾卵母细胞之前和成熟后卵母细胞成熟后，清晰的白点出现在动物半球和毛囊细胞的顶部被剥离。每个非洲爪蟾卵母细胞中大约为1毫米的直径。 <img alt="图3" src="/files/ftp_upload/52496/52496fig3highres.jpg" /> 图3:ICSI胚胎， 体外成熟卵母细胞生产的发展。 ICSI胚胎各阶段（A）的发展进行了总结。卵母细胞被注射以控制加扰的反义寡核苷酸（对照寡注射）或不注射（非喷射）中，随后IVM和ICSI。胚胎在每个阶段对应的数量显示上述杆。平均值±SEM表示。 N = 4-13独立的实验/不同的Females。（B）尚存ICSI胚胎的例子。这些tailbud阶段的胚胎生产了一个实验，其中约200的卵母细胞作为起始材料。

Watch this Scientific Journal Video about Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development at JoVE.com

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 ...

manipulation-vitro-maturation-xenopus-laevis-oocytes-followed

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

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

22.4K 次观看.  University of Cambridge. 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.

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

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

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, ...

Functional Cloning Using a Xenopus Oocyte Expression System

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in abundance, may not be secreted but tethered to the signaling cell(s), or may require the presence of binding partners or upstream regulatory molecules. Thus in a search for gene products capable of eliciting an early lens-inductive response in competent ectoderm, we utilized an expression cloning system that would allow identification of paracrine or juxtacrine factors as well as transcriptional or other regulatory proteins. Pools of mRNA were injected into Xenopus oocytes, and responding tissue placed directly on the oocytes and co-cultured. Following functional cloning of ldb1 from a neural plate stage cDNA library based on its ability to elicit the expression of the early lens placode marker foxe3 in lens-competent animal cap ectoderm, we characterized the mRNA expression pattern, and assayed developmental progression following overexpression or knockdown of ldb1. This system is suitable in a very wide variety of contexts where identification of an inducer or its upstream regulatory molecules is sought using a functional response in competent tissue.

Functional Cloning Using a Xenopus Oocyte Expression System

We describe a Xenopus oocyte and animal cap system for the expression cloning of genes capable of inducing a response in competent ectoderm, and discuss techniques for the subsequent analysis of such genes. This system is useful in the functional identification of a wide range of gene products.

功能性克隆使用<em&gt;爪</em&gt;卵母细胞表达系统

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in ...

Technique to Target Microinjection to the Developing Xenopus Kidney

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 embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

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 ...

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into the developing oocyte. Because these events rely on maternal products which typically act very soon after fertilization-that preexist inside the egg, standard approaches for expression and functional reduction involving the injection of reagents into the fertilized egg are typically ineffective. Instead, such manipulations must be performed during oogenesis, prior to or during the accumulation of maternal products. This article describes in detail a protocol for the in vitro maturation of immature zebrafish oocytes and their subsequent in vitro fertilization, yielding viable embryos that survive to adulthood. This method allows the functional manipulation of maternal products during oogenesis, such as the expression of products for phenotypic rescue and tagged construct visualization, as well as the reduction of gene function through reverse-genetics agents.

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here.

产妇基因产品的官能化操作使用<em&gt;体外</em斑马鱼&gt;卵母细胞成熟

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into ...

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

Fertilization of Xenopus oocytes using the Host Transfer Method

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to generate maternal-effect mutant animals. Additionally, many of the techniques to overexpress or inhibit gene function in organisms such as Xenopus and zebrafish fail to sufficiently target critical maternal signaling pathways, such as Wnt signaling. In Xenopus, manipulating gene function in cultured oocytes and subsequently fertilizing them can ameliorate these problems to some extent. Oocytes are manually defolliculated from donor ovary tissue, injected or treated in culture as desired, and then stimulated with progesterone to induce maturation. Next, the oocytes are introduced into the body cavity of an ovulating host female frog, whereupon they will be translocated through the host's oviduct and acquire modifications and jelly coats necessary for fertilization. The resulting embryos can then be raised to the desired stage and analyzed for the effects of any experimental perturbations. This host-transfer method has been highly effective in uncovering basic mechanisms of early development and allows a wide range of experimental possibilities not available in any other vertebrate model organism.

Fertilization of Xenopus oocytes using the Host Transfer Method

Procedure for fertilizing Xenopus oocytes by the host transfer method.

施肥爪蟾卵母细胞使用主机传输方法

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

活细胞成像和胚胎上皮细胞的定量分析非洲爪蟾</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

生产转基因非洲爪蟾限制性内切酶介导的整合和核移植

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

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利用共聚焦分析

干细胞样

功能性克隆使用

技术目标显微注射到显影

产妇基因产品的官能化操作使用

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

施肥爪蟾卵母细胞使用主机传输方法

活细胞成像和胚胎上皮细胞的定量分析非洲爪蟾

生产转基因非洲爪蟾限制性内切酶介导的整合和核移植