作者想感谢休斯顿实验室的关键读的手稿成员。研究是支持的美国国立卫生研究院授予DWH（GM083999）

1。手术切除的卵巢组织<ol class="lalpha"><li>准备一批新的卵母细胞培养液中（OCM;材料），根据实验的大小之间的400-800毫升。调整pH值至7.6-7.8，直到酚红变成深红色（5N氢氧化钠6个左右的小滴）。倒出少量检查在一个单独的管的pH值，因为pH电极可能污染媒体。 OCM应存放在18 ° C，并在一个星期内使用。 </li><li> 〜2L的缓冲麻醉溶液（材料）中选择3-5名女性和地方之一。我们尽量避免使用，美联储在当天或手术前的青蛙。虽然青蛙是屈服于麻醉剂，用70％乙醇消毒手术区，并准备手术器械。 </li><li>消毒手术器械浸泡在炎热的珠灭菌20秒，在250 ° C。下列文书应手：手术刀处理（＃3）和刀片（＃10或11），杜蒙钳，波恩虹膜剪刀（弯曲或直），哈尔西或奥尔森Hegar微持针器，并缝合几双。广场上保持无菌缝合包内无菌培养皿或消毒工具。把握针持针器开始前所需的方向。我们喜欢奥尔森Hegar针持有人，因为在处理包含剪刀刀片，可用于调整缝线，而无需切换文书。 </li><li> 10分钟后，检查已经取得了手术的麻醉平面。麻醉青蛙会停止移动，并不会响应一个脚趾捏或上缴。麻醉应持续15分钟，这是足够的时间来执行操作。唐合适的手术装束。这可以包括清洁（非乳胶）手套，实验室外套，和口罩，根据个人的喜好和机构动物照顾指引。 </li><li>注意无菌技术操作，青蛙的皮肤分泌物中含有抗菌肽，一般在手术过程中减少污染的发生，虽然无菌技术操作应尽可能。可用于外科窗帘，但可能并不需要的体制IACUCs和可能无法提供多少好处。一般“尖端技术”使用，其中的手做触摸仪器，缝合材料或切口部位的无菌针头。仪器可resterilized（珠灭菌）以下的皮肤切口，无菌区应保持台式放置工具（如培养皿或缝合包）。体制要求，可以显着差异，所以请按照您的机构“的指导方针，并收到相应的指令执行手术前。 </li><li>从麻醉中取出的青蛙和背斜卧在潮湿的手术擦拭。注。 povidine碘稀释（1:20）或洗必泰（0.75％）冲洗可以清除皮肤。然而，要避免商业外科磨砂，肥皂和70％的异丙醇，因为这些东西会损伤娇嫩的肌肤两栖动物。 </li><li>使用手术刀或虹膜小剪刀，一个小（1 - 1.5厘米）切口在腹部，侧中线下部的皮肤。备用双方应执行相同的青蛙的后续切口，互相间隔。切口可平行或垂直于中线。 </li><li>请在两个阶段的皮肤和腹部肌肉的切口。切割后的皮肤，解除肌肉层和周围的白色筋膜与钳，并提出肌肉的切口。用剪刀和剪直下，尝试作出一个迅速切断暴露体腔，留下干净的伤口边缘。如果只是筋膜被切断，它会收回，并将使缝合更加困难。解除肌肉将有助于避免无意中伤人任何内部器官。另外，尽量避免通过任何可见的淋巴结心脏或血管的肌肉层内切割。扩大与剪刀的切口长度，使卵巢可以很容易地渡过难关。 </li><li>一旦切口，卵巢应清晰可见。促使卵巢，抓钳和外部的卵巢组织。组织所需的金额被删除，在体壁的水平和修剪。不要东西的组织，进入体腔腔，因为这可能是感染或其他并发症的源。 </li><li>立即在解剖显微镜下观察卵巢的小块defolliculate几个卵母细胞，以测试他们的素质。他们应该坚定和统一的大小和着色。如果他们是可以接受的的，取出所需数量的卵巢组织，缝合切口。足以装满一个90毫米的培养皿通常足以满足大多数实验。如果存在质量问题的卵母细胞，缝合青蛙和重复使用不同的女性。 </li><li>关闭切口缝合肌肉和筋膜层。插入的N<html> eedle一个外侧切口几毫米，一定要通过以及通过筋膜层。刚刚通过的肌肉缝合撕裂组织和松动。使用镊子，以帮助从下面插入切口对面针。释放和重新把握针从体内外，渡过难关，直到几英寸左右的尾巴仍然缝合，照顾，不要把它拉一路过关斩将。 </li><li>关闭伤口缝合模式用一个简单的中断。我们使用一个基本的仪器配合，使外科医生的平方海里（礁节）。循环各地的持针器缝合的长端，抓住尾巴，并通过循环拉向你并拧紧。循环和再次转危为安，此时远离你拉持针器。第三扔掉，可用于增加安全性（在同一方向作为第一）。修剪缝合，留短尾巴，并在另一缝合肌肉几毫米远。两个缝线足够小缝线;较大的3个。重复的皮肤层。基本缝合技术的视频演示，YouTube上搜索 - “仪器领带”，也有不少选择，但基本技术是相同的。 </li><li>用去离子水冲洗掉的女性，并把她放在阴凉（18℃）水填补了回收桶。虽然在实验室中，青蛙是保存在与Amquel处理，以去除氯的自来水。每隔几分钟，轻轻从水中青蛙，并期待囫囵吞枣或眼胀的动作，从麻醉中复苏的指标。 </li><li>注：生存手术是不是绝对必要的，以取得卵巢组织，虽然女性将继续产生良好的卵母细胞后，许多操作。执行手术的生存也将减少使用动物的数量。一定要按照动物保健单位的指导方针，在脊椎动物生存的手术方法，术后护理和监测，并允许个人手术数量。 </li></ol> 2。培养和Defolliculating卵母细胞<ol class="lalpha"><li>右后手术完成后，细分成小块（1〜2厘米2），并存储在新鲜OCM使用5-6件，每90毫米菜卵巢。个人裂片卵巢剖开，夷为平地，并切成见方的小块。这些冲洗干净OCM，并放置在培养皿。扁平化和分裂的卵巢，将扩大在文化生活和defolliculation容易。 </li><li>移动叶小碟的OCM和手动defolliculate 300-500卵母细胞。使用无菌，火抛光的吸管转移卵母细胞。我们发现，用棉花堵塞各种，是尽量减少污染的文化的关键。商店的卵母细胞，在18 ° C在8毫升OCM（中期菜（猎鹰1007））在100-150组。通过练习，你应该能够defolliculate足够的卵母细胞，在1-2小时内。 Collagenased卵母细胞不适合主机的传输，因为他们不能fertilizable。 </li><li> Defolliculating方法。我们defolliculate使用制表匠钳（杜蒙＃4S或5S，精细科学的工具），Smith 等人描述的基本方法，（1991）。使用一对钳（在你的惯用手），掌握卵泡膜的结缔组织层，周围完全成熟的阶段六，卵母细胞卵泡卵巢（柄）不远的地方。随着对其他​​钳，把握第一对相邻的卵巢。轻轻撕开拉动整个卵母细胞卵泡层。轻轻梳理出组织的卵母细胞。成功defolliculated的卵母细胞卵母细胞比简单拉不去除毛囊层，将没有可见的血管floppier。 </li><li>注：重要的是保持很轻的压力钳提示，提示，以满足仅够。夹持得太紧，是一种常见的初学者，并会导致卵母细胞的卵泡完好被拉。专门为defolliculating保持镊子一双好。提示应符合恰恰应略高于钳爪蟾胚胎（图1）植粗短。 </li><li>我们定期测试约20黄体酮和中止实验，如果成熟的成熟率差（&lt;50％）在室温下6-7小时后。 </li><li>此时，卵母细胞可以被操纵和培养的需要，如寡核苷酸，吗啉或mRNA显微注射。注射程序而有所不同，实验室，所以我们不会在这里介绍这些。卵母细胞可以维持到一个星期在OCM，但一般健康的下降，所以最好是使用隔离96小时内转移。 </li><li>请记住，只有5至6组（包括控制），可以转移到一个单一的女性，由于重要的染料的限制，所以你的实验计划。 </li></ol>“&gt; 3。卵母细胞成熟和刺激未来东道国女性<ol class="lalpha"><li>如果执行寡枯竭的救援，注入约24-48小时后寡核苷酸基因的RNA（通常为50-300 PG）。寡核苷酸将有这个时间退化，不应影响注入的RNA。 </li><li>在执行转移前的晚上，加16μL孕酮工作液（乙醇1mm）的8毫升OCM，在中菜（最后的孕激素的浓度是1〜2微米）的卵母细胞。孵育10-12小时，18 ° C至使卵母细胞成熟。 </li><li>注入1000个单位的hCG青蛙3-5女性诱发产蛋。我们的实验似乎更好地工作，如果卵母细胞成熟和注射HCG大约在同一时间完成，约12个小时到主机（例如，10日下午-10上午）植入前。让女性在室温或18 ° C过夜。 </li></ol> 4。生命的卵母细胞的制备及染料染色<ol class="lalpha"><li>在上午的转移，使〜45-60“建立和执行程序。在这一点上，成熟的卵母细胞可以被冻结后的基因敲除或蛋白表达的疗效分析。同时，检查的卵母细胞没有受到感染，并有一个很好的成熟率（&gt; 50％）。受感染的卵母细胞，将不施肥。解冻重要的染料（材料），并在5分钟最大转速旋转。 </li><li>颜色不同的实验组加入80μL适当重要的染料（S）的卵母细胞的菜肴，并培育15&#39;摇摆。在这段时间内，选择一台主机和女性开始麻醉。选择了一只青蛙，慢慢地产卵正​​常或轻度挤压产生丰富的鸡蛋。从主机的鸡蛋应质量好。避免铺设的鸡蛋或粉碎鸡蛋串的女性。 </li><li>彩蛋传输大量的新鲜OCM，菜，漩涡简单地洗，事先放在一边转移。 </li><li>准备一个无菌的巴斯德吸管与孔宽度足以容纳一个用于移植的卵母细胞的卵母细胞（1〜1.5毫米）。分数与钻石铅笔吸管和打破干净。消防波兰，直到边缘平滑，注意不要融化开幕闭幕。 </li></ol> 5。表演的卵母细胞移植<ol class="lalpha"><li>卵母细胞移植使用在第I部所述的相同的手术过程中，虽然，操纵，而不是消除卵巢卵母细胞进入体腔用移液器介绍。理想的情况下，过户手续应尽快与切口的大小应保持尽可能小。 </li><li> Anesthesize主机女性和准备手术器械，在第二部分所述一和切口部分中描述的一切口可较小，但需要足够大的移液管（图2A），以适应孔。 </li><li>用镊子移植的卵母细胞，掌握和提高肌肉和筋膜瓣，介绍使用巴斯德吸管准备上述实验的卵母细胞。漩涡中心收集到的卵母细胞的菜他们和尽可能多的吸。允许进入移液器年底定居的卵母细胞被引入，以防止多余的液体，切口插入吸管尖，慢慢让卵母细胞滴入到体腔（图2B）。重复，直到所有的卵母细胞已被转移。不要随时发布的肌肉层，直到你已经开始缝合，或卵母细胞，将切口溢出。 </li><li>后的卵母细胞已经转移，掌握与钳切口的两侧，并带他们一起，使卵母细胞进入人体腔定居。开始缝合描述的那样，照顾上的肌肉和筋膜保持向上的张力，直到第一个结绑（图2C）。这将让被驱逐出卵母细胞，将允许边缘切口愈合均匀。添加另一肌肉/筋膜层缝合，然后缝合皮肤。 </li><li>用去离子水冲洗掉的女性，她放置在一个充满冷却（18℃）Amquel处理过的水回收桶。上述监视她从麻醉中复苏。她应该开始铺设蛋通常在手术后。 </li><li>获得使用正常程序的青蛙一个男性睾丸。商店在OCM或L15睾丸，直到需要。施肥转移实验时，我们更喜欢新鲜的睾丸。 </li></ol> 6。回收的卵母细胞体外受精 <ol class="lalpha"><li>腹膜纤毛将驱动器传输的蛋（和主机的正常体腔鸡蛋）在体腔的前输卵管的开口。彩蛋可以收回，转让后2-3小时开始正常挤压。蛋可每半小时左右的女性，直到停止铺设。 </li><li>施肥在一个SPERM悬浮液（〜4毫升1X孕产妇死亡率）为4分钟。洪水和冲洗与0.1倍的MMR广泛。鸡蛋应激活正常，将开始裂解〜1.5-2小时受精后。转蛋往往会切割略晚于主机鸡蛋。 </li><li>半胱氨酸的正常和排序胚胎成单独的菜肴取出果冻大衣。这是可以做到在任何阶段，根据实验的需要。它通常最容易识别周围的4细胞阶段的各种颜色和晚期囊胚阶段，在最困难的。文化的鸡蛋在一个干净的0.1倍的MMR低密度（&lt;50％中等菜）。从这一点来说，可以被视为胚胎和操纵正常。 </li><li>将青蛙的动物设施和显示器在未来数天的并发症。 </li><li>替代的施肥方法;产蛋高盐缓冲液<ol class="lroman"><li>青蛙从麻醉中恢复后，她到〜1L高盐MMR（1.2倍）。允许女性奠定蛋约4-6小时到缓冲区。挤压剩下的鸡蛋，排水1.2倍MMR和清洁碟的彩蛋排序;删除所有可能的液体。 </li><li>洗净广泛0.3X孕产妇死亡率和受精鸡蛋，直到激活（10分钟）在最小的体积0.3X的MMR /精子悬液。洪水与0.1倍MMR和以上文化。这个选项是非常有用的，如果在同一个舞台，或者如果鸡蛋手册表达损害捐助卵母细胞所需的许多鸡蛋。 </li></ol></li></ol>

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D., Ecker, R. E., Subtelny, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+induction+of+physiological+maturation+in+Rana+pipiens+oocytes+removed+from+their+ovarian+follicles.">In vitro induction of physiological maturation in Rana pipiens oocytes removed from their ovarian follicles.</a> Dev Biol. 17, 627-643 (1968).</li><li>Smith, L. D., Xu, W., Varnold, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oogenesis+and+oocyte+isolation.">Oogenesis and oocyte isolation.</a> Methods Cell Biol. 36, 45-60 (1991).</li><li>Woolf, T. M., Jennings, C. G., Rebagliati, M., Melton, D. A. <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,+toxicity+and+effectiveness+of+unmodified+and+phosphorothioate+antisense+oligodeoxynucleotides+in+Xenopus+oocytes+and+embryos.">The stability, toxicity and effectiveness of unmodified and phosphorothioate antisense oligodeoxynucleotides in Xenopus oocytes and embryos.</a> Nucleic Acids Res. 18, 1763-1769 (1990).</li><li>Wylie, C., Kofron, M., Payne, C., Anderson, R., Hosobuchi, M., Joseph, E., Heasman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+beta-catenin+establishes+a+'dorsal+signal'+in+early+Xenopus+embryos.">Maternal beta-catenin establishes a 'dorsal signal' in early Xenopus embryos.</a> Development. 122, 2987-2996 (1996).</li><li>Zhang, J., Houston, D. W., King, M. L., Payne, C., Wylie, C., Heasman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+maternal+VegT+in+establishing+the+primary+germ+layers+in+Xenopus+embryos.">The role of maternal VegT in establishing the primary germ layers in Xenopus embryos.</a> Cell. 94, 515-524 (1998).</li><li>Zuck, M. V., Wylie, C. C., Heasman, J., Richter, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+mRNAs+in+Xenopus+embryos:+an+antisense+approach..">Maternal mRNAs in Xenopus embryos: an antisense approach..</a> A comparative methods approach to the study of oocytes and embryos. , 341-354 (1998).</li></ol>

动物实验是由美国爱荷华IACUC委员会大学规定的准则和法规的规定执行。

一个成功的转移，将导致受精和正常的卵母细胞&gt; 50％（图3）裂解。过去一般在30-60％的转让的卵母细胞存活的神经胚阶段。我们通常是75-150％实验组的卵母细胞，这将产生几种类型的分析足够的胚胎（原位RT - PCR）以及可视化的表型转移。植入大幅更多的卵母细胞似乎并不大大提高产量。此外，应至少有30％组的卵母细胞转移到保证，至少有一些通过原肠。重要的染料一般不影响这里描述的浓?...

解决方案和食谱： OCM（卵母细胞培养液中，从卫理等修改，1996年。 ） <table border="0"><tbody><tr><td> 70％的L - 15的Leibovitz的中等（含L -谷氨酰胺） </td><td> （Invitrogen公司11415-064） </td></tr><tr><td> 0.4 mg / ml的牛血清白蛋白（BSA的分数V） </td><td> （SIGMA A - 9418 - 50G） </td></tr><tr><td> 1X青霉素，链霉素</td><td> （Sigma公司的P - 0781） </td></tr><tr><td colspan="2"> 5N氢氧化钠调整pH值7.6-7.8 </td></tr><tr><td colspan="2">在18 ° C，每周新鲜，商店</td></tr></tbody></table> 10X MMR（Marc的改良林格液;扎克等人 ，1998年） <table border="0"><tbody><tr><td> 1M氯化钠</td></tr><tr><td> 20 mM的氯化钾</td></tr><tr><td> 20毫米氯化钙2 </td></tr><tr><td> 10毫米氯化镁2 </td></tr><tr><td> 150毫米的HEPES </td></tr><tr><td>调整pH值7.6-7.8 </td></tr><tr><td>保存在4 ° C </td></tr></tbody></table>麻醉的解决方案<table border="0"><tbody><tr><td> 1 g / L的3 - 氨基苯甲酸乙酯（MS222） </td><td> （Sigma公司A - 5040） </td></tr><tr><td colspan="2"> 0.7 g / L的碳酸氢钠</td></tr><tr><td colspan="2">在Amquel处理过的水溶解</td></tr><tr><td colspan="2">新鲜每周</td></tr></tbody></table>孕酮股票<table border="0"><tbody><tr><td> 100％的乙醇10毫米孕酮（Sigma公司的P - 0130） </td></tr><tr><td>为500X的股票，在100％乙醇稀释至1毫米/工作方案</td></tr><tr><td>储存在-20 ° C </td></tr></tbody></table>重要的染料（Heasman 等 ，1991年修改） <table border="0"><tbody><tr><td>蓝：0.1％耐尔蓝一</td><td> （Aldrich公司121479 - 5G）， </td></tr><tr><td>红色：0.25％中性红</td><td> （西格玛ñ - 6634）， </td></tr><tr><td>布朗：布朗俾斯麦1％ </td><td> （Sigma公司的B - 2759）， </td></tr><tr><td colspan="2">绿色（80μL蓝+ 80μL棕色）， </td></tr><tr><td colspan="2">紫红色（80μL蓝+ 80μL红色）。 </td></tr><tr><td colspan="2">第六个颜色，橙色（80μL红+ 80μL棕色），也可以使用，但往往是很难区分从褐色。 </td></tr></tbody></table>在去离子水蓝色，红色和褐色的股票于50 ml管，孵育20分钟摇摆，并在临床离心机纺。上清aliquotted到微型离心管中，并储存在-20 ° C

fertilization of xenopus oocytes using the host transfer method

往往阻碍了必要的时间和费用，产生产妇效应突变动物研究母系遗传的分子脊椎动物的早期发展的贡献。此外，许多技术如爪蟾和斑马鱼的生物过表达或抑制基因功能未能充分针对危重孕产妇的信号转导通路，如Wnt信号。在非洲爪蟾 ，在培养的卵母细胞的基因功能和操纵，随后施肥可以在一定程度上改善这些问题。手动defolliculated卵母细胞是从供体卵巢组织，注射或视为文化，然后用黄体酮刺激诱导成熟。下一步，卵母细胞引入腔体内的雌蛙排卵主机，届时他们将通过主机的输卵管移位和掌握修改和果冻大衣施肥必要。由此产生的胚胎，然后将筹集到所需的阶段和任何实验扰动的影响分析。这台主机传输方法已发现早期发展的基本机制非常有效，并没有在任何其他脊椎动物模式生物的实验可能允许广泛。

Watch this Scientific Journal Video about Fertilization of Xenopus oocytes using the Host Transfer Method at JoVE.com

Fertilization of Xenopus oocytes using the Host Transfer Method

对施肥的程序<em&gt;爪蟾卵母细胞</em由主机传输方法。

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 ...

fertilization-of-xenopus-oocytes-using-the-host-transfer-method

Research

JoVE Journal

Biology

Fertilization of Xenopus oocytes using the Host Transfer Method

14.5K 次观看.  University of Iowa. 对施肥的程序爪蟾卵母细胞

视频: Fertilization of Xenopus oocytes using the Host Transfer Method

Nuclear Transfer into Mouse Oocytes

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying development, differentiation and aging are epigenetic rather than genetic processes. The reversibility of these processes opens exciting perspectives in basic research, and in the more distant future, in regenerative medicine. In the mouse, embryonic stem cells can be derived from cloned preimplantation stage embryos. Such embryonic stem cells have the ability to give rise to all cell types of the adult organism. Importantly, these cells are genetically identical to the donor. If applicable to human, this would allow the derivation of stem cells from a patient. These cells could then be differentiated into the affected cell type of the patient and studied in vitro, or used to replace the damaged or missing cells. The study of nuclear transfer in the mouse remains important as it can inform us about the principles of nuclear reprogramming. This movie and the accompanying protocol are intended to help learning nuclear transfer in the mouse, a method initially developed in the group of Prof. Yanagimachi (WAKAYAMA et al. 1998).

This movie and the protocol are intended to help learning nuclear transfer.

小鼠卵母细胞的细胞核移植到

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying ...

科研

生物学

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

补丁钳爪蟾卵母细胞中表达的离子通道

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

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 ...

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

对于斑马鱼精子超低温冷冻保存的一个高通量的方法和<em在体外施肥

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

可视化的RNA中的本地化爪蟾卵母细胞

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development of a larger region of the embryo. This makes defining its role in a particular tissue difficult to analyze. For example, noggin overexpression in Xenopus laevis embryos causes the expansion of the entire anterior region, including the eye1,2. From this result, it is not known if Noggin plays a direct role in eye determination or that by causing an expansion of neural tissue, Noggin indirectly affects eye formation. Having this complex phenotype makes studying its eye-specific role in cell fate determination difficult to analyze. We have developed an assay that overcomes this problem. Taking advantage of the pluripotent nature of the Xenopus laevis animal cap 3, we have developed an assay to test the ability of gene product(s), like noggin or the eye field transcription factors (EFTFs), to transform caps into particular tissue or cell types by transplanting this tissue onto the side of the embryo 4. While we have found either Noggin protein treatment or a collection of transcription factors can determine retinal cell fate in animal caps, this procedure could be used to identify gene product(s) involved in specifying other tissues as well.

Tissue Determination Using the Animal Cap Transplant ACT Assay in Xenopus laevis

Animal caps overexpressing gene product(s) are transplanted to the flank of developing Xenopus laevis embryos in order to establish whether tissue is determined.

组织测定动物第移植（ACT）的含量非洲爪蟾</em

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

膜片钳和灌注技术研究离子通道的表达爪蟾卵母细胞

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage checkpoint signaling as a surveillance mechanism to sense DNA damage and direct cellular responses to DNA damage. There are several groups of proteins called sensors, transducers and effectors involved in DNA damage checkpoint signaling (Figure 1). In this complex signaling pathway, ATR (ATM and Rad3-related) is one of the major kinases that can respond to DNA damage and replication stress. Activated ATR can phosphorylate its downstream substrates such as Chk1 (Checkpoint kinase 1). Consequently, phosphorylated and activated Chk1 leads to many downstream effects in the DNA damage checkpoint including cell cycle arrest, transcription activation, DNA damage repair, and apoptosis or senescence (Figure 1). When DNA is damaged, failing to activate the DNA damage checkpoint results in unrepaired damage and, subsequently, genomic instability. The study of the DNA damage checkpoint will elucidate how cells maintain genomic integrity and provide a better understanding of how human diseases, such as cancer, develop.
Xenopus laevis egg extracts are emerging as a powerful cell-free extract model system in DNA damage checkpoint research. Low-speed extract (LSE) was initially described by the Masui group1. The addition of demembranated sperm chromatin to LSE results in nuclei formation where DNA is replicated in a semiconservative fashion once per cell cycle.
The ATR/Chk1-mediated checkpoint signaling pathway is triggered by DNA damage or replication stress 2. Two methods are currently used to induce the DNA damage checkpoint: DNA damaging approaches and DNA damage-mimicking structures 3. DNA damage can be induced by ultraviolet (UV) irradiation, &gamma;-irradiation, methyl methanesulfonate (MMS), mitomycin C (MMC), 4-nitroquinoline-1-oxide (4-NQO), or aphidicolin3, 4. MMS is an alkylating agent that inhibits DNA replication and activates the ATR/Chk1-mediated DNA damage checkpoint 4-7. UV irradiation also triggers the ATR/Chk1-dependent DNA damage checkpoint 8. The DNA damage-mimicking structure AT70 is an annealed complex of two oligonucleotides poly-(dA)70 and poly-(dT)70. The AT70 system was developed in Bill Dunphy's laboratory and is widely used to induce ATR/Chk1 checkpoint signaling 9-12.
Here, we describe protocols (1) to prepare cell-free egg extracts (LSE), (2) to treat Xenopus sperm chromatin with two different DNA damaging approaches (MMS and UV), (3) to prepare the DNA damage-mimicking structure AT70, and (4) to trigger the ATR/Chk1-mediated DNA damage checkpoint in LSE with damaged sperm chromatin or a DNA damage-mimicking structure.

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

Xenopus egg extract is a useful model system to investigate the DNA damage checkpoint. This protocol is for the preparation of Xenopus egg extracts and DNA damage checkpoint inducing reagents. These techniques are adaptable to a variety of DNA damaging approaches in the study of the DNA damage checkpoint signaling.

研究DNA损伤检验点<em&gt;爪蟾</em&gt;卵提取物

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

在EMBL的MultiBac蛋白质复杂的生产平台

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H+,K+-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb+ or Li+ transport by Na+,K+- or gastric H+,K+-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb+ (Li+) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb+ uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na+/2K+ transport stoichiometry of the Na+,K+-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H+,K+-ATPase. In principle, the assay is not limited to K+-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.

测量离子转运的Na，K，H，K-ATP酶活性的<em&gt;爪蟾</em卵母细胞的原子吸收分光光度法：另一种放射性同位素检测

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating ...