Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R01 GM33397. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. J.G.G. is American Cancer Society Professor of Developmental Genetics.

关于青蛙和蝾螈的一般信息，以及动物的来源，可在以下网站上找到:Xenbase（http://www.xenbase.org）和萨尔站点（http://www.ambystoma.org）。该协议遵循卡内基研究所胚胎学系为科学的动物护理准则。 1.解决方案<ol><li>使10升"蛙水":添加10毫升的1M的NaHCO 3到10升去离子水或脱氯的 H 2ö1M的氯化钙和10毫升并搅拌。 </li><li>使1升20％多聚甲醛:使1升的4毫的 Na 2 CO 3。在化学油烟机加200克粉状多聚甲醛，热到约80℃溶解。凉，并通过滤纸过滤。注意:多聚甲醛是有毒的，必须小心处理。另外，购买商业多聚甲醛溶液。 </li><li>让1升两栖类动物麻醉剂溶液:加入1.5克3- aminoben的zoate甲磺酸盐至1L蛙水。室温保存。 </li><li>使1升的卵母细胞的培养基OR2 14:82.5毫摩尔NaCl，2.5mM的氯化钾，1mM的氯化钙 ，1mM的MgCl 2的，1毫米的 Na 2 HPO 4，5mM的HEPES（500毫库存，pH值8.3）。最终的pH将是约pH 7.8。添加100毫克的氨苄青霉素和100毫克链霉素，以防止细菌的生长。 </li><li>让1升GV隔离解决方案:83毫米氯化钾，17毫米氯化钠，6.5毫米的 Na 2 HPO 4，3.5毫米的KH 2 PO 4，1毫米氯化镁2，1二硫苏糖醇（DTT）。调节pH至7.0。 </li><li>使100毫升的GV分散液:20.7毫米氯化钾，4.3毫摩尔NaCl，1.6毫摩尔的 Na 2 HPO 4，0.9毫摩尔KH 2 PO 4，1mM的MgCl 2的，1毫二硫苏糖醇（DTT），0.1％多聚甲醛。调节pH至7.0。为了便于核凝胶的更迅速扩散，添加10-50微米的CaCl 2。 </li><li>让500毫升胶层的解决方案。膨胀0.5克商业明胶20毫升水2 O 5 - 10分钟。加入500毫升煮沸H 2 O和精心漩涡溶解明胶。酷并添加50毫克CRK（SO 4）2·12 H 2 O.吸和存储滤波器在4℃ </li><li>让10毫升装网上平台。溶解10毫克苯二胺的5ml的PBS（135 mM氯化钠，2.5mM的氯化钾，4.3毫的 Na 2 HPO 4，1.5毫摩尔KH 2 PO 4，调节pH为9）。加入5毫升甘油。存放在250微升等分在-80℃。 </li></ol> 2.材料<ol><li>对于底涂幻灯片，代替3-英寸×1英寸玻璃显微镜载玻片在商业洗涤剂溶液和揉搓它们以除去任何污染物。在幻灯片机架地方，自来水冲洗干净，然后在去离子水。沾在胶层解决方案的幻灯片，在一个角度外流，并在60 摄氏度烘烤过夜</li><li>塑料方形，减少25平方毫米从1毫米厚的聚（甲基丙烯酸甲酯）片材。一个钻5毫米圆孔在每平方米的中心。砂两面和方以及该孔的圆周的边缘。 注意:作为替代自制塑料正方形和良好滑动，如在接下来的段落中所描述，使用原位 PCR和杂交腔室（25μL）的商业粘合剂。一个很可能也使用细胞培养用可重复使用的有机硅隔离器，虽然这并没有被彻底尚未测试。 </li><li>对于好幻灯片，融20 -热板50毫升玻璃烧杯- （57 摄氏度 56）30克石蜡。用20微升移液管，扩散石蜡之一5μL的滴上的底涂滑动的中心的每一侧。按一个塑料方形入石蜡和滑动放置到热板上。石蜡融化，并应在塑料方形下均匀扩散。 <ol><li>取下热板幻灯片，让它冷却。放置支撑滑动的各端下，使滑动不接触TABL在冷却过程中ETOP。如果石蜡冷却过快，有可能拉离中心孔的路程。准备10个或更多以及幻灯片:每个GV准备采用新井幻灯片。 </li></ol></li><li>对于载玻片载体用于离心，使用特殊构造的载体中使用的转子。一些制造商提供的离心机与运营商96 - 以及塑料板。这些载波可以很容易地适于保持显微镜载玻片。 </li></ol> 3.卵母细胞的分离<ol><li>放置在足够麻醉剂溶液中的成年雌性蛙（ 爪蟾或爪蟾热带 ）或蝾（Ambystoma mexicanum）以完全覆盖的动物。当动物停止运动，并将其放置在合适的容器上刨冰床肚皮了。 </li><li>用手术剪使2 - 3厘米切口下腹部横向中线。移动至内脏轻轻放在一边上找到的切口一侧的卵巢（第ERE是两个卵巢，一个在腹部的每一侧）。切出具有足够的卵母细胞用于计划实验卵巢的一部分。因为有成千上万的卵母细胞中的每个卵巢，相对小片的卵巢（1 - 3厘米）通常就足够了。 </li><li>将一个或卵巢几块在OR 2盐水中在室温下在大陪替氏培养皿（100毫米）。卵母细胞可被存储在良好的条件数天，如果损坏的卵母细胞被除去，OR2溶液每天更换。 </li><li>缝合手术丝绸切口和动物返回到个人玻璃碗进行恢复。加入足够的"蛙水"来掩盖动物，直到它恢复意识（约15分钟）。动物展示自主运动后，填补了碗水。虽然淡水应使用，无菌性是没有必要的，因为两栖类几乎不会成为手术后感染。如果需要的话，新霉素可以应用到缝线。伤口通常FE后完全治愈瓦特天，并且在同一动物可以再次约2个月后使用。 </li></ol> 4.一株生发泡（GV）的<ol><li>将一个卵巢的一部分 - 在包含OR 2解决方案的小皮氏培养皿（35×10mm）的（10 20大卵母细胞）。 </li><li>从使用两对＃5珠宝商的镊子撕组织的细柄的卵母细胞连接到子房壁卵巢取出卵母细胞。放入含GV分离培养基另一个小培养皿卵母细胞。 注:LBCs的状态取决于卵母细胞的大小（到期日）。 LBCs达到与著名的环最大长度在比全尺寸略少卵母细胞。最成熟的卵母细胞中通常包含以简约循环短染色体。 </li><li>执行解剖显微镜（场直径约10 - 20毫米）的低放大倍数下的以下步骤。 <ol><li>两手抓，对附近的动物极（暗半球）镊子的卵母细胞，并作出SM所有的眼泪。看在用于透明卵母核被挤出的蛋黄，也被称为胚泡或GV。 </li><li>另外，捅附近的动物极一个大洞，一个钳子，轻轻地用蛋黄（ 图2）沿挤出GV。 </li><li>轻轻摇动GV从散装蛋黄的路程。通常会有少量蛋黄的牢固附着到GV。 </li></ol></li></ol> 5.罢免核膜<ol><li>对于非洲爪蟾和十热带 ，从分离培养基转移到GV蔓延介质中60秒隔离，即使仍然有一点点蛋黄贴壁。 注意:如果留在隔离介质，而不会在随后的步骤中扩散GVS这两个物种的"硬化"在约60秒。因此速度检查爪 GV内容时是极为重要的。 <ol><li>把握靠近GV的顶部核膜带有一对珠宝商的镊子，小心不要按下。把握信封的第二对为靠近第一尽可能钳。拉两对镊子从对方的GV内容将"啪"出免费信封，里面紧紧地坚持一个或两个钳的路程。 </li><li>拿起一个玻璃吸管的GV内容或可调20微升微量。如果使用可调移液管，移液管设置为10微升并切断塑料尖端用刀片的端部。在剩下的5微升拿起GV之前绘制的液体5微升。 </li><li>传输静止胶凝核内容预先填充有扩频溶液良好滑动的中心。扩频溶液必须具有一个凸表面，使得加入盖玻片时气泡未捕获。 </li><li>加盖玻片（18毫米），放置在潮湿小室中的幻灯片。核凝胶将在未来10慢慢分散 - 60分钟，以便在染色体S和核细胞器来撒谎载玻片的表面上。 注:不适当地在隔离介质和蝾螈A. mexicanum的GVS N. viridescens不"变硬"。因此，我们可以继续进行去除比人能爪核膜更加从容。 </li></ol></li><li>删除从蝾螈GV核膜如第5.1.1节中描述，但在分离培养基这样做。 <ol><li>拿起略有胶凝GV内容有吸​​管和转移到包含传播解决方案的培养皿。 </li><li>快速冲洗出的扩频溶液枪头，拿起核凝胶，并将其放置在预先填充有扩频溶液良好滑动的中心。加18毫米盖玻片并将其放置在潮湿小室中的整个滑动。核SAP将在未来10慢慢散去 - 60分钟和染色体和核细胞器会趴在玻璃表面上。 </li> &lt;/ OL&gt; </li></ol> 6. LBCs和细胞器的初步观察<ol><li>看在低倍率的相衬（常规正置显微镜）的LBCs和核细胞器在传播室核内容分散了。因为该腔室是深约1mm和核内容在底部，使用低放大倍数的物镜具有长工作距离（5X - 10X）。 注意:用于检查在这个时候制备的主要原因是，以确定它是否是值得通过接下来的步骤承载。在良好的准备染色体是完整的，并已落户到室的底部。继续进行到使制备的一小时内离心步骤，因为这将保证lampbrush染色体循环到滑动的最佳附接。 </li></ol> 7.离心核内容<ol><li>放置在盖玻片一张滤纸，然后向下按从腔室去除过量的液体。 </li><li>把大约1凡士林毫米3上的盖玻片的每一侧。熔化用金属棒加热到约70凡士林- 80℃至盖玻片密封到底层塑料正方形。 </li><li>地方在滑动载体用于离心，确保相对的载体是平衡的。离心幻灯片在约4800 XG 30分钟 - 1小时。在4℃使用的离心机的慢启动功能。 </li></ol> 8.修复核内容<ol><li>对于大多数的后续步骤，修复与多聚甲醛核内容。 </li><li>将一个滑动架到一个标准的显微镜载玻片染色菜，然后分别放在幻灯片在机架中。添加足够的固定剂以覆盖载玻片（2 - 在OR 2或PBS中的4％多聚甲醛）。 </li><li>一对钝钳的，推盖玻片横向，把它捡起来，并在适当的玻璃垃圾丢弃。离开滑动在固定剂至少15 - 30分钟。用于原位杂交和最抗体污渍，固定的较长时期（H或d）是允许的。 </li><li>要取出塑料方，地方滑一次一个入冰冷的OR2或PBS在染色皿中。在塑料广场边上插入一个锋利的刀片和撬松。 注意:理想情况下，塑料会脱落干净，使所有石蜡在幻灯片上。蜡提供一个有用的目的:因为它包围，其中核内容被安装在滑动的小的中心区，它作为一个坝，允许一个与小容积的工作-试剂免疫染色等 （5 10微升）。幻灯片可以无限期冷OR 2或PBS举行。 </li></ol> 9.免疫染色LBCs核细胞器<ol><li>取下存储在OR2或PBS固定GV准备。擦去多余的支持液体和发生在潮湿的室内。一个方便的chamb尔是含有湿滤纸的90 mm直径圈90×15毫米的培养皿。 注意:此时的核内容被牢固地连接到在自由石蜡的一个5毫米的圆形区域中的幻灯片。因为蜡是拒水，人们可以通过添加和到中心区域的用20微升吸管边缘去除的小体积液体更改的解决方案。主要预防措施是避免接触与枪头无蜡区。 </li><li>开展与小体积的免疫染色 - 试剂（10 20μL）。因为染色体和核细胞器是非常小的，并与加入的试剂直接接触，染色方案也很短。初级和次级抗体染色时间可短至1小时或更少。不要在任何一个步骤清洁剂，因为这会导致准备损坏。如果需要的话，染色用DAPI 10的制备（1微克/毫升） - 20分钟以强调LBCs的轴。 </li><li>在山甘油或商业坐骑ING中等适合免疫。为了避免在安装步骤中俘获的气泡，步骤如下。 <ol><li>拉手安装介质成20μL的吸移管的尖端的15微升。由"芯吸"其关闭与（在一个非常薄的三角形状切口从＃1滤纸的90mm圆）一张滤纸从立即围绕核扩散的区域除去尽可能多的液体尽可能。 </li><li>吸取大部分安装介质上的准备，小心不要碰到核蔓延的区域。放置最后1微升左右的22毫米盖玻片（厚度＃1.5）的中心安装的介质。在反转的准备盖玻片，小心地放下。 </li><li>对于临时坐骑，长达数天，用50％的甘油1毫克/毫升苯二胺。为永久架，特别是对于超分辨的研究，使用该硬化至约1.46的折射率的低荧光介质。 </li></ol></li></ol> 10. 荧光 LBCs的原位杂交（FISH）和核细胞器<ol><li>由于FISH协议通常需要在高温下的一个步骤，请从准备石蜡。用剃刀刀片仔细刮掉，虽然过程是繁琐的，并可能导致在制备的意外损坏。它来标记在滑动的用金刚石点后面的制剂的领域是非常有用的。 <ol><li>可替换地，溶解用二甲苯的石蜡。设置一系列科普林罐子或含有染色皿的:30％，50％，70％，95％和100％的乙醇;二甲苯3容器; 2多个容器100％的乙醇和一种丙酮中。 </li><li>放置在滑板支架幻灯片和脱水他们在乙醇系列，留下幻灯片3 - 5分钟，在每个浓度（更短的时间在搅拌）。 </li><li>通过二甲苯的3容器传递幻灯片溶解石蜡，约5分钟每如果搅拌。去除通过穿过两个100％乙醇二甲苯，最后用丙酮除去乙醇。从丙酮以幻灯片和倾斜他们干。 </li></ol></li><li>使用任何标准的协议进行原位杂交。 15,16 </li></ol> 11.隔离下油一GV的<ol><li>拿起从OR2液卵母细胞与珠宝商的镊子，包括少周围组织成为可能。将卵母细胞在一小块＃1滤纸。过量OR2溶液将转移到滤纸，而使卵母细胞几乎不含液体。拿起"干"的卵母细胞，并在一个小培养皿（35×10mm）的将其放置轻质矿物油（石蜡油）的表面之下。 <ol><li>用细钨针或镊子的一个叉，戳在附近的黑暗动物极卵母细胞的小孔。拿起卵母细胞有一对钳（避免提示），并轻轻挤压。的GV将沿着挤出具有较小或L-在其表面上卵黄arger量（ 图3）。 </li><li>通过与钳轻轻侧扫GV删除尽可能多蛋黄越好。在大多数情况下，这将是难以去除所有的蛋黄。然而，对于大多数目的蛋黄少量是没有问题的。 </li></ol></li><li>观察GV内容，拼合GV到更大或更小的程度。 <ol><li>从井中取出幻灯片塑料方形，用剩余的石蜡作为浅插孔，用于石蜡油。 10拿起GV用石蜡油沿着一个20微升吸管和转移到石蜡区域的中心。添加盖玻片，并观察局部扁平GV的内容。这种技术不损伤LBCs。 </li></ol></li><li>可替换地，压平的GV到约10微米的厚度为核细胞器的观察。 <ol><li>切断塑料吸管尖端的最终以1毫米一把锋利的剃刀刀片。吸GV进入与矿物油的正是5μL尖一起。挤出GV和所有的油到22毫米的玻璃盖玻片的中心。 </li><li>降低标准的3"×1"的玻璃滑动慢慢直到它刚好接触油滴。下降和盖玻片将通过毛细管现象被解除和油会扩散到盖玻片的边缘。当丢弃已经完全传播时，油将大约10微米厚。该LBCs将会损坏和核仁大会稍微压缩，但因为它们在完整GV存在（ 图3）最小核细胞器将出现更多或更少。 </li></ol></li></ol>

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	<li>Purkinje, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Symbolae ad ovi avium historiam ante incubationem. , (1830).</li><li>R&#252;ckert, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zur+Entwickelungsgeschichte+des+Ovarialeies+bei+Selachiern.">Zur Entwickelungsgeschichte des Ovarialeies bei Selachiern.</a> Anat. Anz. 7, 107-158 (1892).</li><li>Callan, H. 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> Lampbrush Chromosomes. 36, (1986).</li><li>Gall, J. G., Callan, H. G., Wu, Z., Murphy, C., Kay, B. K., Peng, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lampbrush+chromosomes.">Lampbrush chromosomes.</a> Xenopus laevis: Practical Uses in Cell and Molecular Biology. Vol. 36 Methods in Cell Biology , 149-166 (1991).</li><li>Gall, J. G., Wu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examining+the+contents+of+isolated+Xenopus+germinal+vesicles.">Examining the contents of isolated Xenopus germinal vesicles.</a> Methods. 51, 45-51 (2010).</li><li>Penrad-Mobayed, M., Kanhoush, R., Perrin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tips+and+tricks+for+preparing+lampbrush+chromosome+spreads+from+Xenopus+tropicalis+oocytes.">Tips and tricks for preparing lampbrush chromosome spreads from Xenopus tropicalis oocytes.</a> Methods. 51, 37-44 (2010).</li><li>Morgan, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Working+with+oocyte+nuclei:+cytological+preparations+of+active+chromatin+and+nuclear+bodies+from+amphibian+germinal+vesicles.">Working with oocyte nuclei: cytological preparations of active chromatin and nuclear bodies from amphibian germinal vesicles.</a> Methods Mol. Biol. 463, 55-66 (2008).</li><li>Paine, P. L., Johnson, M. E., Lau, Y. T., Tluczek, L. J., Miller, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+oocyte+nucleus+isolated+in+oil+retains+in+vivo+structure+and+functions.">The oocyte nucleus isolated in oil retains in vivo structure and functions.</a> Biotechniques. 13, 238-246 (1992).</li><li>Handwerger, K. E., Cordero, J. A., Gall, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cajal+bodies,+nucleoli,+and+speckles+in+the+Xenopus+oocyte+nucleus+have+a+low-density,+sponge-like+structure.">Cajal bodies, nucleoli, and speckles in the Xenopus oocyte nucleus have a low-density, sponge-like structure.</a> Mol Biol Cell. 16, 202-211 (2005).</li><li>Patel, S., Novikova, N., Beenders, B., Austin, C., Bellini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+images+of+RNA+polymerase+II+transcription+units.">Live images of RNA polymerase II transcription units.</a> Chromosome Res. 16, 223-232 (2008).</li><li>Gall, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Octagonal+nuclear+pores.">Octagonal nuclear pores.</a> J Cell Biol. 32, 391-399 (1967).</li><li>L&#246;schberger, A., 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=Super-resolution+imaging+visualizes+the+eightfold+symmetry+of+gp210+proteins+around+the+nuclear+pore+complex+and+resolves+the+central+channel+with+nanometer+resolution.">Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution.</a> J Cell Sci. 125, 570-575 (2012).</li><li>Gottfert, F., 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=Coaligned+dual-channel+STED+nanoscopy+and+molecular+diffusion+analysis+at+20+nm+resolution.">Coaligned dual-channel STED nanoscopy and molecular diffusion analysis at 20 nm resolution.</a> Biophys J. 105, L01-L03 (2013).</li><li>Wallace, R. A., Jared, D. W., Dumont, J. N., Sega, 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=Protein+incorporation+by+isolated+amphibian+oocytes:+III.+Optimum+incubation+conditions.">Protein incorporation by isolated amphibian oocytes: III. Optimum incubation conditions.</a> J. Exp. Zool. 184, 321-333 (1973).</li><li>Bridger, J., Spector, D. L., Goldman, R. D., Leinwand, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+in+situhybridization+to+DNA.">Fluorescence in situhybridization to DNA.</a> Cells: A Laboratory Manual. 3, 111.111-111.136 (1998).</li><li>Singer, R. H., Spector, D. L., Goldman, R. D., Leinwand, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization+to+RNA.">In situ hybridization to RNA.</a> Cells: A Laboratory Manual. 3, 111-116 (1998).</li><li>Gardner, E. J., Nizami, Z. F., Talbot, C. C., Gall, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+intronic+sequence+RNA+(sisRNA),+a+new+class+of+noncoding+RNA+from+the+oocyte+nucleus+of+Xenopus+tropicalis.">Stable intronic sequence RNA (sisRNA), a new class of noncoding RNA from the oocyte nucleus of Xenopus tropicalis.</a> Genes Dev. 26, 2550-2559 (2012).</li><li>Talhouarne, G. J., Gall, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lariat+intronic+RNAs+in+the+cytoplasm+of+Xenopus+tropicalis+oocytes.">Lariat intronic RNAs in the cytoplasm of Xenopus tropicalis oocytes.</a> RNA. 20, 1476-1487 (2014).</li><li>Duryee, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+nuclei+and+non-mitotic+chromosome+pairs+from+frog+eggs.">Isolation of nuclei and non-mitotic chromosome pairs from frog eggs.</a> Arch. Exp. Zellforsch. 19, 171-176 (1937).</li></ol>

The authors have nothing to disclose.

从青蛙和蝾螈的手隔离GVS"生活"LBCs第一个观测到了近80年前由美国生物学家威廉Duryee，19引进相衬和DIC的显微镜前，荧光免疫前和鱼了。 LBCs的在技术的LBCs重视载玻片个体基因水平所需的开发研究染色体结构和转录的细节，来解决他们在一个栩栩如生的方式，并没有破坏他们的基本形态应用分子生物学技术的优势。几乎每一个技术，实现这些目标需要一定的适应被调查的种类和逼真的保存和分子特征之间的一些妥协。例如，GVS和尾两栖类LBCs都异常容易的工作，因为其巨大的规模和与该GV内容在合适的介质中分散的难易程度。 üNTIL然而，最近很少有人知道他们的基因组，使得难以用分子细节的细胞学特征的财富相关。相反地， 爪蟾的LBCs是相当小的相比，这些尾两栖类（ 图5）的，但两者十热带和爪蟾的基因组已经被测序，并合理地注明。 对于那些首先用水陆两用GVS，主要的挑战将是隔离GV并删除核膜不破坏LBCs。到目前为止，还没有人发现了一个方法，以消除信封，除了与珠宝商的镊子的手。这将是一个重大的技术进步，如果一个方法被发现"解散"或"消化"信封，但留下的染色体和其他核细胞器损坏。这样的技术将是分子研究同样有用。在我们的细胞核和细胞质RNA的调查，我们发现它必要核RNA（ 图2）分析之前删除核膜。 17,18如果信封不去除，核RNA的少量由污染附着在外壳的外细胞质RNA的淹没。 另一项主要的改进是一些方法来更紧密地连接到LBCs载玻片发现。编制离心是至关重要的，但即使长时间离心（1小时在4500 XG）并不能保证附件。人们通常可以告诉时LBCs深受中等放大倍率下相衬检查它们连接。那么附加的染色体不显示布朗运动的。如果循环任何布朗运动是检测，随后的免疫染色或鱼的协议会导致广泛的损害或重大损失。

大多数脊椎动物，与有袋类动物和胎盘哺乳动物显着的例外，产生大量yolky鸡蛋。尽管他们有时巨大的规模，这些蛋是在女性的卵巢达到其最终尺寸，同时还单细胞。卵巢卵子被称为卵母细胞，每个通常包含一个巨大的细胞核，自19 世纪初的生发泡或干脆GV闻名。在普通实验室青蛙， 非洲爪蟾和爪蟾热带的1卵母细胞，达到1.2和0.8mm分别（ 图1）的最大直径。来自这两个青蛙成熟卵母细胞的GVS是0.3 -在0.4mm直径（ 图2,3）。蝾通常有更大的卵母细胞和GVS。墨西哥螈，Ambystoma mexicanum的完全成熟的卵母细胞，是大于2毫米直径的GV是约0.5毫米。这样，这些晶核是肉眼并且可以容易地看到在许多方面是不可能的典型体细胞的细胞核被操纵。 同样引人注目的是GV，在19 世纪的最后已经承认的事实中染色体的巨大规模。 Ambystoma等山椒个体染色体可高达1毫米的长度（ 图4，图5）。这些爪的相当小的，虽然与长度可达100微米或更多，他们相形见绌大多数生物体的典型体细胞染色体。卵母细胞的染色体的一个重要特征是其非凡的转录活性，这导致其最具特征的形态学特征之一-数百成对的横向环（ 图5）。每个循环由一个或几个转录单位即积极地合成RNA。该循环给卵母细胞染色体一个模糊的外表，这导致了名为"lampbrush"染色体后，他们的肤浅相似之处在更早的时候用来清洁煤油灯烟囱刷。 2 本文的重点是采用隔离GVS来研究LBCs和核细胞器（核仁蛋白基因体和斑点）。两个而不同的技术进行说明。在第一，更常见的技术，GVS在盐溶液使用珠宝商的镊子分离，简要地漂洗，以除去附着的蛋黄，和核膜被去除，再次用珠宝商的钳子。凝胶状内容，将含有LBCs和核细胞器，被允许沉降到玻璃显微镜载玻片或盖玻片。这些制剂可直接通过相衬或DIC显微镜进行检查。或者，制剂可被离心以LBCs和细胞器附加到幻灯片或盖玻片。这些制剂随后可以用于核酸和蛋白质的详细的分子分析处理，主要是通过免疫荧光和fluorescen吨原位杂交（FISH）。 3-7 第二种技术涉及在矿物油中的GV的分离。 8油隔离GVS留许多小时转录活性并且是研究其中一个想要的核内容尽可能逼真可能有用。 9,10由于核"汁液"的折射率接近于LBCs和其他核细胞器（ 图3）的，显微技术可与油分离GVS一个挑战。 最后，由于它们的尺寸和易于操纵的，GVS是用于在核膜研究理想材料。核孔复合物首先从电子显微镜的研究描述在水陆两用车辆GV信封11和最近的超分辨率观察已使用了相同的材料。 12,13

<table><tbody><tr><td>Paraformaldehyde (reagent grade, crystalline)</td><td>Sigma-Aldrich</td><td>P6148-500G</td><td>Despite warnings in many protocols, a concentrated solution can be stored indefinitely at RT</td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfonate</td><td>Sigma-Aldrich</td><td>A5040-100G</td><td>Sometimes referred to as MS-222</td></tr><tr><td>Ethicon RB-1 1/2 circle taper point 3-0 sutures</td><td>VWR&amp;nbsp;</td><td>95057-000</td><td></td></tr><tr><td>Paraplast (paraffin wax)</td><td>Sigma-Aldrich</td><td>P3558-1KG</td><td></td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-Phenylenediamine</td><td>Sigma-Aldrich</td><td>P6001</td><td></td></tr><tr><td>Gelatin</td><td>Grocery Store</td><td></td><td>Commercial Knox gelatin works fine</td></tr><tr><td>ProLong Gold antifade mountant</td><td>ThermoFisher Scientific</td><td>P10144</td><td></td></tr><tr><td>Gold Seal cover glass&amp;nbsp; 22 x 22 mm #1 1/2 (0.16 - 0.19 mm thick)</td><td>Electron Microscopy Sciences</td><td>63786-01</td><td>These coverslips are the recommended thickness for superresolution microscopy</td></tr><tr><td>Dumont forceps #5</td><td>Electron Microscopy Sciences</td><td>72700-D</td><td>http://www.emsdiasum.com/microscopy/products/tweezers/dumont_positive_action.aspx</td></tr><tr><td>Paraffin oil (light)&amp;nbsp;</td><td>EMD Chemicals</td><td>PX0047-1</td><td>For isolating GVs in oil</td></tr><tr><td>Adhesive &lt;em&gt;in situ&lt;/em&gt; PCR and hybridization chambers (25 &amp;micro;L)</td><td>BioRad</td><td>Frame-Seal Slide Chambers #SLF0201</td><td>http://www.bio-rad.com/en-us/sku/slf0201-frame-seal-slide-chambers</td></tr><tr><td>Silicone isolators</td><td>Grace-Biolabs</td><td>select from catalog link</td><td>http://www.gracebio.com/life-science-products/microfluidics/silicone-isolators.html</td></tr><tr><td>Coplin jars and staining dishes</td><td>Electron Microscopy Sciences</td><td>select from catalog link</td><td>http://www.emsdiasum.com/microscopy/products/histology/staining.aspx</td></tr></tbody></table>

isolation of giant lampbrush chromosomes from living oocytes of frogs and salamanders

我们描述研究在卵母细胞，或遗腹卵，青蛙和蝾螈的发现巨人转录活性lampbrush染色体（LBCs）方法。个别LBCs可高达1毫米的长度和它们驻留在一个巨大细胞核，本身高达0.5毫米的直径。染色体的大尺寸允许通过光学显微镜活性基因的无与伦比的观测，但在同一时间都需要分离细胞核，除去核膜，并在显微镜载玻片上扩散染色体特殊技术。卵母细胞核，也称为生发泡（GV），在培养基中，允许核肌动蛋白的部分胶凝并保留LBCs的微妙结构是分离的。此步骤是在解剖显微镜下使用珠宝商的镊子手动进行。接下来，核膜被删除，请手动再次与珠宝商的镊子。核内容被迅速转移到dispe介质RSES肌动蛋白凝胶并允许未损坏LBCs沉降到显微镜载玻片。此时LBCs和其他核细胞器可以通过相差或差分干涉相差显微镜观察时，虽然更精细细节由布朗运动模糊。对于高分辨率显微镜观察或分子分析，整个编制离心牢固安装微妙LBCs的幻灯片。然后在多聚甲醛的简要定影后跟免疫荧光染色或原位杂交。 LBCs处于转录活性状态，其巨大的尺寸允许在利用共聚焦或超高分辨率显微镜个体基因水平分子分析。

要检查巨头lampbrush染色体我们先隔离蛙或蝾螈的卵母细胞。 图1示出了一组从青蛙， 蟾蜍卵巢切除后缓冲盐溶液成熟的卵母细胞。这样的卵母细胞保持良好状态，在室温下天。的核（或胚泡）随后从与珠宝商的镊子的卵母细胞中删除，或者在盐溶液（ 图2）或在油中（ 图3）。油分离核的核内容可以通过轻轻地挤压细胞核和通过相差或差分干涉对比（DIC）显微镜观察进行检查。审查已在盐溶液被分离出的核中的内容，首先必须删除与珠宝商的镊子核膜和允许内容沉降到显微镜载玻片。然后将制剂离心附着吨他牢牢染色体到滑动，在此之后，染色体可以与抗体（ 图4）进行染色或原位杂交进行。山椒的lampbrush染色体比青蛙（ 图5）的大得多。在这两种情况下单独的转录单位（基因）可以被看作是从染色体轴线横向伸出的染色质环。 <img alt="图1" src="/files/ftp_upload/54103/54103fig1.jpg" /> 图1: 成熟的卵母细胞来自青蛙 热带十 。一个成熟的雌蛙的卵巢中含有数千成熟的不同阶段卵母细胞。最小的是体细胞的大小和已经达到第一次减数分裂的前期。作为卵母细胞的增长，它逐渐积累蛋黄，这使电池的不透明的白色外观。最大垫URE卵母细胞，在这里显示，获得较深的上限，由于黑色素积聚。 十热带的最大的卵母细胞是在直径约0.8毫米，那些X的蟾约1.4毫米，而那些螈的是一个巨大的2.2毫米。除了规模，三者在整体外观相似。比例尺= 1毫米。 <img alt="图2" src="/files/ftp_upload/54103/54103fig2.jpg" /> 图2: 从 X的热带 的卵母细胞的GV的去除 。 剩下。一个小孔在卵母细胞的暗动物极有珠宝商的镊子制作和GV轻轻挤压。在这个例子中，GV为几乎不含蛋黄的，因为它出来的卵母细胞。粘附蛋黄可以通过用尖端直径只比GV本身的直径稍大抽吸GV进出吸管除去。 对。 ŧ十重稀土热带的GVS。这些GVS分别在稍微酸性介质（pH调至5.8 GV隔离溶液），这将导致在核膜溶胀从凝胶状核内容远左几分钟。以这种方式对待GVS不会蔓延细胞学检查。然而，这样的GVS是理想的分子分析:信封可以与珠宝商镊子去除，提供了核内容完全不含细胞质污染的样本。 17,18比例尺= 0.5毫米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54103/54103fig2large.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/54103/54103fig3.jpg" /> 图3: 在油隔离 X.热带 的GV。 剩下。经过一个小刺在卵母细胞做出附近的暗动物婆乐时，GV开始挤出。在这种情况下，GV出来，几乎没有附着蛋黄。 中间。该GV现在是从卵母细胞胞浆内完全免费的。这样的GVS继续转录RNA几个小时。 对。的GV是轻轻油盖玻片下压扁后，核细胞器可以通过DIC查看，如这里所示，或者通过相衬。整个GV比这里显示的小面积大得多。 HLB =组蛋白基因座体与在其表面的三个斑点。比例尺= 0.5毫米第一两个面板，10微米的三分之一。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54103/54103fig3large.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/54103/54103fig4.jpg" /> 图4:Lampbrush 染色体（LBCs）从蝾螈 A. mexicanum。 剩下。 </st荣&gt;从在第一次减数分裂的前期单GV的14成对染色体，免疫染色用抗磷酸化的RNA聚合酶II的抗体。这种微小的染色"点"是组蛋白基因的身体。 对。同样准备通过暗场照明观察。现在人们可以看到无数的未染色放大核仁。比例尺= 250微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54103/54103fig4large.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/54103/54103fig5.jpg" /> 图5:从蝾螈 A. mexicanum 与青蛙 热带十 的单LBC， 在相同的放大倍率。这些图像表明蝾螈的LBCs和青蛙（插图）之间的极端大小差异。 differen大小CE与基因组的总DNA含量（约30 GBP代表A. mexicanum VS 1.7 GBP代表X.热带 ）相关。个别侧回路（转录单位）也都在蝾远长于在青蛙。比例尺= 100微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54103/54103fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Isolation of Giant Lampbrush Chromosomes from Living Oocytes of Frogs and Salamanders at JoVE.com

Isolation of Giant Lampbrush Chromosomes from Living Oocytes of Frogs and Salamanders

我们提出简单的技术从活青蛙和蝾螈卵母细胞中分离巨头转录活性lampbrush染色体。我们描述了如何通过相位对比或微分干涉对比，以及如何解决这些问题原位杂交和免疫荧光染色荧光观察这些染色体"活着"。

从青蛙和蝾螈的卵母细胞活巨Lampbrush染色体的分离

We describe methods for studying the giant transcriptionally active lampbrush chromosomes (LBCs) found in the oocyte, or unlaid egg, of frogs and ...

isolation-giant-lampbrush-chromosomes-from-living-oocytes-frogs

Nanyang Technological University

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JoVE Journal

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Molecular Biology

Genetics

Unit 1

DNA and Chromosome Structure

SCIENTISTS IN ACTION

12.9K 次观看.  Carnegie Institution for Science. 我们提出简单的技术从活青蛙和蝾螈卵母细胞中分离巨头转录活性lampbrush染色体。我们描述了如何通过相位对比或微分干涉对比，以及如何解决这些问题原位杂交和免疫荧光染色荧光观察这些染色体"活着"。 

视频: Isolation of Giant Lampbrush Chromosomes from Living Oocytes of Frogs and Salamanders

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

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

Real-time measurement of oscillations at the single-cell level is important to uncover the mechanisms of biological clocks. Although bulk extracts prepared from Xenopus laevis eggs have been powerful in dissecting biochemical networks underlying the cell-cycle progression, their ensemble average measurement typically leads to a damped oscillation, despite each individual oscillator being sustained. This is due to the difficulty of perfect synchronization among individual oscillators in noisy biological systems. To retrieve the single-cell dynamics of the oscillator, we developed a droplet-based artificial cell system that can reconstitute mitotic cycles in cell-like compartments encapsulating cycling cytoplasmic extracts of Xenopus laevis eggs. These simple cytoplasmic-only cells exhibit sustained oscillations for over 30 cycles. To build more complicated cells with nuclei, we added demembranated sperm chromatin to trigger nuclei self-assembly in the system. We observed a periodic progression of chromosome condensation/decondensation and nuclei envelop breakdown/reformation, like in real cells. This indicates that the mitotic oscillator functions faithfully to drive multiple downstream mitotic events. We simultaneously tracked the dynamics of the mitotic oscillator and downstream processes in individual droplets using multi-channel time-lapse fluorescence microscopy. The artificial cell-cycle system provides a high-throughput framework for quantitative manipulation and analysis of mitotic oscillations with single-cell resolution, which likely provides important insights into the regulatory machinery and functions of the clock.

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

We present a method for the generation of in vitro self-sustained mitotic oscillations at the single-cell level by encapsulating egg extracts of Xenopus laevis in water-in-oil microemulsions.

无细胞爪卵萃取物微乳液细胞周期振荡的重建

Real-time measurement of oscillations at the single-cell level is important to uncover the mechanisms of biological clocks. Although bulk extracts ...

生物化学

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Much information about the coupling of presynaptic ionic currents with the release of neurotransmitter has been obtained from invertebrate preparations, most notably the squid giant synapse1. However, except for the preparation described here, few vertebrate preparations exist in which it is possible to make simultaneous measurements of neurotransmitter release and presynaptic ionic currents. Embryonic Xenopus motoneurons and muscle cells can be grown together in simple culture medium at room temperature; they will form functional synapses within twelve to twenty-four hours, and can be used to study nerve and muscle cell development and synaptic interactions for several days (until overgrowth occurs). Some advantages of these co-cultures over other vertebrate preparations include the simplicity of preparation, the ability to maintain the cultures and work at room temperature, and the ready accessibility of the synapses formed2-4. The preparation has been used widely to study the biophysical properties of presynaptic ion channels and the regulation of transmitter release5-8. In addition, the preparation has lent itself to other uses including the study of neurite outgrowth and synaptogenesis9-12, molecular mechanisms of neurotransmitter release13-15, the role of diffusible messengers in neuromodulation16,17, and in vitro synaptic plasticity18-19.

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

This video demonstrates the procedures used to grow primary cultures of embryonic Xenopus nerve and muscle cells and the usefulness of this preparation for making simultaneous pre- and post-synaptic patch clamp recordings.

同时前和突触后电生理记录<em&gt;爪蟾</em神经肌肉文化

Much information about the coupling of  presynaptic ionic currents with the release of neurotransmitter has been obtained  from invertebrate preparations, ...

神经科学

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 ...

生物学

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 ...

Analysis of Transforming Growth Factor &#223; Family Cleavage Products Secreted Into the Blastocoele of Xenopus laevis Embryos

The two arms of the Transforming Growth Factor &#223; (Tgf&#223;) superfamily, represented by Tgf&#223;/Nodal or Bone morphogenetic protein (Bmp) ligands, respectively, play essential roles in embryonic development and adult homeostasis. Members of the Tgf&#223; family are made as inactive precursors that dimerize and fold within the endoplasmic reticulum. The precursor is subsequently cleaved into ligand and prodomain fragments. Although only the dimeric ligand can engage Tgf&#223; receptors and activate downstream signaling, there is growing recognition that the prodomain moiety contributes to ligand activity. This article describes a protocol that can be used to identify cleavage products generated during activation of Tgf&#223; precursor proteins. RNA encoding Tgf&#223; precursors are first microinjected into X. laevis embryos. The following day, cleavage products are collected from the blastocoele of&#160;gastrula stage embryos and analyzed on Western blots. This protocol can be completed relatively quickly, does not require expensive reagents and provides a source of concentrated Tgf&#223; cleavage products under physiologic conditions.

Analysis of Transforming Growth Factor &#223; Family Cleavage Products Secreted Into the Blastocoele of Xenopus laevis Embryos

When Transforming Growth Factor &#223; family precursor proteins are ectopically expressed in Xenopus laevis embryos, they dimerize, get cleaved and are secreted into the blastocoele, which begins at the late blastula to early gastrula stage. We describe a method for aspirating cleavage products from the blastocoele cavity for immunoblot analysis.

转变生长因子的分析 - 家族产品分泌到 异种胚胎 的爆炸

The two arms of the Transforming Growth Factor &#223; (Tgf&#223;) superfamily, represented by Tgf&#223;/Nodal or Bone morphogenetic protein (Bmp) ligands, ...

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

研究膜蛋白的构象动力学原位网站的荧光标记

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Many organisms localize mRNAs to specific subcellular destinations to spatially and temporally control gene expression. Recent studies have demonstrated that the majority of the transcriptome is localized to a nonrandom position in cells and embryos. One approach to identify localized mRNAs is to biochemically purify a cellular structure of interest and to identify all associated transcripts. Using recently developed high-throughput sequencing technologies it is now straightforward to identify all RNAs associated with a subcellular structure. To facilitate transcript identification it is necessary to work with an organism with a fully sequenced genome. One attractive system for the biochemical purification of subcellular structures are egg extracts produced from the frog Xenopus laevis. However, X. laevis currently does not have a fully sequenced genome, which hampers transcript identification. In this article we describe a method to produce egg extracts from a related frog, X. tropicalis, that has a fully sequenced genome. We provide details for microtubule polymerization, purification and transcript isolation. While this article describes a specific method for identification of microtubule-associated transcripts, we believe that it will be easily applied to other subcellular structures and will provide a powerful method for identification of localized RNAs.

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

We describe the collection of unfertilized Xenopus tropicalis eggs and production of a meiosis II-arrested egg extract. This egg extract can be used to purify microtubules and microtubule-associated RNAs.

生产<em&gt;热带爪蟾</em&gt;卵提取物识别微管相关的RNA

Many  organisms localize mRNAs to specific subcellular destinations to spatially and  temporally control gene expression. Recent studies have demonstrated ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

非洲爪蟾作为一种模型来确定翻译减值

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...

A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod shaped chromosomes which are moved individually by the mitotic spindle apparatus. Mitotic chromatin condensation yields chromosomes compacted fifty-fold denser as in interphase. During exit from mitosis, chromosomes have to re-establish their functional interphase state, which is enclosed by a nuclear envelope and is competent for replication and transcription. The decondensation process is morphologically well described, but in molecular terms poorly understood: We lack knowledge about the underlying molecular events and to a large extent the factors involved as well as their regulation. We describe here a cell-free system that faithfully recapitulates chromatin decondensation in vitro, based on mitotic chromatin clusters purified from synchronized HeLa cells and X. laevis egg extract. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its co-ordination with processes simultaneously occurring during mitotic exit such as nuclear envelope and pore complex re-assembly.

The molecular mechanisms of the decondensation of highly compacted mitotic chromatin are ill-defined. We present a cell-free assay based on mitotic chromatin clusters isolated from HeLa cells and Xenopus laevis egg extract that faithfully reconstitutes the decondensation process in vitro.

无细胞化验，研究染色质解聚在有丝分裂的结束

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod ...

从青蛙和蝾螈的卵母细胞活巨Lampbrush染色体的分离

摘要

探索更多视频

此视频中的章节

操作和

无细胞爪卵萃取物微乳液细胞周期振荡的重建

同时前和突触后电生理记录

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

转变生长因子的分析 - 家族产品分泌到异种胚胎的爆炸

研究膜蛋白的构象动力学原位网站的荧光标记

生产

非洲爪蟾作为一种模型来确定翻译减值

无细胞化验，研究染色质解聚在有丝分裂的结束

从青蛙和蝾螈的卵母细胞活巨Lampbrush染色体的分离

摘要

探索更多视频

此视频中的章节

操作和

无细胞爪卵萃取物微乳液细胞周期振荡的重建

同时前和突触后电生理记录

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

转变生长因子的分析 - 家族产品分泌到 异种胚胎 的爆炸

研究膜蛋白的构象动力学原位网站的荧光标记

生产

非洲爪蟾作为一种模型来确定翻译减值

无细胞化验，研究染色质解聚在有丝分裂的结束

转变生长因子的分析 - 家族产品分泌到异种胚胎的爆炸