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>

<ol>
	<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?...

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 border="0" cellpadding="0" cellspacing="0" width="1562">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Paraformaldehyde (reagent grade, crystalline)</td>
			<td style="width:301px;">Sigma-Aldrich</td>
			<td style="width:213px;">P6148-500G</td>
			<td style="width:643px;">Despite warnings in many protocols, a concentrated solution can be stored indefinitely at room temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethyl 3-aminobenzoate methanesulfonate</td>
			<td>Sigma-Aldrich</td>
			<td>A5040-100G</td>
			<td>Sometimes referred to as MS-222</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethicon RB-1 1/2 circle taper point 3-0 sutures</td>
			<td>VWR&#160;</td>
			<td>95057-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraplast (paraffin wax)</td>
			<td>Sigma-Aldrich</td>
			<td>P3558-1KG</td>
			<td style="width:643px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">p-Phenylenediamine</td>
			<td>Sigma-Aldrich</td>
			<td>P6001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gelatin</td>
			<td>Grocery Store</td>
			<td></td>
			<td>Commercial Knox gelatin works fine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ProLong Gold antifade mountant</td>
			<td>ThermoFisher Scientific</td>
			<td>P10144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gold Seal cover glass&#160; 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 height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Paraffin oil (light)&#160;</td>
			<td>EMD Chemicals</td>
			<td>PX0047-1</td>
			<td>For isolating GVs in oil</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adhesive in situ PCR and hybridization chambers (25 &#181;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 height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">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...

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

Research

Education

JoVE Journal

JoVE Core

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

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

机器人平台的高通量原生质体分离和转型

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

科研

遗传学

Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an excellent model for rapid assessment of whole transcriptome from whole animal or individual cell populations due to the ease of isolation of RNA from large numbers of animals. Here a protocol for global gene expression analysis in zebrafish embryos using RNA sequencing (RNASeq) is presented. We describe preparation of RNA from whole embryos or from cell populations obtained using cell sorting in transgenic animals. We also describe an approach for analysis of RNASeq data to identify enriched pathways and Gene Ontology (GO) terms in global gene expression data sets. Finally, we provide a protocol for validation of gene expression changes using quantitative reverse transcriptase PCR (qRT-PCR). These protocols can be used for comparative analysis of control and experimental sets of zebrafish to identify novel gene expression changes, and provide molecular insight into phenotypes of interest.

This protocol presents an approach for whole transcriptome analysis from zebrafish embryos, larvae, or sorted cells. We include isolation of RNA, pathway analysis of RNASeq data, and qRT-PCR-based validation of gene expression changes.

斑马鱼 RNASeq-based 基因表达数据的样品制备与分析

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an ...

Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly defined states of chromatin organization. The dynamic architecture of the nucleus is controlled by multiple mechanisms and shapes the transcriptional output programs. It is, therefore, important to determine locus-specific chromatin accessibility in a reliable fashion that is preferably independent from antibodies, which can be a potentially confounding source of experimental variability. Chromatin accessibility can be measured by various methods, including the Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) assay, that allow the determination of general chromatin accessibility in a relatively low number of cells. Here we describe a FAIRE protocol that allows simple, reliable, and fast identification of genomic regions with a low protein occupancy. In this method, the DNA is covalently bound to the chromatin proteins using formaldehyde as a crosslinking agent and sheared to small pieces. The free DNA is afterwards enriched using phenol:chloroform extraction. The ratio of free DNA is determined by quantitative polymerase chain reaction (qPCR) or DNA sequencing (DNA-seq) compared to a control sample representing total DNA. The regions with a looser chromatin structure are enriched in the free DNA sample, thus allowing the identification of genomic regions with lower chromatin compaction.

The plasticity of nuclear architecture is controlled by dynamic epigenetic mechanisms including non-coding RNAs, DNA methylation, nucleosome repositioning as well as histone composition and modification. Here we describe a Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) protocol which allows the determination of chromatin accessibility in a reproducible, inexpensive, and straightforward manner.

甲醛辅助分离测定哺乳动物细胞染色质可及性的调控元素

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly ...

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

小鼠卵母细胞在减数分裂过程中对人类基因功能的评估

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such as initial massive cell death and low therapeutic effects, impaired their broad clinical translation. Genetic engineering of stem cells prior to transplantation emerged as a promising method to optimize therapeutic stem cell effects. However, safe and efficient gene delivery systems are still lacking. Therefore, the development of suitable methods may provide an approach to resolve current challenges in stem cell-based therapies.
The present protocol describes the extraction and characterization of human dental follicle stem cells (hDFSCs) as well as their non-viral genetic modification. The postnatal dental follicle unveiled as a promising and easily accessible source for harvesting adult multipotent stem cells possessing high proliferation potential. The described isolation procedure presents a simple and reliable method to harvest hDFSCs from impacted wisdom teeth. Also this protocol comprises methods to define stem cell characteristics of isolated cells. For genetic engineering of hDFSCs, an optimized cationic lipid-based transfection strategy is presented enabling highly efficient microRNA introduction without causing cytotoxic effects. MicroRNAs are suitable candidates for transient cell manipulation, as these small translational regulators control the fate and behavior of stem cells without the hazard of stable genome integration. Thus, this protocol represents a safe and efficient procedure for engineering of hDFSCs that may become important for optimizing their therapeutic efficacy.

This protocol describes the transient genetic engineering of dental stem cells extracted from the human dental follicle. The applied non-viral modification strategy may become a basis for the improvement of therapeutic stem cell products.

人牙小泡干细胞的分离、鉴定及微 rna 遗传修饰

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such ...

Generation of Cancer Cell Clones to Visualize Telomeric Repeat-containing RNA TERRA Expressed from a Single Telomere in Living Cells

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in telomere biology, including heterochromatin formation and telomere length homeostasis. Recent findings revealed that TERRA molecules also interact with internal chromosomal regions to regulate gene expression in mouse embryonic stem (ES) cells. In line with this evidence, RNA fluorescence in situ hybridization (RNA-FISH) analyses have shown that only a subset of TERRA transcripts localize at chromosome ends. A better understanding of the dynamics of TERRA molecules will help define their function and mechanisms of action. Here, we describe a method to label and visualize single-telomere TERRA transcripts in cancer cells using the MS2-GFP system. To this aim, we present a protocol to generate stable clones, using the AGS human stomach cancer cell line, containing MS2 sequences integrated at a single subtelomere. Transcription of TERRA from the MS2-tagged telomere results in the expression of MS2-tagged TERRA molecules that are visualized by live-cell fluorescence microscopy upon co-expression of a MS2 RNA-binding protein fused to GFP (MS2-GFP). This approach enables researchers to study the dynamics of single-telomere TERRA molecules in cancer cells, and it can be applied to other cell lines.

Here, we present a protocol to generate cancer cell clones containing a MS2 sequence tag at a single subtelomere. This approach, relying on the MS2-GFP system, enables visualization of the endogenous transcripts of telomeric repeat-containing RNA (TERRA) expressed from a single telomere in living cells.

从活细胞中的单端粒表达的癌症细胞克隆的生成, 以可视化含有端粒重复的 rna terra

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in ...

Isolation of Papillary and Reticular Fibroblasts from Human Skin by Fluorescence-activated Cell Sorting

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have traditionally been attributed to the superficial papillary or lower reticular dermis according to their histological localization. In mouse dermis, papillary and reticular fibroblasts originate from two different lineages with diverging functions regarding physiological and pathological processes and a distinct cell surface marker expression profile by which they can be distinguished.
Importantly, evidence from explant cultures from superficial and lower dermal layers suggest that at least two functionally distinct dermal fibroblasts lineages exist in human skin dermis as well. However, unlike for mouse skin, cell surface markers enabling the discrimination of different fibroblast subsets have not yet been established for human skin. We developed a novel protocol for the isolation of human papillary and reticular fibroblast populations via fluorescence-activated cell sorting (FACS) using the two cell surface markers Fibroblast Activation Protein (FAP) and Thymocyte antigen 1 (Thy1)/CD90. This method enables the isolation of pure fibroblast subsets without in vitro manipulation, which was shown to affect gene expression, thus permitting accurate functional analysis of human dermal fibroblast subsets in regard to tissue homeostasis or disease pathology.

This manuscript describes a FACS-based protocol for isolation of papillary and reticular fibroblasts from human skin. It circumvents in vitro culture which was inevitable with the commonly used isolation protocol via explant cultures. The emanating fibroblast subsets are functionally distinct and display differential gene expression and localization within the dermis.

荧光活化细胞分选分离人皮肤状和网状成纤维细胞

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have ...

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, and was deployed to West Africa during the recent Ebola virus epidemic, highlighting this possibility. In addition to its practical advantages (low cost, ease of equipment transport and use), this technology also provides fundamental advantages over second-generation sequencing approaches, particularly the very long read length, ability to directly sequence RNA, and real-time availability of data. Raw read accuracy is lower than with other sequencing platforms, which represents the main limitation of this technology; however, this can be partially mitigated by the high read depth generated. Here, we present a field-compatible protocol for sequencing of the mRNAs encoding for Niemann-Pick C1, which is the cellular receptor for ebolaviruses. This protocol encompasses extraction of RNA from animal blood samples, followed by RT-PCR for target enrichment, barcoding, library preparation, and the sequencing run itself, and can be easily adapted for use with other DNA or RNA targets.

Nanopore sequencing is a novel technology that allows cost-effective sequencing in remote locations and resource-poor settings. Here, we present a protocol for sequencing of mRNAs from whole blood that is compatible with such conditions.

纳米测序全血 mRNA 测序

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, ...

Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify and Localize mRNAs in Murine Oocytes

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), quantitative, real-time RT-PCR (RT-qPCR) and RNA sequencing. When these techniques are performed using a single oocyte or embryo, low-copy mRNAs are not reliably detected. To overcome this problem, oocytes or embryos can be pooled together for analysis; however, this often leads to high variability amongst samples. In this protocol, we describe the use of fluorescence in situ hybridization (FISH) using branched DNA chemistry. This technique identifies the spatial pattern of mRNAs in individual cells. When the technique is coupled with Spot Finding and Tracking&#160;computer software, the abundance of mRNAs in the cell can also be quantified. Using this technique, there is reduced variability within an experimental group and fewer oocytes and embryos are required to detect significant differences between experimental groups. Commercially available branched-DNA SM-FISH kits have been optimized to detect mRNAs in sectioned tissues or adherent cells on slides. However, oocytes do not effectively adhere to slides and some reagents in the kit were too harsh resulting in oocyte lysis. To prevent this lysis, several modifications were made to the FISH kit. Specifically, oocyte permeabilization and wash&#160;buffers designed for the immunofluorescence of oocytes and embryos replaced the proprietary buffers. The permeabilization, washes, and incubations with probes and amplifier were performed in 6-well plates and oocytes were placed on slides at the end of the protocol using mounting media. These modifications were able to overcome the limitations of the commercially available kit, in particular, the oocyte lysis. To accurately and reproducibly count the number of mRNAs in individual oocytes, computer software was used. Together, this protocol represents an alternative to PCR and sequencing to compare the expression of specific transcripts in single cells.

Use of Single Molecule Fluorescent In Situ Hybridization SM-FISH to Quantify and Localize mRNAs in Murine Oocytes

To reproducibly count the numbers of mRNAs in individual oocytes, single molecule RNA fluorescence in situ hybridization (RNA-FISH) was optimized for non-adherent cells. Oocytes were collected, hybridized with the transcript specific probes, and quantified using an image quantification software.

单分子荧光原位杂交 (SM-FISH) 在小鼠卵母细胞中 Mrna 的定量和定位

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), ...

High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often performed at low throughput, it is moreover time-consuming and error-prone, even more so when multiplexing several plasmids. We developed an easy, fast, and accurate method to perform cell transfection in a 384-well plate layout using acoustic droplet ejection (ADE) technology. The nanodispenser device used in this study&#160;is based on this technology and allows precise nanovolume delivery at high speed from a source well plate to a destination one. It can dispense and multiplex DNA and transfection reagent according to a predesigned spreadsheet. Here we present an optimal protocol to perform ADE-based high-throughput plasmid transfection which makes it possible to reach an efficiency of up to 90% and a nearly 100% cotransfection in cotransfection experiments. We extend initial work by proposing a user-friendly spreadsheet-based macro, able to manage up to four plasmids/wells from a library containing up to 1,536 different plasmids, and a tablet-based pipetting guide application. The macro designs the necessary template(s) of the source plate(s) and generates the ready-to-use files for the nanodispenser and tablet-based application. The four-steps transfection protocol involves i) a diluent dispense with a classical liquid handler, ii) plasmid distribution and multiplexing, iii) a transfection reagent dispense by the nanodispenser, and iv) cell plating on the prefilled wells. The described software-based control of ADE plasmid multiplexing and transfection allows even nonspecialists in the field to perform a reliable cell transfection in a fast and safe way. This method enables rapid identification of optimal settings for a given cell type and can be transposed to higher-scale and manual approaches. The protocol eases applications, such as human ORFeome protein (set of open reading frames [ORFs] in a genome) expression or CRISPR-Cas9-based gene function validation, in nonpooled screening strategies.

This protocol describes high-throughput plasmid transfection of mammalian cells in a 384-well plate using acoustic droplet ejection technology. The time-consuming, error-prone DNA dispensing and multiplexing, but also the transfection reagent dispensing, are software-driven and performed by a nanodispenser device. The cells are then seeded in these prefilled wells.

使用声学纳米分配技术实现高通量DNA质粒多路复用和转染

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often ...