我们要感谢塔尔Nawy和马丁拜耳他们的意见，在胚胎隔离。地铁，VG，UG和CB的设计胚胎隔离设备。地铁，VG和CB的发展胚胎隔离协议。地铁，VG和CB成立的协议，分离出的胚胎，产生的胚胎基因，VG进行地铁GUS染色，PCR，JJ鱼实验。地铁，VG，CG和UG写的手稿。这项工作是由苏黎世大学，罗氏研究基金会（地铁）名衔，以及从瑞士国家基金会（UG和CB）补助。

概述图1中所示的流程图中的程序。微毛细管和仪器的设置示于图2和图3，和胚胎隔离的典型步骤示于图4。 1。材料和缓冲液制备 1.1硅涂层玻璃微毛细管<ol><li>将微毛细管〜5毫升的Sigmacote（Sigma公司）在一个15毫升的Falcon管中，颠倒几次。 </li><li>移除该溶液中，放置在Falcon管中的毛细血管的铝箔和烘烤3小时，在60℃下在室温下保存。 </li></ol> 1.2获取〜50-100微米直径的微细提示<ol><li>可以手动地在本生灯或通过使用市售的拉出器（垂直丝拔出器或微量拉马），拉直径为1毫米的玻璃毛细管。 </li><li>使用钻石尖笔或刀片切割的前端的上拉毛细管以创建所需的开口。选择最佳形立体显微镜下毛细血管。的开口应为50-100微米（ 图2）。 </li></ol> 1.3玻片制备<ol><li>硅化干净的幻灯片，涵盖了所有井与Sigmacote（〜1毫升/幻灯片）5分钟，取出。烘烤3小时铝箔。在室温下保存。 </li><li>洗10分钟，在10％的SDS，2×2分钟在无核酸酶的水（高压灭菌DEPC-DDH 2 O的），在70％乙醇2分钟，2分钟，在100％乙醇，多井的玻璃显微镜载玻片空气干燥。所有步骤都做了蒸压科普林氏罐子。幻灯片可以重新使用多次，每次使用之间提供彻底的清洁。 </li><li>之前，为了胚胎隔离扩散〜0.5微升10毫克/毫升牛血清白蛋白（BSA）与吸移管的整个表面上的各孔中，风干。 </li></ol> 1.4显微镜和毛细血管设置， &lt;醇&gt; <li>使用倒置显微镜，10倍和20倍的放大倍率目标。优化光的对比度（胚胎是相当透明的）。 </li><li>放置一个显微持有旁边的显微镜的玻璃毛细管。玻璃毛细管连接到微量注射器（ 图3A）或一个普通P-200的移液管，通过橡胶管（ 图3B，请参阅2001的详细说明，在此设置中）。 </li><li>将毛细管在〜70°的角度（ 图3）以上的显微镜载片上，并调整位置的视场中的开口。 </li><li>只要胚胎隔离前占用和释放〜5-10微升BSA（1毫克/毫升），涂层的毛细管。 </li></ol> 1.5缓冲器 表1列出了根据下游应用的隔离和目标缓冲区。 <ol><li>准备〜1毫升缓冲隔离每个样品在使用前新鲜，并保持它在冰上。 </li><li>准备目标缓冲区在0.5毫升的Eppendorf低约束力的管冰（分子），或在潮湿室在显微镜幻灯片（细胞学）。 </li></ol> 2。种子解剖和胚胎提取 2.1种子发展同步<ol><li class="jove_content">阉割鲜花和保持他们2天的生长室，同时避免接触的暴露与其他花的雌蕊，然后授粉（ 例如 11）。 </li><li class="jove_content">清除种子的微观调查，在你的成长条件下测试的发展阶段。随着我们的成长条件（16小时光照在21°C，8小时黑暗在18°C和70％的湿度）收集种子2.5天授粉后（DAP）的产生主要有2-4个细胞的胚胎和种子收集了350 - 4 DAP产生球状胚胎。 </li></ol> 2.2种子解剖和破裂<ol><li>删除的s10-15角果立体显微镜下用镊子和胰岛素针（〜2.5 DAP）的电火工品。 </li><li>种子浸入在20μl的隔离缓冲液在2 ml圆底的Eppendorf管中，在冰上放置。 </li><li>轻轻粉碎的种子用塑料杵（预清洗的10％的SDS，DEPC-DDH 2 O的漂洗，并用70％乙醇洗涤）籽提取物是混浊的胚胎，直到释放。武力申请将由审判后，每一个用户。 </li><li>冲洗洗杵杵用300微升的隔离缓冲和稀释样品。 </li><li>分拆下来的提取物在5000 XG 5秒。轻轻悬浮颗粒的提取物通过吹打向上和向下的2-3倍。 </li><li>提取过滤30微米尼龙网（安装管适配器， 例如从PartecCelltricks）。冲洗网带一个额外的200微升隔离缓冲区。 </li></ol> 3。胚胎隔离 3.1玻片制备<ol> &lt;LI&gt;将一个干净，BSA的涂层和硅多井幻灯片倒置显微镜的舞台上，通过向上和向下轻轻吹打悬浮过滤籽提取物和吸管2 40-50微升籽提取物的液滴成1或2口井。在一个时间只有1或2滴筛选防止样品的蒸发。 </li><li>将50微升新鲜的隔离缓冲液（1洗ST下降）在不同的投影片的井前准备。在一个有盖的，湿热试验箱，防止水分蒸发，保持这张幻灯片。 </li></ol> 3.2屏幕，清理，收集<ol><li>筛选胚胎籽提取物的液滴，在10倍的放大倍率目标所需的舞台。如果有必要，确认阶段，与20倍的放大倍率。胚胎通常会沉至底部的滑动。 </li><li>手动清除杂物围绕胚胎的钨针，胰岛素针或类似设备。 </li><li>将玻璃毛细管附近的胚胎，使用显微操作器，吨阿克了胚胎，用尽可能小的解决方案。 </li><li>收集几个胚胎（ 例如，所有的液滴），并释放他们在洗ST下降（分子应用程序）或在目标缓冲区（细胞学）。每个收集轮应保持在5-10分钟和胚胎必须被收集在（所有收集轮的总体积应为&lt;5微升）最小体积。 </li><li>重复筛选和收集在第一洗涤下拉，直到所需量的胚胎被收集（集中，如果可能的话，以方便回忆）。 </li><li>记得所有的胚胎从洗降（如果碎片结转，用针之前删除它们回忆）。 </li><li>松开胚胎成第二洗涤下拉为50μl。重复3.2.6。 </li><li>松开目标缓冲区中的胚胎。该转让应涉及接触，而不是浸泡，毛细管末端。 </li><li>更换microcapil下一个样品LARY。 </li></ol>

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作者宣称，他们有没有竞争经济利益。

我们开发了一个胚胎隔离协议，它是快速，有效，并可以很容易地转移到其他实验室。 这里所叙述的设备包括倒置显微镜，显微操作，玻璃微毛细管，垂直灯丝拉出器和微量注射器（ 图3A）。其设置与一个单一的动物细胞分离的转录组学分析17。我们还成功地与玻璃微毛细管手动伸过来的火焰（切带菱形刀片），并通过标准实验室移液器（P-200的Pipetman）（ 图3B），而不是一个微量经营一个更基本的设置。在这种情况下，微细管，通过橡胶管连接到吸移管。可以用来做的交界处，用封口膜包裹，以使它们气密10微升filtertips的。 MICR固定在毛细管 - 枪头举行 -omanipulator臂采用聚苯乙烯块和磁带（ 图3B）。这些基本设置被证明是有效和可靠的8。然而，存在的主要困难之一是维持微细管和移液管的空气之间的紧密连接，特别是当改变毛细管。验证和调整的压力在毛细管 - 以确保微调胚胎收集在一个最小的体积 - 可以使用这些基本设置，费时。改进的安装与液压控制的显微操纵器和一个垂直的灯丝车夫是一个相当大的改进，并节省时间。 这种方法的成功应用所需的技能很容易转移。五个用户在我们的实验室的方法在一个相对较短的时间内成功地了解到。某些简化的协议，可能存在根据用户的操纵容易。例如，它可能会跳过步骤2.2.5和2.2.6，而解剖只有5角果（而不是15）和胚胎周围彻底清洗碎片钨针（此过程适用于8）。通过去雄和授粉延迟种子发育兼同步，但建议增加在相同的发展阶段，特别是在早期胚胎的数量。此外，胚胎隔离可以被加速洗涤步骤被省略， 例如细胞学应用。此外，滑动的硅化（步骤1.3.2）的用户快速地工作，使得胚胎粘在玻璃表面，随着时间的推移是没有问题的，是没有必要的。 无论是过滤或手工清洗应用时，这是非常重要的，以避免结转，为下游的RNA或DNA的应用程序创建的潜在污染的组织碎片。这个问题是最近提出的在转录组分析研究，采用不同的方法胚胎隔离18。批量胚胎隔离这里描述的协议相比， 拟南芥胚胎解剖，从个别种子手动使用细针，一个漫长的过程，需要出色的清扫能力。此过程显然需要2个或更多的清洗，以避免种子周围的组织，可能遇到的情况的可能的污染细胞结转使用此方法时，考虑到小尺寸的胚胎，在访问它们与解剖针的困难，和密合性周围的细胞。与此相反，我们的程序（i）允许稀释后的胚胎从种子中挤出破碎和周围的碎片胚胎收集在一个大体积（500微升），然后，（ii）该视觉上选择没有粘合剂的胚胎，污染碎屑，及（iii）的稀释可能的RNA污染泄漏的破裂细胞的洗涤步骤。虽然有可能只使用一个洗涤步骤中，如果在胚胎显得非常干净，并收集在不到5微升8，洗第二个步骤是建议，尤其是对于初次使用的用户可能会收集的胚胎在几轮（ 即添加剂容积）。应收集在胚胎尽可能少尽可能溶液，允许的最大稀释的洗涤步骤，每一潜在的污染物。每个收集轮应尽量短（5-10分钟内）释放后，放入洗净下降（不再存在在毛细管中趋于减少的回收率）允许的最大恢复。此外，收集到的胚胎转移到提取介质（以下洗涤步骤）应只涉及微细尖端的开放，而不是浸泡的尖端接触，因为这可能也将碎片粘在上面的微细壁开幕。最后，毛细管应改变之间的每一个新的种子提取物。 我们的协议允许几个起伏tream应用程序，通过简单地使用适当的隔离缓冲区保存RNA，DNA，染色质，酶或荧光报道分子的完整性。我们成功地分离出RNA保护性隔离缓冲器，用于RNA的提取及转录分析8，1为DNA甲基化分析的TE缓冲液，100mM磷酸缓冲盐水（PBS）进行FISH和免疫染色（詹尼士Grossniklaus和Baroux的，未发表的， 图中的胚胎5C），染色溶液没有GUS报告基因分析的底物（19， 图5B）。应用最敏感的隔离条件/胚胎质量肯定是RNA提取和扩增。从孤立的胚胎中提取的RNA的数量是相当低的。从〜30 2-4细胞阶段的胚胎（因此60-120细胞总数），我们估计金额〜纳克两个安捷伦2100生物分析仪（安捷伦科技）和量子比特（Invitrogen公司）测量的基础上的总RNA。 Followi吴RNA提取，RNA的质量验证的Agilent RNA 6000 Pico芯片（安捷伦科技）。但是，低提取的RNA的量并不总是足以产生一个可靠的档案中。扩增产物（cDNA）的（ 例如 PCR检测的低表达基因的胚胎特异性和/或生物分析仪配置文件），因此，必须做进一步的测试。 最后，能够隔离胚胎的视觉标准，在我们的协议是一笔巨大的财富。从相同的拟南芥角果（水果）胚胎不同步发展。因此，工作在整个的长角果提取物（ 例如，用于RT-PCR分析或定量的报告基因分析）不允许与一个给定的发育阶段很好的相关性。隔离在一个特定的发育阶段的胚胎，解决了这个问题。另外，一个类似的问题提出使用时，其中的长角果包含不同的胚胎基因型的杂合突变体。隔离基于视觉标准到d的胚胎例如 istinguish野生型突变胚胎的表型与基因型允许下游关联分析。我们已经这样做了，成功地分析DNA甲基化特异位点不同基因型20。

开花植物的胚胎被包围的胚乳，营养组织来自第二施肥事件。胚和胚乳种皮包围几层细胞。总的来说，这些组织形成了一个种子，这里面的水果开发。因此，组织和细胞特异性分析拟南芥胚胎强烈受损，由于他们无法进入。然而，胚胎后期球状或更高的阶段是相对适合人工清扫使用细钨针，体视显微镜下，或使用镊子提取它们施加轻微压力种子。这种技术已成功用于转录或表观基因组的分析，如分析芯片杂交，亚硫酸氢盐测序，RNA测序（ 如 1-3）。相反，研究胚胎在球状早期受精卵保持技术上的挑战。至目前为止，只有少数的研究有代表转录组分析orted固定的种子第4或个人从种子的胚胎，用细的工具5手动提取的胚胎组织可以使用激光捕获显微切割（LCM）的幼胚。然而，LCM设备是不常用的和手动提取胚胎在早期阶段是耗费时间和需要优秀的清扫技能是不会轻易转让。除的全基因组分析， 原位基因表达分析也难以进行，年轻，整个安装拟南芥胚胎。在一定程度上被释放幼胚在种子上的温和的压力来在显微镜玻片上，并用于报告基因检测或蛋白的检测通过免疫组化（例如见6,7）。然而，该技术中，不允许高通量胚胎隔离，从而阻碍了定量分析。 因此，我们开发了一个高效，快速的协议从拟南芥种子，设置简单，很容易转移，适合各种下游应用的早期胚胎隔离。其基本原理是轻轻压碎种子 - 从解剖幼荚Eppendorf管中，使用塑料杵在适当的隔离缓冲区。籽提取物被放置在液滴上的多井滑动的存在下释放的胚胎进行筛选使用倒置显微镜在所需的阶段。用玻璃微毛细管连接到微量注射器或标准实验室吸液管收集胚胎。对于分子的应用，胚胎洗涤两次，反复滴释放到新的隔离缓冲，然后将他们转移到目标缓冲区在一个最小的体积。进行细胞学的应用程序（报告基因分析，免疫染色，FISH），洗涤步骤可以省略。 该方法有几个优点:（一）〜45分钟进行细胞学的应用程序产生25-40个胚胎的lications或在3-4小时的分子的应用程序（包括清洗步骤），（ii）其允许隔离特异性胚胎阶段，（ⅲ）是很容易转移到其他人的实验室由于其简单的设置，（ⅳ）需要可负担得起的设备的基本设置，这是可以升级，以及（v）被成功地用于各种下游应用，如RNA测序，基因特异性的DNA甲基化分析9，报告基因分析（10和Raissig 等。准备），鱼（J.詹尼士，U. Grossniklaus，C. Baroux未发表的，请参阅图5）。

<table border="1">
	<tbody>
 <tr>
 <td>Name of the Reagent</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>REAGENTS</td>
 </tr>
 <tr>
 <td>Sigmacote</td>
 <td>SIGMA </td>
 <td>SL2-100 ml</td>
 <td></td>
 </tr>
 <tr>
 <td>RNAse OUT</td>
 <td>Invitrogen (life technologies)</td>
 <td>10777-019</td>
 <td></td>
 </tr>
 <tr>
 <td>First- strand buffer</td>
 <td>Invitrogen (life technologies)</td>
 <td>18064-022</td>
 <td>contained in Superscript II package</td>
 </tr>
 <tr>
 <td>DTT</td>
 <td>Invitrogen (life technologies)</td>
 <td>18064-022</td>
 <td>contained in Superscript II package</td>
 </tr>
 <tr>
 <td>Bovine serum albumin (BSA) 100x =10 mg/ml </td>
 <td>New England Biolabs Inc.</td>
 <td></td>
 <td>Different suppliers will also work </td>
 </tr>
 <tr>
 <td>Thin wall Capillaries 1.0 mm</td>
 <td>World Precision Instruments</td>
 <td>TW100F-4</td>
 <td></td>
 </tr>
 <tr>
 <td>DNA LoBind tube 0.5 ml</td>
 <td>Vaudaux-Eppendorf</td>
 <td>0030108.035</td>
 <td></td>
 </tr>
 <tr>
 <td>CellTrics&Delta; 30 &mu;m</td>
 <td>PARTEC</td>
 <td>04-0042-2316</td>
 <td></td>
 </tr>
 <tr>
 <td>5wells 10 mm diameter slides</td>
 <td>Electron Microscopy Sciences</td>
 <td>63421-10</td>
 <td></td>
 </tr>
 <tr>
 <td>Formaldehyde Solution</td>
 <td>Sigma-Aldrich</td>
 <td>F1635</td>
 <td></td>
 </tr>
 <tr>
 <td>Superfrost Plus slide</td>
 <td>Thermo Fisher</td>
 <td>J1800AMNZ</td>
 <td>Menzel-Gl&auml;ser</td>
 </tr>
 <tr>
 <td>Tris</td>
 <td>Amaresco</td>
 <td>0497</td>
 <td></td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>Applichem</td>
 <td>A2937</td>
 <td></td>
 </tr>
 <tr>
 <td>Glycin</td>
 <td>Fluka</td>
 <td>50050</td>
 <td></td>
 </tr>
 <tr>
 <td>SDS pellets</td>
 <td>Roth</td>
 <td>CN30.3</td>
 <td></td>
 </tr>
 <tr>
 <td>Micro Pestle</td>
 <td>VWR</td>
 <td>431-0094</td>
 <td></td>
 </tr>
 <tr>
 <td>Microfine insulin syringes</td>
 <td>BD</td>
 <td>U-100</td>
 <td></td>
 </tr>
 <tr>
 <td>DEPC</td>
 <td>Sigma-Aldrich</td>
 <td>D5758</td>
 <td></td>
 </tr>
 <tr>
 <td>Ethanol</td>
 <td>Schaurlau</td>
 <td>ET00102500</td>
 <td></td>
 </tr>
 <tr>
 <td>Forceps N5</td>
 <td>Dumont</td>
 <td>0108-5</td>
 <td></td>
 </tr>
 <tr>
 <td>Bioanalyzer Pico Chip</td>
 <td>Agilent Technologies</td>
 <td>5067-1513</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 <td>Inverted microscope</td>
 <td>Nikon TMS (Japan),</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Micromanipulator</td>
 <td>Leitz</td>
 <td></td>
 <td>Leica</td>
 </tr>
 <tr>
 <td>Micomanipulator Post mount LH1 probe</td>
 <td>Leica microsystems</td>
 <td>39430101</td>
 <td>Different brand will also do the work</td>
 </tr>
 <tr>
 <td>Vertical filament puller</td>
 <td>Sutter instrument</td>
 <td>P-20 model</td>
 <td>Other model are also suitable </td>
 </tr>
 <tr>
 <td>Cell Tram vario</td>
 <td>Vaudaux-Eppendorf</td>
 <td>5176.000.033</td>
 <td></td>
 </tr>
 <tr>
 <td>Bioanalyzer</td>
 <td>Agilent Technologies</td>
 <td>2100</td>
 <td></td>
 </tr>
 <tr>
 <td>Qubit Fluorometer</td>
 <td>Invitrogen (life technologies</td>
 <td></td>
 <td></td>
 </tr>
	 </tbody>
 </table>

efficient and rapid isolation of early-stage embryos from arabidopsis thaliana seeds

开花植物，胚胎发育滋补组织 - 胚乳 - 经产妇种子珠被包围（或种子大衣）。其结果是，隔离植物胚胎早期阶段（1细胞球形期）在技术上具有挑战性，由于它们的相对位置偏远。在早期阶段高效的手动剥离强烈影响年轻的拟南芥种子的小尺寸和对周围组织的密合性的胚胎。在这里，我们描述了一种方法，使拟南芥幼胚的高效分离，得到在1小时至4小时，根据下游应用的40个胚胎。通过略微粉碎250-750种子用塑料杵在Eppendorf管中，的胚胎被释放到隔离缓冲器。一种玻璃微毛细管连接到一个标准实验室吸液管（通过橡胶管）或液压控制的微量注射器是用来收集胚胎从液滴位上多井倒置显微镜上滑动。所需的技术很简单，很容易转移，并基本安装不需要昂贵的设备。收集胚胎适合用于各种下游应用如逆转录-聚合酶链反应（RT-PCR），RNA测序，DNA甲基化分析， 荧光原位杂交（FISH），免疫荧光，和报告基因检测。

洗涤胚胎分离过程（ 图1），允许多达隔离如果洗涤液进行的分子的应用程序， 例如在4小时中的40个胚胎，或在不到一个小时，如果省略， 例如用于细胞学应用。 图2显示高和低品质微细的提示和图3示出的胚胎分离机的安装，在倒置显微镜上， 图4显示的过程中，胚胎隔离。 我们成功地应用于我们最近几篇文章发表在各种应用程序。该方法最初是开发早期胚胎转录分析父母的贡献。通过两种不同的拟南芥加入（兰茨贝格万寿菊 （L ER）和哥伦比亚（COL-0））杂交产生的杂交胚胎中分离出2-4细胞阶段。中提取总RNA，cDNA的库采用线性扩增并测序了坚实的平台上。等位基因特异性转录生成基于SNP ​​分析8。为了控制我们的胚胎cDNA文库测序之前扩增胚胎特定的成绩单，WUSCHEL同源盒2，8和9（WOX2 WOX8 WOX9）和肌动蛋白11（13,14， 图5A）。 此外，这个方法是用来隔离幼胚，胚胎在特定的位点在基因组中的DNA甲基化模式分析。胚胎中分离出，并在1×TE缓冲液洗涤。进行小规模的亚硫酸氢盐测序的说明9,15。 同样，胚胎隔离已经被证明是非常有用的报告基因检测与胚胎低表达，在产妇的种皮混淆检测或屏蔽周围的胚胎（胚乳，种皮）组织记者表达。胚胎CARR<html>英记者转基因染色（β-葡萄糖醛酸酶; GUS）或直接进行隔离（绿色或红色萤光蛋白GFP，RFP）。 MEDEA启动子（对MEA）10的控制下表达GUS报告基因的胚胎在图5B中给出了一个例子。相对缓解可以分离出许多胚胎也可以进行定量分析（ 例如在不同的遗传背景，Raissig中，Grossniklaus等染色的胚胎数。准备中）。 最后，我们成功地应用荧光原位杂交（FISH）和免疫组化技术来研究核架构隔离嵌入丙烯酰胺垫在胚胎的幻灯片上。的FISH探针对的着丝粒重复序列和的核仁的组织区域，例如， 如图5C所示。 <html> 1"FO:内容宽度="5英寸"FO:SRC ="/ files/ftp_upload/50371/50371fig1highres.jpg"SRC ="/ files/ftp_upload/50371/50371fig1.jpg"/&gt; 图1。胚胎分离过程的流程图。协议分为三个部分:1 -材料准备; 2 -种子解剖3 -胚胎移植隔离。 <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/50371/50371fig2highres.jpg" src="/files/ftp_upload/50371/50371fig2.jpg" /> 图2。的微细提示。A.高品质的顺利开幕，大约100微米的微细提示与。B.低质量的扩大开放和轮廓不规则微细提示（仅作细胞学应用这些技巧是可以接受的）。比例尺:2毫米。 ghres.jpg"SRC ="/ files/ftp_upload/50371/50371fig3.jpg"/&gt; 图3。设置的胚胎隔离显微镜。A.和B.筛选是在显微镜玻片上，在倒置显微镜下。收集胚胎，用玻璃微毛细管固定在显微操作器能够精确地控制它的位置（参见图4）和连接到一个微量注射器（A）或标准实验室吸液管（B）。A.玻璃微细管与液压控制的微量注射器（ 例如离心细胞电车Vario的）的一个精确的控制过程中B胚集合。玻璃微毛细管连接到一个标准的P-200移液管通过橡胶柔性管。胚胎收集和释放的控制转动的移液管的校准轮。作为连接器和切割枪头路口用封口膜密封。英里crocapillary显微操作器通过聚苯乙烯嵌段，Falcon管中，磁带上的固定。 <img alt="图4" fo:content-width="5in" fo:src="/files/ftp_upload/50371/50371fig4highres.jpg" src="/files/ftp_upload/50371/50371fig4.jpg" /> 图4。胚胎隔离过程。答 2-4细胞胚胎（箭头）被确定在种子提取物（筛选滴），被包围的碎片（种皮碎片）。用针管周围的杂物被手动删除。C.右旁的胚胎（箭头所示）移动的玻璃毛细管，D.胚胎收集于毛细管（箭头）。E.几个胚胎最后一滴后洗涤。规模= 50微米。 <img alt="图5" src="/files/ftp_upload/50371/50371fig5.jpg" /> F<html> IGURE 5。下游应用胚胎隔离。A.基因表达分析:PCR扩增WOX9 WOX2 WOX8 ACTIN11 13,14 2-4细胞期胚胎cDNA文库（描述8）洗1X次（第一车道）和基因组DNA（2车道）。B.记者测定:从植物中分离出携带AP MEA :: GUS结构是在一个标准的GUS染色解决方案（从Raissig 等人，2011年许可后转载幻灯片上染色的8细胞胚胎植物细胞 ，ASPB版权）。 C.荧光原位杂交（FISH）:8细胞胚胎对抗着丝粒重复（红色），和45S rDNA重复的间接免疫检测前16（绿色）与探针杂交。双色FISH图像叠加与DAPI复染和DIC图像（的DM6000B落射荧光显微镜，莱卡，德国）的。 内容"的FO:保持together.within"="总是"&gt; <table border="1"><tbody><tr><td> 应用 </td><td> 隔离缓冲 </td><td> 目标缓冲区 </td></tr><tr><td> RNA的提取（ 例如扩增和转录） </td><td> 1X第一链缓冲液（Invitrogen公司），1毫米DTT，4％RNAseOUT </td><td> RNA提取缓冲液</td></tr><tr><td> DNA提取（ 如亚硫酸氢盐测序） </td><td> 100毫摩尔Tris，pH为8的10mM EDTA（TE） </td><td> DNA提取缓冲液，或1倍TE缓冲液</td></tr><tr><td> GFP记者分析</td><td> 1 M甘氨酸0.5倍MS 1 </td><td> 1 M甘氨酸0.5倍MS </td></tr><tr><td> GUS记者分析</td><td>染色缓冲液2 无 X-GLUC（基板） </td><td>染色缓冲液与 X-GLUC（基片） </td></tr><tr><td> 鱼及免疫组化染色</td><td> 100 mM磷酸盐缓冲液（PBS） </td><td>激活亚克力:Bisacryl的混合物（33:3）的Superfrost加幻灯片聚合的上30分钟</td></tr></tbody></table> 表1中。的分离和不同的下游应用的目标缓冲区的缓冲区可以适应具体的实验要求1 Murashig＆Skoog培养基。2 例如 Jefferson 等人，:EMBO J. 6（13），3901-3907（1987）。

Watch this Scientific Journal Video about Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds at JoVE.com

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

我们报告了一种简单有效的方法分离胚胎在早期发展阶段<em&gt;拟南芥</em&gt;种子。多达40个胚胎可以在1小时至4小时，分离根据下游应用。该程序是适用于转录，DNA甲基化，报告基因的表达，免疫荧光<em在原位</em&gt;杂交分析。

高效，快速的早期胚胎分离<em&gt;拟南芥</em&gt;种子

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

efficient-rapid-isolation-early-stage-embryos-from-arabidopsis

Nanyang Technological University

Research

JoVE Journal

Biology

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

17.7K 次观看.  University of Zürich. 我们报告了一种简单有效的方法分离胚胎在早期发展阶段拟南芥种子。多达40个胚胎可以在1小时至4小时，分离根据下游应用。该程序是适用于转录，DNA甲基化，报告基因的表达，免疫荧光

视频: Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

The neural tube expresses many proteins in specific spatiotemporal patterns during development. These proteins have been shown to be critical for cell fate determination, cell migration, and formation of neural circuits. Neuronal induction and patterning involve bone morphogenetic protein (BMP), sonic hedgehog (SHH), fibroblast growth factor (FGF), among others. In particular, the expression pattern of Fgf8 is in close proximity to regions expressing BMP4 and SHH. This expression pattern is consistent with developmental interactions that facilitate patterning in the telencephalon.

Here we provide a visual demonstration of a method in which an in ovo preparation can be used to test the effects of Fgfs in the formation of the forebrain. Beads are coated with protein and placed in the developing neural tube to provide sustained exposure. Because the procedure uses small, carefully placed beads, it is minimally invasive and allows several beads to be placed within a single neural tube. Moreover, the method allows for continued development so that embryos can be analyzed at a more mature stage to detect changes in anatomy and in neural patterning. This simple but useful protocol allows for real time imaging. It provides a means to make spatially and temporally limited changes to endogenous protein levels.

A variety of growth factors and proteins interact to induce cells to take on different cell fates during development. Here we demonstrate the use of an in ovo preparation to address possible interactions between different proteins in development by placing beads on E2.5 chick embryos.

配售早期鸡胚生长因子包被的磁珠

The neural tube expresses many proteins in specific spatiotemporal patterns during development.   These proteins have been shown to be critical for cell ...

科研

生物学

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

从14日龄的幼苗组织原生质体的分离拟南芥</em

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method1-2 has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, &beta;-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit3, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&#946;-glucuronidase Reporter Gene Expression

This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::&beta;-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.

花香浸改造拟南芥检查 pTSO2：β-葡萄糖醛酸酶报告基因的表达

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography (TLC) Coupled with Gas-Liquid Chromatography (GLC)

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as phosphatidylcholine or phosphatidylethanolamine, form bilayer membranes which act as both boundaries and interfaces for chemical exchange between cells and their surroundings. Unlike animals, plant cells have a special organelle for photosynthesis, the chloroplast. The intricate membrane system of the chloroplast contains unique glycerolipids, namely glycolipids lacking phosphorus: monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG)4. The roles of these lipids are beyond simply structural. These glycolipids and other glycerolipids were found in the crystal structures of photosystem I and II indicating the involvement of glycerolipids in photosynthesis8,11. During phosphate starvation, DGDG is transferred to extraplastidic membranes to compensate the loss of phospholipids9,12.
Much of our knowledge of the biosynthesis and function of these lipids has been derived from a combination of genetic and biochemical studies with Arabidopsis thaliana14. During these studies, a simple procedure for the analysis of polar lipids has been essential for the screening and analysis of lipid mutants and will be outlined in detail. A leaf lipid extract is first separated by thin layer chromatography (TLC) and glycerolipids are stained reversibly with iodine vapor. The individual lipids are scraped from the TLC plate and converted to fatty acyl methylesters (FAMEs), which are analyzed by gas-liquid chromatography coupled with flame ionization detection (FID-GLC) (Figure 1). This method has been proven to be a reliable tool for mutant screening. For example, the tgd1,2,3,4 endoplasmic reticulum-to-plastid lipid trafficking mutants were discovered based on the accumulation of an abnormal galactoglycerolipid: trigalactosyldiacylglycerol (TGDG) and a decrease in the relative amount of 18:3 (carbons : double bonds) fatty acyl groups in membrane lipids 3,13,18,20. This method is also applicable for determining enzymatic activities of proteins using lipids as substrate6.

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC

Composition of polar lipid extracts and the fatty acid composition of individual glycerolipids are determined in a simple and robust lipid profiling experiment. For this purpose, glycerolipids are isolated by thin layer chromatography and subjected to transmethylation of their acyl groups. Fatty acyl methylesters are quantified by gas-liquid chromatography.

拟南芥极地甘油脂分析薄层色谱（TLC）再加上气相色谱（GLC）

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

使用早期的小鼠胚胎组织特异性基因的染色质免疫沉淀检测

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

kinesin的分离和纯化果蝇胚胎

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

人类多能干细胞的神经元在一个Multititre的板格式的快速和高效地生成

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

代谢标记和膜分馏的比较蛋白质组学分析<em&gt;拟南芥</em&gt;悬浮细胞培养

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

组织化学染色<em&gt;拟南芥</em&gt;次生壁元素

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of microbial contaminants present on the seed surface. Currently, Arabidopsis seeds are sterilized using two distinct sterilization techniques in conditions that differ slightly between labs and have not been standardized, often resulting in only partially effective sterilization or in excessive seed mortality. Most of these methods are also not easily scalable to a large number of seed lines of diverse genotypes. As technologies for high-throughput analysis of Arabidopsis continue to proliferate, standardized techniques for sterilizing large numbers of seeds of different genotypes are becoming essential for conducting these types of experiments. The response of a number of Arabidopsis lines to two different sterilization techniques was evaluated based on seed germination rate and the level of seed contamination with microbes and other pathogens. The treatments included different concentrations of sterilizing agents and times of exposure, combined to determine optimal conditions for Arabidopsis seed sterilization. Optimized protocols have been developed for two different sterilization methods: bleach (liquid-phase) and chlorine (Cl2) gas (vapor-phase), both resulting in high seed germination rates and minimal microbial contamination. The utility of these protocols was illustrated through the testing of both wild type and mutant seeds with a range of germination potentials. Our results show that seeds can be effectively sterilized using either method without excessive seed mortality, although detrimental effects of sterilization were observed for seeds with lower than optimal germination potential. In addition, an equation was developed to enable researchers to apply the standardized chlorine gas sterilization conditions to airtight containers of different sizes. The protocols described here allow easy, efficient, and inexpensive seed sterilization for a large number of Arabidopsis lines.

The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.

拟南芥种子高通量灭菌的标准化方法

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of ...