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>

<ol>
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L., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+demethylation+of+repetitive+elements+during+seed+development+underlies+gene+imprinting.">Extensive demethylation of repetitive elements during seed development underlies gene imprinting.</a> Science. 324, 1447-1451 (2009).</li><li>Muller, B., Sheen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokinin+and+auxin+interaction+in+root+stem-cell+specification+during+early+embryogenesis.">Cytokinin and auxin interaction in root stem-cell specification during early embryogenesis.</a> Nature. 453, 1094-1097 (2008).</li><li>Gehring, M., Missirian, V., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+analysis+of+parent-of-origin+allelic+expression+in+Arabidopsis+thaliana+seeds.">Genomic analysis of parent-of-origin allelic expression in Arabidopsis thaliana seeds.</a> PLoS One. 6, 23687-23 (2011).</li><li>Xiang, D., 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=Genome-wide+analysis+reveals+gene+expression+and+metabolic+network+dynamics+during+embryo+development+in+Arabidopsis.">Genome-wide analysis reveals gene expression and metabolic network dynamics during embryo development in Arabidopsis.</a> Plant Physiol. 156, 346-356 (2011).</li><li>Nawy, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+GATA+factor+HANABA+TARANU+is+required+to+position+the+proembryo+boundary+in+the+early+Arabidopsis+embryo.">The GATA factor HANABA TARANU is required to position the proembryo boundary in the early Arabidopsis embryo.</a> Dev Cell. 19, 103-113 (2010).</li><li>Baroux, C., Pecinka, A., Fuchs, J., Schubert, I., Grossniklaus, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+triploid+endosperm+genome+of+Arabidopsis+adopts+a+peculiar,+parental-dosage-dependent+chromatin+organization.">The triploid endosperm genome of Arabidopsis adopts a peculiar, parental-dosage-dependent chromatin organization.</a> Plant Cell. 19, 1782-1794 (2007).</li><li>Autran, D., 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=Maternal+epigenetic+pathways+control+parental+contributions+to+Arabidopsis+early+embryogenesis.">Maternal epigenetic pathways control parental contributions to Arabidopsis early embryogenesis.</a> Cell. 145, 707-719 (2011).</li><li>Wohrmann, H. 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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）?...

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...

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

Research

JoVE Journal

Biology

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

17.6K 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 ...

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 ...

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 ...

通过共聚焦成像对 ERL1 动力学进行时空研究

Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane system. This includes the secretory transport of newly synthesized proteins to the cell surface or the outside of the cell, the endocytic transport of extracellular cargoes or plasma membrane components into the cell, and the recycling or shuttling transport of cargoes between the subcellular organelles, etc. Membrane trafficking events are crucial to the development, growth, and environmental adaptation of all eukaryotic cells and, thus, are under stringent regulation. Cell-surface receptor kinases, which perceive ligand signals from the extracellular space, undergo both secretory and endocytic transport. Commonly used approaches to study the membrane trafficking events using a plasma membrane-localized leucine-rich-repeat receptor kinase, ERL1, are described here. The approaches include plant material preparation, pharmacological treatment, and confocal imaging setup. To monitor the spatiotemporal regulation of ERL1, this study describes the co-localization analysis between ERL1 and a multi-vesicular body marker protein, RFP-Ara7, the time series analysis of these two proteins, and the z-stack analysis of ERL1-YFP treated with the membrane trafficking inhibitors brefeldin A and wortmannin.

作者聚焦：研究气孔谱系细胞中膜运输事件的基于图像的方法  

Several commonly used methods are introduced here to study the membrane trafficking events of a plasma membrane receptor kinase. This manuscript describes detailed protocols including the plant material preparation, pharmacological treatment, and confocal imaging setup.

基于图像的方法研究气孔谱系细胞中的膜运输事件

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane ...

一种测量拟南芥花粉水合作用曲线的改进方法

A High-Resolution, Single-Grain, In Vivo Pollen Hydration Bioassay for Arabidopsis thaliana

Sexual reproduction in flowering plants requires initial interaction between the pollen grain and the stigmatic surface, where a molecular dialog is established between the interacting partners. Studies across a range of species have revealed that a series of molecular checkpoints regulate the pollen-stigma interaction to ensure that only compatible, generally intraspecific pollen is successful in effecting fertilization. In species that possess a 'dry stigma', such as the model plant Arabidopsis thaliana, the first post-pollination, prezygotic compatibility checkpoint is the establishment of pollen hydration. 
This phase of pollination is tightly regulated, whereby signals from the pollen grain elicit the release of water from the stigma, thus permitting pollen hydration. The ability to accurately measure and track pollen hydration over time is key to the design of experiments directed at understanding the regulation of this critical step in reproduction. Published protocols frequently utilize flowers that have been excised from the parent plant, maintained on liquid or solid media, and bulk pollinated. 
This paper describes a noninvasive, in vivo pollination bioassay that permits minute-by-minute hydration tracking of individual A. thaliana pollen grains at high resolution. The assay is highly reproducible, able to detect very subtle variations of pollen hydration profiles, and thus is suitable for the analysis of mutants that affect pathways regulating pollination. Although the protocol is lengthier than those described for bulk pollinations, the precision and reproducibility it provides, along with its in vivo nature, make it ideal for the detailed dissection of pollination phenotypes.

作者聚焦：拟南芥的高分辨率、单粒、体内花粉水合生物测定  

An improved method to measure pollen hydration profiles in Arabidopsis thaliana is described here. The new method offers higher resolution, is noninvasive, and is highly reproducible. The protocol represents a new tool for a finer dissection of the processes that regulate the early stages of pollination.

拟南芥的高分辨率、单粒、体内花粉水合生物测定

Sexual reproduction in flowering plants requires initial interaction between the pollen grain and the stigmatic surface, where a molecular dialog is ...