The authors thank Nicolas Allegre, Maud Peyny and Jean-Philippe Da Ponte for their excellent technical assistance. This work was supported by the Agence Nationale de la Recherche (ANR-JC-CARDIAC-SPE), the INSERM starting grant, ANR ID-CELL-SPE grant, Equipe FRM and the AFM 15845 grant.

1.飞线代<ol><li>产生两个不同的构建体。在第一构建体中，RpL10A的cDNA（核糖体蛋白亚基L10A）被克隆GFP下游入pUASP-PL-GFP-NTER质粒（从32修改后的版本）。使用种系启动子允许转基因系的更多价用法。 注意：通过克隆使用pPTGAL4质粒的调控序列驱动懒散基因的GAL4（TF）的上游的组织特异性表达获取第二构建体。 </li><li>分别注入质粒导入瓦特1118苍蝇和插入构建成由P-元件插入在飞行基因组（按标准程序）33。 </li><li>为了选择转化收回飞线，在两个不同的染色体结构。最终的TRAP线由为了得到一个双转基因稳定线组合这两条线（遗传杂交）创建。 F0：W-; CYO，UAS EGFP :: RpL10A; MKRS / TM6Bx W-; CYO / SCU;没精打采的Gal4 / TM6B。 F1：W-; CYO，UAS EGFP :: RpL10A;没精打采的Gal4 / TM6B。大规模扩大这条线，并收集所需上演胚胎。 </li></ol> 2.胚胎收集<ol><li>准备含有约40克年轻蝇每笼8大圆筒人口笼（约18万蝇/笼）。 </li><li>继续执行所谓的预层次感，让苍蝇清除发育中的胚胎从他们的输卵管。为此，躺在苍蝇1小时于含固化琼脂和葡萄汁的混合物2 11个厘米直径的陪替氏培养皿并覆盖表面的⅓与新制备的酵母膏。经过3交往葡萄果汁板收集类似的新鲜制作的板真正的胚胎。 注：孵育时间将根据发展的时间窗口影响而有所不同。 </li><li>从笼中取出板并离开它们在相同的温度下需要获得正确发育阶段（例如，3小时产卵+ 10小时ADDIT的时间有理孵化给出了从胚胎期10-13小时后产蛋（AEL））。重悬板内容（胚，酵母膏，死蝇）用50毫升的水用刷子。 </li><li>经过一系列的筛（700微米，355微米，112微米）以分离从死苍蝇胚胎和剩余的身体部位，并收集胚胎保持在较小直径筛子，然后在去离子水中简单冲洗的。不要用每收集超过18个板块，因为它会堵塞滤网。 </li><li>在4.5％的漂白粉Dechorionate胚胎在去离子水中2分钟，用去离子水彻底冲洗30-60秒。孵育在PBS 0.01％吐温20，100微克/毫升放线菌酮的胚胎，在室温下搅拌5-10分钟。 </li><li>纤维素吸水张干胚，并将其转移到离心管。称重管和通过浸入液氮中闪冻胚胎。在这个阶段，将干燥的胚胎可存放数个月，在-80℃。 </li></ol><p类=“jove_title”&gt; 3。再加以绿色荧光蛋白抗体的磁性珠体的制备<ol><li>彻底重悬蛋白G磁珠在原瓶通过温和搅拌。 </li><li>转移90微升珠不含RNA酶的管和收集它们上的磁体（30-60秒）。 90微升珠粒需要进行免疫沉淀（IP）与1ml裂解物含60-80毫克/毫升蛋白质。尊重比例为防止珠饱和。根据蛋白质的量使用几个管。 </li><li>在磁体洗涤珠粒以1×PBS中0.01％吐温20（500微升），并收集珠丢弃上清液。加30微克的GFP抗体在350微升PBS中0.01％吐温20的至珠孵育缓慢端超过端混合30分钟，在室温。 </li><li>收集在磁体（30-60秒）珠，弃去上清液，并冲洗在500μl的多核糖体提取缓冲液（HEPES 10mM的，氯化钾150mM的，MgCl 2的5mM的，DTT为0.5mM，加入Triton 1％，放线菌酮100微克/毫升，镨otease抑制剂1×，RNA酶抑制剂100 U）。 </li><li>收集在磁铁珠和加入500μl封闭缓冲液（BSA 0.1微克/微升，酵母tRNA 0.1微克/微升，糖原0.1微克/微升在多核糖体提取缓冲液）。 </li><li>孵育在室温30分钟，并用新鲜封闭缓冲液重复此步骤一次。 </li><li>收集的磁珠和冲洗的500微升多聚核糖体提取缓冲液，然后回忆珠立即进行免疫纯化步骤（第6）。 </li></ol> 4.裂解液的制备<ol><li>在开始之前，设立程序上的多方位速度快珠磨机：2×10秒，5000转，15秒暂停。转印干燥胚胎（1.5克）至15ml管中预冷对含有多核糖体提取缓冲液冰，并立即均匀。均质1.5克的胚胎在4毫升的多核糖体提取缓冲液。 </li><li>两种传播胚胎匀浆预冷的2 mL管和研磨安莉芳操作系统通过运行均化程序。转移溶解物到新鲜预冷的微量离心管在冰上，并通过离心在4℃下以2000 克制备postnuclear上清液10分钟。 </li><li>上清转移到新鲜的预冷的微量离心管在冰上，加入10％的Nonidet P-40的1/100样品体积向上清液（最终浓度= 0.1％），并通过反转管轻轻混合。 </li><li>脉冲离心机在minifuge样品收集液体在管的底部，添加300mM的1,2-Diheptanoyl -sn-甘油基-3-磷酸胆碱（DHPC）（终浓度1/9样品体积= 30毫），轻轻混合它通过颠倒试管，并通过孵育期间颠倒几次在冰上孵育混合物5分钟（混合）。 </li><li>制备后线粒体清液离心在4℃下进行10分钟，以20,000 克 。测量蛋白质浓度通过Bradford方法：在裂解物浓度应介于60-80毫克/毫升。 ŤAKE上清液至新鲜新预冷的微量离心管中，并立即进行预吸收步骤（第5部分）。 </li></ol> 5.预吸收<ol><li>彻底缓慢搅拌重悬磁珠并转移30微升珠粒成微量离心管在RT（30微升珠粒/ ml的1胚胎裂解物）。 </li><li>收集珠上的磁铁，吸管上清，重悬在多核糖体提取缓冲液（500微升）的珠。收集珠上的磁铁，加入胚胎裂解物，并孵育1小时，在4℃下在旋转器上。 <ol><li>除去珠，并保持上清冰上的免疫纯化步骤。免疫纯化前，保持100微升上清液（输入）对全球RNA样品与免疫纯化后收集的RNA样品，通过RT-qPCR的，例如比较。 </li></ol></li></ol> 6.免疫纯化<ol><li>加入1毫升（相当于60-80毫克/毫升的蛋白质）的预吸收裂解物至含有90微升耦合到GFP抗体，封闭的珠不含RNA酶的管。孵育样品在4℃下2小时，轻轻端过端混合在试管旋转器中。另外，进行孵化O / N在4℃。 </li><li>培养结束后，收集的磁珠。使用minifuge降速的珠，然后重悬它们在500μl洗涤缓冲液（HEPES 10mM的，氯化钾350MM，MgCl 2的5mM的，诺乃洗涤剂P-40 1％，DTT 0.5mM的，放线菌酮100微克/毫升，RNA酶抑制剂为100U ），并收集他们的磁铁。重复此步骤两次。 </li><li>改变珠粒到一个新的预冷的无RNase管和洗涤两次。收集上的磁铁珠和通过TRIZOL直接加入1ml至珠进行到RNA提取，并按照制造商的说明。 </li></ol> 7. RNA清理与质量评价<ol><li>使用的RNeasy微型试剂盒按照制造商的说明。在完成后，ELUTËRN​​A与（X）微升RNA酶的水。暖2分钟，在60℃，和存储单元冷冻样品在-80℃下。使用生物分析仪检查RNA的质量（按照制造商的说明）。 </li><li>为了测试TRAPed RNA的特异性，根据制造商的说明（上标III）的3纳克纯化的RNA（输入和IP）执行反向转录。使用定量PCR使用基因特异性引物获得的基因。 </li></ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+transformation+of+Drosophila+with+transposable+element+vectors.">Genetic transformation of Drosophila with transposable element vectors.</a> Science. 218, 348-353 (1982).</li><li>Ingolia, N. 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=Ribosome+profiling+reveals+pervasive+translation+outside+of+annotated+protein-coding+genes.">Ribosome profiling reveals pervasive translation outside of annotated protein-coding genes.</a> Cell reports. 8, 1365-1379 (2014).</li></ol>

作者什么都没有透露。

本文介绍一种改性的翻译核糖体亲和纯化协议（称为“陷阱-RC”）致力于在果蝇胚胎罕见的细胞群的研究。信息被设置在用于特定核糖核酸成功分离适于微阵列或RNA-SEQ分析，所需的生物材料，即 ，1）的数量和优化过程为胚胎裂解和多核糖体提取的关键步骤，收率2）珠/抗体对裂解物的比率为最佳免疫沉淀; 3）步骤允许减少的背景，包括洗涤缓冲液组合物，以便运行的TRAP的rc实验以高特异性和灵敏度。 这种协议产生再生的数据使得它容易地应用到任何其他类型的细胞。这种技术使得可以分析差异基因表达在积极-翻译mRNA的水平，从而铺路理解在一个规范表达<html> IFIC细胞类型在一个特定的时间窗口。但是请注意，蛋白丰度将取决于翻译和退化过程的速率。为TRAP方法的主要限制是它不能测量蛋白质含量以精确定量的方式或以检测翻译后修饰。通过测量沿mRNA的核糖体体密度提高量化，并且在这样做时，在一个细胞类型特异性的方式可以使TRAP结合核糖体足迹一个非常强大的工具。在最近的论文34，在人胚胎肾293细胞中进行这种组合。作者跑核酸酶足迹接着RpL10A的使用链亲和素珠诱导型生物素化形式的亲和纯化。这是一个原则证明，有可能在整个有机体而靶向特定的细胞类型，所述的限制是需要的目的生物物质的量进行核糖体足迹。 “&gt;表演TRAP实验在具有全局转录分析并行将使得有可能给新转录和翻译actively-的RNA。这将是深深信息发生在特定的发育上下文或特定细胞群的潜在转录后机制之间跟踪比例 - 用微RNA的例子。 总之，TRAP是一种高效，特异性和灵敏的用于识别的RNA结合到在细胞特异性方式积极-翻译核糖体方法。这种方法可以在各种生物和组织类型的使用。这里所描述的新的TRAP的rc协议稀有细胞群（总细胞数小于1％）和最终材料的足够用于在全基因组水平的后续分析的量进行了优化。 这种方法的一个可能限制是一个强有力的司机的要求，以产生标记的核糖体在足够量的小样ETE与内源的未标记的人在靶细胞类型。在最近的一项研究25，作者估计10-30％的标记与未标记的核糖体的比例。 为了克服这个问题增加UAS-RpL10A-EGFP拷贝数应大大改进这种平衡。备选地，添加到所描述的遗传背景一个UAS-GAL4基因将放大生产GAL4蛋白质的和间接增加RpL10A-EGFP的表达。这应该有利于更好地占用mRNA上标记的核糖体。 注意，这已经有效的方法，可以更有力通过适应互补的方法带来了更多的定量方面或以发现和解开，这对基因表达的控制至关重要的分子机制作出。

了解发育过程中细胞如何获得特殊性能是至关重要的，以欣赏器官的复杂性，以及他们对病理状况的潜在变化。有一个大的研究推动破译的基因调控网络，通过unrevealing全球基因表达谱underly细胞规范和分化，聚合酶II绑定状态，在调控序列和组蛋白翻译后修饰的转录因子占用。 为了评估基因表达在全球范围内芯片和RNA测序方法的使用。微阵列容许检测mRNA的丰度，而核糖核酸-SEQ是更好地面向通知上的不同类型的RNA包括编码和非编码像微RNA，lncRNAs或snRNAs的RNA。然而，这些技术不能区分积极转录，稳态mRNA的，积极-mRNA翻译，和mRNA进入降解过程。测量稳定ST吃了mRNA水平是已知的蛋白质成分1-3的不良估计。在相反，识别积极-mRNA翻译给出蛋白产生的更精确的图像。 然而，转录研究主要被引导在整个生物体水平通过比较增益或的特定因子的功能丧失与野生型条件4-7或解剖组织，使得基因表达谱难以解释。在细胞靶向工具，亲和纯化和灵敏度的改进的最新进展现在允许执行的全基因组分析在活生物体小细胞群体或甚至单个细胞。 在果蝇中，转基因株系大型集合经由GAL4 / UAS系统8-10使不同的组织和细胞亚群的具体时间和空间定向，得到所需的细胞功能的分子机制的更准确的定量。 11。基于蔗糖梯度离心该方法允许多核糖体级分和测定mRNA的的选择翻译的全基因组时与微阵列或深度测序进行分析。多核糖体隔离可与核酸酶消化，以确定对应于在翻译过程12由核糖体保护RNA片段核糖体的脚印。核糖体足迹增加核糖核酸翻译和RNA序列级别分辨率和核糖体定位定量的准确性，使得能够测量翻译的速率。但是，到目前为止还没有被应用到translatomes的小区固有的隔离，其主要原因是在核糖体足迹过程结束时获得的RNA产量的急剧下降。鉴于RNA的大小选择可产生潜在的错误 - 对从也可以以类似的方式保护不同的RNA的RNP ositives，需要进一步的发展以提高核糖体足迹并使其适应小区特有的方法。 这种方法之一，被称为翻译核糖体亲和纯化（TRAP）已在2008年已描述由海曼等人 。并应用于从小鼠的神经元的一个子集隔离translatome。通过以细胞类型特异性的方式表位标记核糖体蛋白质，核糖可以选择性地纯化无细胞解离和排序的费力的工序。在这种情况下，60S核糖体蛋白L10A在核糖体的表面，具有标记的eGFP 13。自2008年以来，一些研究已经用这种方法在不同的物种，从小鼠14-19 为 X. 蟾 20,21，斑马鱼22,23和拟南芥24。该陷阱的方法也已经适应了果蝇模型，并使用普里FY细胞类型特异性从神经元细胞25和星形胶质细胞26的mRNA。多功能二进制GAL4 / UAS系统被用来表示在一个组织特异性方式GFP标记RpL10A。成蝇首脑进行解剖，以执行从（由泛神经ELAV-Gal4的驱动程序有针对性的）神经细胞和孤立的RNA核糖体的选择进行了测序。大量的上调基因对应于那些已知待表达在神经系统，这表明该方法具有良好的特异性和促使我们以使其适应稀有细胞群在显影果蝇胚胎本（小于1细胞的％） 。 在果蝇模型中，胚胎阶段是首选发育过程的研究模型，作为分子的行动者和形态发生运动是进化上保守。在这个模型中迄今开展的唯一的组织特异性转录的方法使用电池或核这样已执行rting并只能够研究稳态转录27-31，创造一个需要专用于组织/细胞特异性translatome分析在果蝇胚胎的方法。在这里，我们报告奉献给果蝇胚胎的第一个陷阱协议。用这种方法，从事合mRNA翻译在每个胚胎周围100肌细胞的一个非常有限的细胞群被成功分离高质量和特异性。二进制GAL4 / UAS系统用于驱动在肌肉的亚群GFP标记RpL10A的表达无任何毒性，没有明显的表型或发育迟缓如前所示25。 果蝇胚胎具有每hemisegment 30的肌肉，这将引起肌肉组织幼虫。通过使用发现的懒散身份基因的附近的调节区，6出来的30多成核的肌肉的有针对性的。在该测定中，核糖体使用的是翻译延伸固定在mRNA上抑制剂放线菌酮。胞质提取物随后用于亲和纯化用GFP抗体 - 包被的磁珠。纯化的RNA质量利用生物分析仪进行了验证。定量逆转录PCR（RT-qPCR的）被用来确定特异性和mRNA分离灵敏度和证明我们的优化的协议是高效率的。

<table border="0" cellpadding="0" cellspacing="0">
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			<td>HEPES</td>
			<td>Sigma&#160;</td>
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			<td>UC DAVIS/NIH Neuromab Faculty Antibodies Incorporated</td>
			<td>75-132&#160;</td>
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			<td>Applied Biosystem&#160;</td>
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			<td>Applied Biosystem&#160;</td>
			<td>AM95306</td>
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			<td>Rnase free tube 1,5 mL&#160;</td>
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			<td>BP337-500</td>
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trap-rc, translating ribosome affinity purification from rare cell populations of drosophila embryos

Measuring levels of mRNAs in the process of translation in individual cells provides information on the proteins involved in cellular functions at a given point in time. The protocol dubbed Translating Ribosome Affinity Purification (TRAP) is able to capture this mRNA translation process in a cell-type-specific manner. Based on the affinity purification of polysomes carrying a tagged ribosomal subunit, TRAP can be applied to translatome analyses in individual cells, making it possible to compare cell types during the course of developmental processes or to track disease development progress and the impact of potential therapies at molecular level. Here we report an optimized version of the TRAP protocol, called TRAP-rc (rare cells), dedicated to identifying engaged-in-translation RNAs from rare cell populations. TRAP-rc was validated using the Gal4/UAS targeting system in a restricted population of muscle cells in Drosophila embryos. This novel protocol allows the recovery of cell-type-specific RNA in sufficient quantities for global gene expression analytics such as microarrays or RNA-seq. The robustness of the protocol and the large collections of Gal4 drivers make TRAP-rc a highly versatile approach with potential applications in cell-specific genome-wide studies.

果蝇胚胎体细胞肌肉系统由每hemisegment 30的肌肉，并且每个肌肉具有特定的组属性：身份基因，位置，融合事件，附着位点和神经支配多个代码。识别翻译基因在特定的肌肉子会给形成这些特定的肌肉特性所需的蛋白质的信息。从肌肉表达身份基因懒散的亚群使用陷阱基础的方法，纯化RNA（图1A-B）。这种方法的一个主要问题是分离的RNA的质量和特异性。这里报告的优化的协议启用的评估与生物分析仪分析高质量的RNA系统恢复。轮廓获得的节目的RNA没有退化（图1C）。 在周围的TRAP方法开发等的研究，有人提出了数据的特异性。在这里，我们我的mProve抑制背景链接到非特异性核糖核酸至珠或管的结合和通过洗涤缓冲液的优化的方法。为了评估本底水平，并分离懒散表达细胞的效率，我们导致同胚胎阶段的3个重复的RT-qPCR的试验。折叠式富集的4种不同的基因（MEF2，懈怠，普洛斯彼罗，soxNeuro）进行了计算相比输入和归上日RPL32基因（图1D）。这些结果显示泛肌肉基因MEF2和懒散基因的5.6倍富集 2.3倍富集。与此相反，表达在神经系统中的两个基因被耗尽相比输入。非常相似的倍数变化值上观察到3次生物学重复，表明协议是健壮。随着没精打采-Gal4的驱动程序，并开始用1.5克，周围包含100 GFP细胞/胚胎（共150×10 ^ 6阳性1.5×10 ^ 6胚胎获得细胞）。 运行的TRAP实验后，产率为约25-45纳克的视阶段的感兴趣特异性RNA。这种材料是足以运行微阵列分析或使用扩增构建RNA测序文库的RNA序列。效率将用于表达RpL10A-EGFP司机的实力在很大程度上取决于。 <img alt="图1" src="/files/ftp_upload/52985/52985fig1.jpg" /> 图1.质量和从懒散-GAL4&gt; UASRpL10A-EGFP胚胎TRAP隔离RNA特异性评估。 （AB）的阶段-16胚胎表示RpL10A-EGFP表达特别是在每hemisegment（A）中的六个懒散肌肉细胞的共聚焦图像。共定位RpL10A-EGFP的与一般肌肉标记Beta3tubulin上观察到的合并图像 （B）。 （ 三）TRAPed RNA RA的质量控制氮对生物分析仪显示的rRNA 18S和28S完美的完整性。 （ 四）RT-qPCR的分析显示在舞台上，16个胚胎的3生物学重复的TRAP隔离基因实验特异性高。倍数变化的计算方法相比，输入和标准化对RPL32基因存在于所有的肌肉谱系MEF2成绩单是2.3倍富集比输入，而更多的限制没精打采的成绩单是5.6倍富集。神经细胞表达普洛斯彼罗和soxN基因被耗尽的2.2倍和5.5倍，分别为。误差线表示标准偏差（n = 3）。 <a href="https://www.jove.com/files/ftp_upload/52985/52985fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos at JoVE.com

TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos

Translating Ribosome Affinity Purification (TRAP) is able to capture cell-type-specific translation of mRNA. Here we report the first TRAP protocol dedicated to isolation of mRNA in rare cell populations of Drosophila embryos.

TRAP-RC，翻译核糖体亲和纯化的稀有细胞群<em&gt;果蝇</em&gt;胚胎

Measuring levels of mRNAs in the process of translation in individual cells provides information on the proteins involved in cellular functions at a given ...

trap-rc-translating-ribosome-affinity-purification-from-rare-cell

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Biology

TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos

17.7K 次观看.  University of Clermont-Ferrand. Translating Ribosome Affinity Purification (TRAP) is able to capture cell-type-specific translation of mRNA. Here we report the first TRAP protocol dedicated to isolation of mRNA in rare cell populations of Drosophila embryos. 

视频: TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

从Midgastrula舞台果蝇胚胎神经文化的制备

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

科研

生物学

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

果蝇胚胎的体内离心

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

直立成像果蝇胚胎

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

原代细胞培养从果蝇原肠胚

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

影像生活细胞的形状变化果蝇胚胎

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical studies are substantial, therefore it is essential that more predictive toxicology screens are developed and deployed early on in drug development (Greenhough et al 2010). Human hepatocytes represent the current gold standard model for evaluating drug toxicity, but are a limited resource that exhibit variable function. Therefore, the use of immortalised cell lines and animal tissue models are routinely employed due to their abundance. While both sources are informative, they are limited by poor function, species variability and/or instability in culture (Dalgetty et al 2009). Pluripotent stem cells (PSCs) are an attractive alternative source of human hepatocyte like cells (HLCs) (Medine et al 2010). PSCs are capable of self renewal and differentiation to all somatic cell types found in the adult and thereby represent a potentially inexhaustible source of differentiated cells. We have developed a procedure that is simple, highly efficient, amenable to automation and yields functional human HLCs (Hay et al 2008 ; Fletcher et al 2008 ; Hannoun et al 2010 ; Payne et al 2011 and Hay et al 2011). We believe our technology will lead to the scalable production of HLCs for drug discovery, disease modeling, the construction of extra-corporeal devices and possibly cell based transplantation therapies.

This article will focus on the generation of human hepatic endoderm from human embryonic stem cell populations.

肝细胞样细胞从人类胚胎干细胞群的鲁棒代

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical ...

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 ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

鉴定蛋白质相互作用的合作伙伴使用串联亲和纯化

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

高通量的亲和标签的重组蛋白的纯化

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

成绩单和代谢产物的分离纯化<em&gt;果蝇</em&gt;头

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...