作者衷心感谢加拿大卫生研究所（CIHR）和加拿大癌症协会的资金在他们的实验室中的细胞周期研究学院。 MJC是一架MD /博士研究生奖学金从CIHR收件人。 MJC和MA CaRTT培训计划的成员。

1。标签和固定细胞<ol><li>加入1μL细胞增殖标记试剂（BrdU）细胞培养液中每毫升（1到1000稀释）收获前1小时。标签期间可能需要加长增长较慢细胞。 </li><li>收获细胞，吸出培养基，用磷酸盐彻底清洗缓冲液（PBS）。重复，以彻底清除介质的痕迹。 </li><li>快速洗净文化与用含3mm的EDTA的第三次和彻底吸。 </li><li>含3MM EDTA每道菜添加的PBS体积小，是理想的0.5毫升为6 cm培养皿。在22℃，约5分钟，以分离细胞培养。转移到15 mL锥形管。 </li><li>离心5分钟，以颗粒在500 XG细胞，去除上清，重悬在100μL的PBS彻底。 </li><li>加入5 mL 95％乙醇，滴加中，而震荡，修复细胞。在这一步，细胞可以储存至少在4 ° C一个月。 </li></ol> 2。变性和染色的BrdU和DNA <ol><li>离心5分钟沉淀细胞在500 XG的细胞和删除95％乙醇。在1毫升2N盐酸和0.5％TX - 100悬浮在下降明智的方式加入，而震荡。在室温下孵育30分钟。 </li><li>在2.1节之前离心，小心吸上清因为在这一步非常松散的颗粒细胞形成。轻轻悬浮在1毫升0.1M NAB 4 O 7的 （pH值8.5），并在室温下至少30分钟孵育。 </li><li>上述第2.1节和0.5毫升的抗体溶液与鼠抗BrdU抗体（用含1％BSA和0.2％Tween - 20的）悬浮颗粒细胞稀释1到50。在室温下孵育30分钟。 </li><li>沉淀细胞再悬浮在50毫升含兔抗鼠FITC标记中学抗体的抗体解决方案稀释1至25。孵育30分钟台式和避光。 </li><li>最后的时间和离心细胞悬浮于0.5 mL的碘化丙啶和RNase溶液（PI -核糖核酸酶的解决方案，PBS，用1％BSA，10 mg / mL的碘化丙啶，0.25 mg / mL的RNA酶A）和在黑暗中在37 ° C 30分钟。或者可以被消化的RNA过夜4 ° C黑暗环境中。 </li><li>穿过一个细胞过滤器的解决方案，以消除总量和收集单细胞在流式细胞仪对样品吸收适当管。同样，样品应得到保护，免受光线直接照射。 </li></ol> 3。通过流式细胞仪分析<ol><li>细胞应该由一个标准的流式细胞仪，对双峰（两个单元作为一个流动池，通过传递）歧视与碘化丙啶和FITC相应的检测能力进行分析。基于前向散射和侧向散射弹出的事件，在此流式细胞仪应用程序可以检测单个细胞通过门控ulations，并用碘化丙啶DNA染色性能。流式细胞仪和依靠碘化丙啶染色性能之间的一个双重歧视点图的外观有所不同，请参阅咨询您当地的流式细胞仪检测设施，设立在您的分析鉴别双峰。异步细胞增殖的人口，在一般情况下，单细胞通常是两个最丰富的山峰（2N和4N DNA）的双重歧视情节和门应该围绕他们制订。这将选择单细胞用于以下的事件，所有后续的分析。 </li><li>第一个样本进行分析，应该是一个异步增殖的文化，作为阳性对照染色及流式细胞仪设置。碘化丙啶染色等，从单线细胞2N和4N峰在200和400（任意单位）围绕X轴调整光电倍增管的灵敏度。我们常染色丰富的小号upply这些细胞，以确保所有参数流式细胞仪已适当调整，而无需使用此范例。这种积极的控制，也有利于新用户与流式细胞仪的操作变得更加熟悉。 </li><li>调整上述背景下，G1和G2 / M人口的FITC标记的检测灵敏度的光电倍增管。 BrdU阳性细胞应约10倍亮，和Y轴应为对数刻度显示。它有时是非常有用，包括明确区分阳性染色细胞的BrdU染色为阴性对照（非特异性IgG在2.3以上步取代抗BrdU抗体）。 </li><li>调整之间的碘化丙啶和FITC补偿，流式细胞仪的形式从G1在较低的一个马蹄状弧捕获单一事件留给了S期，并分解成右下方为G2 / M请看到下面有一个更深入DIS的参考cussion的流式细胞仪和参考的原则和使用有2具体应用。 </li></ol> 4。数据分析<ol><li> 5 000 10 000与DNA含量的理想范围内的单细胞事件应收集每个样品，以有信心，已经彻底的抽样人口中的细胞培养。 </li><li>一旦细胞已被测量碘化丙啶和BrdU的内容，他们需要被分配到的G1，S或G2 / M期。盖茨周围绘制在200和400（分别为G1和G2 / M期）为中心的两个BrdU阴性人群。以上这些框的一切都应该包括在一个单一的措施S期（图1A）的门。每个门中的细胞百分比表示的相对数量的细胞在G1，S和G2 / M期（图1B）。 </li></ol> 5。分析细胞周期的细胞混合人口的备用协议<ol><li>这是替代协议最有用的表达载体转染时，经常在利益表达基因产物的细胞比例偏低的结果，或药物治疗时选择一个纯粹的细胞人口是不切实际的。这样的结果在一个人口众多的污染细胞的利益的比例相对较小untransduced细胞。在这种情况下，共同转染质粒，表达质粒表示如挂靠 3绿色荧光蛋白的膜，流式细胞仪，或外国的细胞表面标志如CD19 + 4或用于检测转染细胞标记基因或shRNA的利益CD20的5。 </li><li>然而，对于本协议的目的，我们将使用一个例子作为CD20的染色，CD19染色，本质上是相同的。在与CD20的转染的情况下，有没有必要BrdU标签，简单地收获细胞中描述的步骤1.2至1.5以上和我的细胞加入20μL，FITC标记的抗CD20抗体染色行政长官为20分钟，在黑暗中。修复步骤1.6中所述的细胞。细胞可以再次保存至少一个月，在4 ° C黑暗环境中。 GFP的检测，省略这一步，修复抗体染色和存储所述的细胞。 </li><li>重新水合物的细胞在离心5分钟500 XG造粒，并在5毫升含1％BSA的PBS resuspending。 </li><li>重新沉淀5分钟，在0.5毫升碘化丙啶RNase的解决方案，并在上述第2.5节所述悬浮细胞在500 XG细胞。 </li><li>继续按照2.6和分析，在3.1和3.2的指示细胞制备的说明。 </li><li>调整上述背景下，未染色细胞的FITC标记的检测灵敏度的光电倍增管。理想的情况下，CD20 - FITC阳性细胞将10X 100X更激烈的比背景染色和Y轴的这个情节，应被视为对数刻度显示（图2A）。 CD20阳性细胞，比backg 10X更明亮圆（200和400之间的碘与碘化丙啶染色），应选择在碘化丙啶与细胞计数直方图如图显示。 2C。细胞株的表达，易于染色鲜艳（即10倍到100倍以上的背景）的CD20阳性切断应在10倍的背景设置的标记。列入较弱的染色细胞增加在这些实验中的变化，大概是因为感兴趣的基因也弱在这些细胞中表达。对于细胞株，不仅产量弱染色CD20阳性（即小于10倍的背景），应确定一个切断使用CD20的染色，未转染细胞组成的一个阴性对照。 </li><li>应收集每个样品至少有1000 CD20阳性事件。少，1000事件的实验会产生高变异。 ModFit LT（Verity的软件），多周期AV（凤凰流动系统），流量JO（树星），如软件使用的数学估计G1，S和G2 / M的人群，如在图中所示的直方图曲线的形状。 2B和C </li></ol> 6。代表性的成果： 我们提供了三个实验细胞周期分析，用我们的方法的例子。首次采用在小鼠胚胎成纤维细胞的细胞周期蛋白依赖性激酶抑制剂p27kip1的逆转录病毒表达。二十四小时后，病毒感染的药物选择完成后，细胞用BrdU脉冲标记为一小时。在这个实验中异位表达的抑制剂是用于逮捕细胞（图3A和B）的扩散。如图。 3B小BrdU阳性事件在S期的大门到P27。同样，P27表达细胞在S期细胞的比例是相当低的，因为在图框图。 3C。这种类型的分析已经非常有效，在细胞周期控制细胞来自不同菌株克的缺陷特征烯有针对性的小鼠6，7，8。 在第二个实验中，未转换的乳腺上皮细胞（MCF10A）生长抑制因子治疗，转化生长因子-β（TGF -β1）为24小时。四个小时到收获前立即用BrdU细胞标记。如图。 4 BrdU标签在S期细胞TGF -β1信号大大减少，也验证了我们的染色特异性IgG阴性控制（图4C）。细胞周期的不同阶段的量化证实，转化生长因子-β1主要抑制细胞周期的G1期的扩散，导致在这一阶段的积累。 在我们的最后一个例子，pRB的缺陷SAOS - 2细胞转染与CMV - CD20的表达载体和CMV - RB“或作为控制CMV -β-半乳糖。转染后的三天收获，染色和固定。这些细胞的流量流式细胞仪分析S是图所示。 5。这表明，在72小时pRB的表达与分布（图5B）比较阴性对照转染细胞（图5A）的细胞周期分布。一个细胞周期的比例直接比较多周期软件拟合曲线，如图。 5C。这揭示了细胞在G1以下pRB的表达积累和从S和G2 / M期细胞相对减少。我们已经使用了这个实验的变化探测G1期阻滞的机制广泛9，10，11，12， 13。 这些例子说明如何控制细胞周期，可以在各种刺激或单元操作测量。因此，这种方法可以适应许多应用程序需要的细胞周期的位置在文化类型的实验测量。 <img alt="图1" src="/files/ftp_upload/3491/3491fig1.jpg" /> 图1。Quantit细胞周期ATION结合的碘化丙啶和BrdU染色（PI - BrdU）。 （a）本面板显示三个维流式细胞仪碘化丙啶和BrdU染色细胞。注意：2N和4N DNA含量的细胞，碘化丙啶染色强度X轴的规模为200和400马克中心。 BrdU染色强度是衡量一个Y轴对数刻度。注意用于定量在细胞周期的G1，S和G2 / M期细胞的门的位置。 （二）从A G1，S和G2 / M门中的细胞相对比例，这个图上显示。 <img alt="图2" src="/files/ftp_upload/3491/3491fig2.jpg" /> 图2：在选定的单元格，使用碘化丙啶和细胞表面标志CD20的细胞周期的定量分析。 （一）此面板显示碘化丙啶和CD20染色细胞的混合物，其中一些异位表达CD20和PRB。需要注意的是与细胞2N和4N DNA（最丰富的人口）都集中在X轴的200和400分。 CD20 +门的位置选择染色至少10倍以上，比背景明亮的细胞。 （二）与碘化丙啶染色细胞计数的图形显示CD20的负面面板中的细胞，一个是异步增殖。 （三）一个类似的图表显示CD20阳性细胞从面板一个逮捕与pRB的表达，包含主要2N DNA含量的细胞已被诱导。这表明，被逮捕的亚群可以从使用​​这种染色技术在这种文化中的其他细胞区分开来。 <img alt="图3" src="/files/ftp_upload/3491/3491fig3.jpg" /> 图3。抑制细胞增殖的p27Kip1的。 （一）PI - BrdU分析是用来衡量一个空pBABE逆转录病毒vecto转人口异步增殖细胞的细胞周期阶段河（B）类似的细胞分析，转pBABE - P27。注意细胞在S期和更大力度在G1和G2 / M门事件的情况下。 （三）定量细胞异步增殖控制和P27表达细胞在细胞周期的相关阶段。 <img alt="图4" src="/files/ftp_upload/3491/3491fig4.jpg" /> 图4：由TGF -β1抑制细胞增殖（一）PI - BrdU异步增殖MCF10A细胞的分析。 （b）分析细胞24小时与100时的TGF -β1处理。注意主要是在细胞周期的G1期细胞积累。 （三）审定的BrdU取代非特异性IgG控制在异步增殖细胞的抗BrdU抗体染色。 （四）从各自的细胞周期的图形定量A和B。 <img alt="图5" src="/files/ftp_upload/3491/3491fig5.jpg" /><br/&gt; 图5。pRB的细胞增殖抑制。 （一）与CD20的碘化丙啶染色和β-半乳糖苷酶转染细胞的细胞计数显示。这是一个重要的控制，作为转染可以部分同步细胞，呈现未转染的人口（CD20 - 细胞）作为转染子人口的不适当的控制。需要与其他类似于转染细胞相比，转染细胞。 （二）转染细胞的细胞计数与CD20和PRB的碘化丙啶图。请注意一个2N峰几乎完全存在的。 （三）图形表示细胞周期的比例，确定从A和乙使用曲线拟合方法在多周期软件。

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+regions+of+the+pRB+pocket+domain+affect+its+inactivation+by+human+papillomavirus+E7+proteins.">Three regions of the pRB pocket domain affect its inactivation by human papillomavirus E7 proteins.</a> J. Virol. 76, 6224-6224 (2002).</li></ol>

作者什么都没有透露。

根据我们的经验，这些技术的成功取决于几个关键控制和实验条件。其一是建立在这些实验中使用的异步增殖控制。这种控制有三个重要的目的。首先，它确保所有实验样品的培养条件足以支持在没有治疗的情况下不断扩散。这个范例，也是为阳性对照染色的方法，以确保BrdU或CD20阳性细胞时，可以检测到它们存在的目的。最后，这个范例是用于校准流式细胞仪。此对照样品可用于调整碘化丙啶染色强度检测2N和4N的细胞分别在200和400。此外，检测灵敏度可调节的BrdU或CD20的消极和积极的信号，使中心在图1A和2A所示。当所有样品有类似的细胞浓度PI - R激酶解决方案，则需要一些调整的流式细胞仪，随后样品运行。图的原因，这一点很重要。 4B和5B强烈的G1积累创建本质上是一个单一的G1峰可能被曲解为G2 / M 有代表性的实验，也使其他重要事项。首先，他们证明BrdU吸收和标签会有所不同类型的细胞。基于这个原因，重要的是要凭经验确定的脉搏和需要充分检测S期细胞的染色条件的长度。在一般情况下，，在文化双在24小时或更少的细胞类型可以在一个小时内用BrdU标记。增长较慢的细胞类型，可能需要较长的脉冲抗体染色的条件和期限，并改建。 MCF10A细胞在这方面一个很好的例子，因为他们用BrdU染色标记的四个小时，两次只要四倍的标准浓度的抗体。调查应小心不超过6个小时的BrdU的标签，即使非常缓慢生长的细胞，因为这可能会错误地导致G2 / M期细胞在S期的人口纳入。其次，在比较p27kip1蛋白表达的TGF -β1的影响，它是明确的，P27能诱导G1或G2 / M期的积累，而TGF -β1信号诱导G1期阻滞。如3 H - TdR掺入和闪烁计数检测增殖的传统手段是无法区分这些可能性。 在我们的备用协议，我们将演示在一个较大的未转染人口的细胞亚群检测。这是很重要的经验确定最合适的检测转染细胞记者。在我们的经验，一些类型的细胞无论是明示的细胞表面标志物不佳，或不能正确交通质膜，导致无法检测转染细胞。大号ikewise，并非所有的细胞容忍的绿色荧光蛋白的膜结合形式。选择最合适的记者应通过评估转染效率的记者以及表达的相对强度比未转染控制。理想的情况下，这些分子的异源表达会被很好的耐受性，这是暗示性的标志几乎没有细胞周期分布的影响。 这些类型的流式细胞仪检测方法的一个限制是，他们只能够建立到另一个细胞周期的相对丰度相比。出于这个原因，它是模糊的，如果图3 p27表达真正导致在G1和G2 / M，逮捕或如果它只是减缓相对S期通过这些阶段的进展。这些可能性可以进行进一步的调查，由一个平行样细胞治疗，如噻氨酯哒唑的有丝分裂抑制剂，或G1 / S期抑制剂如aphidicolin。由于这些药物创建一个多米纳NT逮捕M期或S早期阶段，分别缓慢增殖细胞会聚集在药物诱导逮捕点。对于被捕是因为在G1 pRB的表达，例如细胞将保持在G1尽管噻氨酯哒唑治疗，而对照组细胞会积聚在M 期 13 。 两者合计，这些实验方法提供了一个灵活的方法，可应用于广泛的哺乳动物细胞周期的研究问题。他们可以很容易地发现在细胞周期进程的改建和量化与对照相比，差异。

<table border="1"><tbody><tr><th>试剂/设备名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td>细胞增殖标记试剂</td><td>通用电气公司Amersham公司</td><td> RPN201 </td><td></td></tr><tr><td>抗BrdU抗体</td><td> BD公司</td><td> 347580 </td><td></td></tr><tr><td>抗鼠FITC标记抗体</td><td>媒介实验室</td><td> FI - 2000 </td><td></td></tr><tr><td>细胞株过滤器</td><td>屋宇署猎鹰</td><td> 352235 </td><td></td></tr><tr><td>抗CD20抗体</td><td> BD公司</td><td> 347673 </td><td></td></tr><tr><td>多周期软件</td><td>凤凰流系统</td><td> N / A </td><td></td></tr></tbody></table>

analysis of cell cycle position in mammalian cells

调节细胞增殖，是中央组织的形态发生过程中的多细胞生物体的发展。此外，细胞增殖失控基础的癌症等疾病的病理。因此有很大的需要，可以研究细胞的增殖和定量细胞的细胞周期的每个阶段中的比例。也不加区别地识别细胞，他们的DNA复制在一个较大的人口，这是至关重要的。由于细胞的增殖的决定是在G1期前立即启动DNA的合成，并通过对细胞周期的其余部分进展，检测DNA合成在这个阶段允许在细胞培养中生长调节状态明确的决心实验。 可以很容易地在细胞DNA含量定量流式细胞仪碘化丙啶，荧光DNA插层染料染色的细胞。同样，活跃的DNA合成，可以定量培养细胞中存在放射性胸腺嘧啶，收获细胞，并测量放射性掺入到酸不溶性部分。我们有相当的专业知识与细胞周期分析，并推荐了不同的方法。我们使用溴脱氧尿苷/ fluorodeoxyuridine（简单缩写为BrdU）染色检测到最近合成的DNA的胸腺嘧啶类似物纳入研究细胞增殖。用BrdU标记和染色细胞相结合，与总DNA染色碘化丙啶和分析，流式细胞仪1细胞在细胞周期的各个阶段提供最准确的测量。这是我们的首选方法，因为它结合了活跃的DNA合成的检测，通过基于抗体的BrdU染色碘化丙啶的总DNA含量。这可以明确分离的细胞在G1，S早期阶段或后期小号从G2 / M期此外，这种方法可以利用调查的进展，通过这些不同的细胞周期调控的许多不同的细胞刺激和药物制剂的影响。 在这份报告中，我们描述标签和染色培养细胞，以及他们用流式细胞仪分析的方法。这种方法可用于测量生长抑制信号从细胞因子，如TGF -β1和增殖抑制剂，如细胞周期蛋白依赖性激酶抑制剂p27kip1的影响，我们还包括实验例子。我们还包括一个备用协议，允许细胞在一个较大的文化 5的一个亚群分析细胞周期的位置。在这种情况下，我们将演示如何检测即使未转染细胞大大多于在相同的文化与视网膜母细胞瘤基因转染细胞，一个细胞周期阻滞。这些例子说明，DNA染色的方法很多，可以利用流式细胞仪和适应研究哺乳动物细胞周期调控的根本问题。

Watch this Scientific Journal Video about Analysis of Cell Cycle Position in Mammalian Cells at JoVE.com

Analysis of Cell Cycle Position in Mammalian Cells

确定的人口细胞的细胞周期的位置，或理解信号是如何影响扩散，可以很容易地通过使用此协议的流流式细胞仪测量。我们报告一个简单的实验方法，染色细胞和量化其在细胞周期中的位置。

在哺乳动物细胞中细胞周期位置分析

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

analysis-of-cell-cycle-position-in-mammalian-cells

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Unit 1

Cell Proliferation

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59.7K 次观看.  University of Western Ontario. 确定的人口细胞的细胞周期的位置，或理解信号是如何影响扩散，可以很容易地通过使用此协议的流流式细胞仪测量。我们报告一个简单的实验方法，染色细胞和量化其在细胞周期中的位置。

视频: Analysis of Cell Cycle Position in Mammalian Cells

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

长期使用宽视场显微镜的成像哺乳动物细胞

科研

生物学

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-3H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2. In contrast, unstable ERAD substrates are trimmed to Man6-5GlcNAc2 and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.

We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC.

N -连接糖链的糖蛋白在哺乳动物细胞中的脉冲大通分析

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells.

在哺乳动物细胞中的DNA双链断裂（DSB）的维修分析

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

在哺乳动物细胞的RNAi筛选使命esiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

异源表达在哺乳动物细胞离子通道的突变和功能分析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

在哺乳动物细胞通过显微注射mRNA的核出口动力学分析

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Expression Analysis of Mammalian Linker-histone Subtypes

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure. H1 is essential for mammalian development1 and regulates specific gene expression in vivo2-4. Among the highly conserved histone proteins, the family of H1 linker histones is the most heterogeneous group. There are 11 H1 subtypes in mammals that are differentially regulated during development and in different cell types. These H1 subtypes include 5 somatic H1s (H1a-e), the replacement H10, 4 germ cell specific H1 subtypes, and H1x5. The presence of multiple H1 subtypes that differ in DNA binding affinity and chromatin compaction ability6-9 provides an additional level of modulation of chromatin function. Thus, quantitative expression analysis of individual H1 subtypes, both of mRNA and proteins, is necessary for better understanding of the regulation of higher order chromatin structure and function.
Here we describe a set of assays designed for analyzing the expression levels of individual H1 subtypes (Figure 1). mRNA expression of various H1 variant genes is measured by a set of highly sensitive and quantitative reverse transcription-PCR (qRT-PCR) assays, which are faster, more accurate and require much less samples compared with the alternative approach of Northern blot analysis. Unlike most other cellular mRNA messages, mRNAs for most histone genes, including the majority of H1 genes, lack a long polyA tail, but contain a stem-loop structure at the 3' untranslated region (UTR)10. Therefore, cDNAs are prepared from total RNA by reverse transcription using random primers instead of oligo-dT primers. Realtime PCR assays with primers specific to each H1 subtypes (Table 1) are performed to obtain highly quantitative measurement of mRNA levels of individual H1 subtypes. Expression of housekeeping genes are analyzed as controls for normalization. 
The relative abundance of proteins of each H1 subtype and core histones is obtained through reverse phase high-performance liquid chromatography (RP-HPLC) analysis of total histones extracted from mammalian cells11-13. The HPLC method and elution conditions described here give optimum separations of mouse H1 subtypes. By quantifying the HPLC profile, we calculate the relative proportion of individual H1 subtypes within H1 family, as well as determine the H1 to nucleosome ratio in the cells.

We describe a set of assays to analyze expression levels of H1 linker histones. mRNA of individual H1 genes are quantitatively measured by random primer based reverse transcription followed by real-time PCR, whereas protein quantification of H1 histones is achieved by HPLC analysis.

哺乳动物链接组蛋白亚型表达分析

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure.  H1 is essential ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

在哺乳动物细胞中的内质网定位的mRNA的可视化

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

中血红素的合成水平在哺乳动物细胞中测量

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

成像本地CA<sup&gt; 2+</sup&gt;在培养的哺乳动物细胞信号

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...