E. M. Winkelbauer的伤害，易卜拉欣P.瓦伦西亚，K.杜甫，郑H.有益的讨论，我们对此表示感谢。 HeLa细胞中获得了来自全国的细胞培养中心（明尼阿波利斯，明尼苏达州）。我们也感谢E7107和哈佛医学院的尼康影像中心提供帮助与光镜卫材有限公司。这项工作是由美国国立卫生研究院授予GM043375支持RR和EGF和NRSA金的JLH。

1。核提取HeLa细胞成长<ol><li> 3 150 mm中厚板板的HeLa细胞DMEM培养基辅以10％胎牛血清和1％青霉素/链霉素。增长5％的CO 2在37℃孵化，直到细胞90％汇合。 </li></ol>提示细胞分裂从合流板和收获后第3天分裂1:10。 提示额外的板可以种植的情况下，有没有足够的细胞（例如，如果细胞小于90％的合流）三大板块。 2。准备和冷藏核提取物解<ol><li>准备好新鲜的解决方案，如表2所列。 </li><li>分装10毫升到15毫升猎鹰管低渗缓冲。等分高盐缓冲和低盐缓冲液1毫升1毫升到1.5毫升的Eppendorf管。冷藏冰的缓冲区。每个缓冲区加入PMSF和DTT的相应卷前使用（见<strong&gt;表1）。 </li></ol> 3。收获HeLa细胞的核提取<ol><li>从细胞吸媒体和室温1X PBS每盘13毫升的洗细胞一次。 </li><li>吸出PBS洗涤。 </li></ol>第一吸气式提示，吸尽可能多的PBS，站在其侧面板，然后等待几秒钟，吸气其余。 <ol start="3"><li>使用手机起苗，刮去电池板的底边，然后将其转移到Eppendorf管中的细胞。避免气泡。 3板刮下细胞放入一个1.5毫升的Eppendorf管。 </li></ol>提示这是比较容易估计细胞的数量，如果用于Eppendorf管。为规模，使用猎鹰管。 <ol start="4"><li>在4°C离心5分钟100 XG颗粒细胞离心。 </li></ol> 4。膨胀HeLa细胞在低渗缓冲</p&gt; <ol><li>小心吸PBS和细胞沉淀在管上的层次看，红细胞压积（PCV）的估计。 HeLa细胞融合板产量约500μL的PCV。 </li><li>加入2倍的圆环低渗缓冲细胞沉淀。摇轻轻地悬浮细胞。使用P1000的Pipetman轻轻悬浮细胞吹打起来，如果有必要的。 </li><li>在4°C离心5分钟100 XG颗粒细胞离心。 *细胞就会开始膨胀，这个时候，细胞体积约750微升1000微升现在应该。 </li><li>小心吸低渗缓冲到最终体积为1.5毫升，并添加新的低渗缓冲。悬浮细胞。 </li><li>再悬浮冰肿细胞孵育10分钟。 </li></ol> 5。裂解细胞，用Dounce均质<ol><li>使用低渗缓冲，冲洗dounce和然后冰上冷却。请用杵干微妙任务制品的呃如Kimwipe。从使用1000μl的扩展微量尖dounce删除剩余的缓冲区。 </li><li>转移到dounce肿细胞。紧杵慢慢移动的dounce的5倍，裂解细胞，小心避免气泡。 </li><li>使用5μL等份从的dounce细胞的细胞裂解法。放置在一个Eppendorf管中的等份，并加入等量的台盼蓝。放在幻灯片的混合物，并用光学显微镜检查细胞。溶解细胞的细胞核会出现蓝色。约90％的溶解是理想的。 </li></ol>提示细胞应dounced倍的数量会有所不同，取决于，dounce的密封性。首次开展此协议时，确定的次数以dounce后每个的dounce的中风进行步骤5.3。不要过dounce，过度douncing会破坏细胞核。 <ol start="4"><li>溶解细胞转移前，chilleðEppendorf管中，并在4℃离心5分钟1500×Ğ自旋。颗粒包含的原子核。小心将上清转移到一个新的管而不破坏细胞核。上清液中含有细胞质。 </li></ol>提示细胞质可以用作是进一步处理到9000使用相同的高速旋转，用于批量提取（见活跃S100的批量提取准备从1）。 6。盐提取细胞核<ol><li>由在管上的层次看，估计可携带核卷（PNV）。通常产生一个圆环的500μL400μLPNV。 </li><li>添加半低盐缓冲X PNV细胞核插入微管尖管底部，缓慢而轻轻搅拌核喷射进缓冲区。不要吸管上升和下降。轻轻一抖管，以确保颗粒完全悬浮在低盐缓冲，然后再继续。 </li> &lt;李&gt;添加8.5×高盐缓冲液，并迅速搅拌1次由反相管PNV。不要动摇。在4°C旋转30分钟。 </li></ol>提示当他们在高盐缓冲液，以避免溶解它们与原子核的温柔。 <ol start="4"><li>在4°C，18,000 xĞ15分钟离心旋转。上清高盐核提取物（HS-NE）的。三板的细胞，通常会产生500-600μLHS-NE的。 </li></ol> 7。集中和透析的核提取物<ol><li>转移到500μL的HS-NE冰鲜小centricons的（Amicon）和自旋为50分钟在4°C，在离心14000 xĞ。反转迷你centricon的（Amicon），并将其放置在一个新的Eppendorf管。在4°C 2分钟旋转1000 XG恢复的HS-NE。该HS-NE将集中约115μL。 </li><li>使用P200的Pipetman，转移到微型透析幻灯片-A Lyzers的HS-NE的45μL等份，他们将在500毫升冰鲜透析液。确保一个幻灯片仪的底部是浮动的底部对齐。搅拌1-2小时，在4°C东北透析后会出现混浊。 </li></ol>提示不要离心除去混浊沉淀，透析后观察，离心，因为这降低了提取物的活性。 <ol start="3"><li> NE可以立即使用或分装。等份应该闪光冻结在液氮中，并储存在-80°C。 NE可以存储和接受至少两个冻融而不失去活性。 </li></ol> 8。代表结果最近， 在体外耦合RNA聚合酶Ⅱ转录拼接系统的高效开发2-5。这些系统采用大量种植HeLa细胞，因此不适合快速测试多个样品的特异性细胞治疗的效果。在此基础上需要ND一般一个小规模的提取协议的实用程序（见讨论），我们建立了一个强大的小规模的核提取方法。散装核提取物在小规模的核提取物的RNA聚合酶II转录/剪接反应比较有代表性的数据如图2所示。一个CMV-DNA的结构，其中包含CMV启动子和编码标准剪接基板（保 ​​税区3， 图2A），用于分析。当这种结构在批量（1-3车道）或小规模（4-6车道）提取物孵育5分钟的时间点（ 图2，1和4车道，新生的mRNA前体的合成类似的水平）。除了​​α-鹅膏蕈碱，以阻止进一步的转录，剪接中间体和拼接产品随着时间的推移累计提取这两种类型（ 图2，泳道2，3，5，6）类似的动力学。这些具有代表性的结果表明，政变效率<html>为首的RNA聚合酶II转录/拼接系统是类似的散装和小规模的核提取物。 为了证明实用的小规模的提取方法，提取准备从拼接抑制剂，E7107，或F 6,7，然后耦合RNA聚合酶II转录/拼接法进行的负控制复合pladienolide的的治疗HeLa细胞。 图3所示，由RNA聚合酶Ⅱ转录发生无论从Pladienolide F和E7107细胞治疗（10分钟时间点）准备的提取物，有效。相比之下，剪接发生通常从Pladienolide的F-处理的细胞准备的提取物，但被取消E7107处理的细胞（ 图3，20-60分钟的时间点）。这些数据提供了证明，使用小规模的核提取物对细胞的特殊待遇，在小规模的概念。 JPG“/&gt; 图1。小规模提取议定书“的原理。步骤P1的。细胞生长单层，收获细胞升降板从使用和低渗缓冲此外肿。加强P2的。 Dounce匀浆用细胞被溶解，离心沉淀细胞核。加强P3的。原子核从细胞质中分离出来，并进行盐提取。加强P4的。核提取物浓缩和透析。加强小五。得到的结果，表明核提取物功能（更详细见图2）。 <img alt="图2" src="/files/ftp_upload/4140/4140fig2.jpg" /> 图2。小规模的核提取物有强劲的耦合RNA聚合酶Ⅱ转录/拼接法。用于耦合RNA聚合酶II转录/剪接的CMV-保税区DNA模板的示意图。 CMV启动子和外显子和内含子的大小表示。 B.耦合RNA聚合酶的比较二转录/剪接的检测，无论是散装的核提取物或小规模的核提取物。 α-鹅膏蕈碱增加了5分钟的转录和剪接后允许出现30和60分钟。 RNA的提取和分馏5％的变性聚丙烯酰胺凝胶检测和phosphoimager。显示拼接和中间体产品。 U6 snRNA中的内源性和tRNA中存在的提取物和32 P标记孵化期间表示。见上耦合RNA聚合酶II转录/拼接系统的详细协议。 <img alt="图3" src="/files/ftp_upload/4140/4140fig3.jpg" /> 图3。 E7107拼接抑制剂药物治疗HeLa细胞准备的小规模的核提取物在剪接缺陷。细胞治疗3微米Pladienolide F或E7107作为描述6，然后用准备小规模的核提取物。 图2使用相同的转录/拼接法进行了一个时间过程。拼接和中间体产品，和U6 snRNA和tRNA的表示。

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Abcam，PLC生产和自由地进入本文主办。

我们已经建立了一个快速和可重复性的方法编制HeLa细胞生长为单层少量的核提取物。我们表明，这些提取物显示，RNA聚合酶II转录/拼接实验的动力学和效率是在小规模和批量的核提取物类似的健壮。我们发现通过展示积极的转录，但在剪接缺陷，从与拼接抑制剂药物治疗的细胞准备提取物提取物的效用。 我们准备小规模的核提取物的方法，建立了结合和优化以前建立的方法从HeLa细胞提取物中悬浮生长在大尺度1,8和方法，为小规模提取物的制备9。我们建立了我们的协议，因为以前的小规模提取物，如一些分析功能耦合的转录/ SP，取得了较好检测。以前的小规模协议和我们之间的一个重要区别是，我们优化裂解细胞，使用一个小型的dounce的条件，而以前的协议裂解细胞，推他们通过一个小针头9。在泡沫的针裂解结果，这也许可以解释为什么提取物无效和/或难以重现一些分析。我们还增加了我们的协议浓度一步。这一步提高提取物的活动，这是极其敏感的浓度，也限制了提取物，是固有的小规模的筹备工作之间的变异。最后，我们准备定期开展与单层细胞只有三个150毫米板，批量提取物相比，没有任何活动的重要作用。因此，这个过程很容易适合小规模的准备，需要昂贵的试剂或细胞的可用性有限。例如，我们已经准备SMALL-RNAi技术敲除细胞和慢性淋巴细胞白血病（CLL）患者细胞中提取，用于这些提取物的功能和/或生化检测（EGF，JLH，TY和RR，未发表）。我们发现，提取有价值的RNAi由特定的生化检测，如immunopreciptations，西部片，银染，其次。建立一种特定的蛋白质击倒的疗效后，可以进行更详细的研究，通过稳定击倒细胞系，它可以用来准备额外的提取物，以较低的成本。质量法的蛋白质存在于这些击倒细胞株获得免疫，也将是一个有用的应用程序的方法。在小规模提取物除了再加转录/拼接法，我们为我们这里的协议描述的例子中使用，应该是普遍适用于众多的功能和生化检测，如那些用于不同的STEPS在基因表达（如封盖，剪接，转录，聚腺苷酸化，microRNA的加工）。该协议也可以适应病人的细胞类型可以得到悬浮（如白血病细胞）或（如病人的成纤维细胞）培养。最后，在手术过程中获得的细胞质部分需要细胞质的功能和生化检测证明非常有用。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 姓名试剂 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>肝素钠</td><td> 4 - （2 - 羟乙基）哌嗪-1 - 甲磺酸</td><td>西格玛</td><td> H3375-500G </td></tr><tr><td>氯化镁2·6H 2 O </td><td>镁六水氯化</td><td>西格玛</td><td> M2670-500G </td></tr><tr><td>氯化钾</td><td>氯化钾</td><td>费舍尔</td><td> P335-212 </td></tr><tr><td> PMSF </td><td> phenylmethanesulfonyl氟</td><td>西格玛</td><td> P7626-100G </td></tr><tr><td>数码地面电视</td><td>二硫苏糖醇</td><td>美国生物分析</td><td> AB00490-5G </td></tr><tr><td> EDTA的</td><td>乙二胺四乙酸二钠，二水</td><td>美国生物分析</td><td> AB00500-01000 </td></tr><tr><td>甘油</td><td></td><td> MP的Biomedicals </td><td> 800689 </td></tr><tr><td> DMEM培养液</td><td> Dulbecco的极小本质中</td><td> Invitrogen公司</td><td> 11995-073 </td></tr><tr><td>胎牛血清</td><td>胎牛血清</td><td> Gibco公司</td><td> 16140 </td></tr><tr><td>青霉素/ </td><td></td><td> Gibco公司</td><td> 15070 </td></tr><tr><td> PBS </td><td>磷酸盐缓冲液</td><td> cellgro </td><td> 21-040-CV </td></tr><tr><td>台盼蓝染色0.4％ </td><td></td><td> Gibco公司</td><td> 15250 </td></tr><tr><td>细胞举重运动员</td><td></td><td>康宁</td><td> 29442-200 </td></tr><tr><td> 150毫米板</td><td></td><td>厂商VWR </td><td> 353025 </td></tr><tr><td>扩展微管提示</td><td></td><td>丹维尔</td><td> P1126 </td></tr><tr><td> Dounce与紧杵均质，1毫升， </td><td></td><td>惠顿</td><td> 357538 </td></tr><tr><td> slidealyzers，截留分子量10kDa </td><td></td><td> ThermoScientific </td><td> 69572 </td></tr><tr><td>迷你Centricons，截留分子量3kDa </td><td></td><td> Millipore公司（Amicon） </td><td> UFC500396 </td></tr></tbody></table> 表1。特定的试剂和设备。 <table border="1"><tbody><tr><td> 解 </td><td> 最后 </td><td> 股票 </td><td> 豁免 </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>低渗</td><td> 10毫米肝素钠，pH值7.9 </td><td> 1M肝素钠，pH值7.9 </td><td> 5毫升</td></tr><tr><td></td> &lt;TD&gt; 1.5毫米MgCl 2的 </td><td> 1M氯化镁2 </td><td> 750μL </td></tr><tr><td></td><td> 10毫米氯化钾</td><td> 3M氯化钾</td><td> 1.67毫升</td></tr><tr><td></td><td> 0.2 mM的PMSF </td><td> 200 mM的PMSF </td><td> 500μL </td></tr><tr><td></td><td> 0.5毫米的DTT </td><td> 2M的DTT </td><td> 125μL </td></tr><tr><td></td><td></td><td></td><td> 带来高达500毫升水 </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>低盐</td><td> 20毫米肝素钠，pH值7.9 </td><td> 1M肝素钠，pH值7.9 </td><td> 2毫升</td></tr><tr><td></td><td> MgCl 2的 1.5毫米</td><td> 1M氯化镁2 </td><td> 150μL </td></tr><tr><td></td><td> 20毫米氯化钾</td><td> 3M氯化钾</td><td> 667μL </td></tr><tr><td> ＆NBSP; </td><td> 0.2 mM的EDTA </td><td> 0.5M EDTA的</td><td> 40μL </td></tr><tr><td></td><td> 25％甘油</td><td>甘油</td><td> 25毫升</td></tr><tr><td></td><td> 0.2 mM的PMSF </td><td> 200 mM的PMSF </td><td> 100μL </td></tr><tr><td></td><td> 0.5毫米的DTT </td><td> 2M的DTT </td><td> 25μL </td></tr><tr><td></td><td></td><td></td><td> 带来高达100毫升水 </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>高盐</td><td> 20毫米肝素钠，pH值7.9 </td><td> 1M肝素钠，pH值7.9 </td><td> 2毫升</td></tr><tr><td></td><td> MgCl 2的 1.5毫米</td><td> 1M氯化镁2 </td><td> 150μL </td></tr><tr><td></td><td> 1.4M氯化钾</td><td> 3M氯化钾</td><td> 48毫升</td></tr><tr><td></td><td> 0.2 mM的EDTA </td><td> 0.5M EDTA的</td><td> 40μL </td></tr><tr><td></td><td> 25％甘油</td><td>甘油</td><td> 25毫升</td></tr><tr><td></td><td> 0.2 mM的PMSF </td><td> 200 mM的PMSF </td><td> 100μL </td></tr><tr><td></td><td> 0.5毫米的DTT </td><td> 2M的DTT </td><td> 25μL </td></tr><tr><td></td><td></td><td></td><td> 带来高达100毫升水 </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>透析</td><td> 20毫米肝素钠，pH值7.9 </td><td> 1M肝素钠，pH值7.9 </td><td> 10毫升</td></tr><tr><td></td><td> 100毫米氯化钾</td><td> 3M氯化钾</td><td> 16.625毫升</td></tr><tr><td></td><td> 0.2 mM的EDTA </td><td> 0.5M EDTA的</td><td> 200μL </td></tr><tr><td></td><td> 20％甘油</td><td>甘油</td><td>百毫升</td></tr><tr><td></td><td> 0.2 mM的PMSF </td><td> 200 mM的PMSF </td><td> 500μL </td></tr><tr><td></td><td> 0.5毫米的DTT </td><td> 2M的DTT </td><td> 125μL </td></tr><tr><td></td><td></td><td></td><td> 带来高达500毫升水 </td></tr></tbody></table> 表2。核提取物制备的解决方案。

small-scale nuclear extracts for functional assays of gene-expression machineries

在体外系统中使用已取得了很大的理解基因表达的进展。对于大多数的研究，功能分析进行了使用散装准备从10-50公升或悬浮细胞生长的提取物。然而，这些大规模的准备工作是不适合快速测试在体外作用，从各种细胞在体内的治疗方法或条件的结果。这日记视频文章显示准备功能的小规模的核提取物的方法，使用HeLa细胞作为一个例子。这种方法进行了用尽可能少3 150毫米作为附着单层细胞生长板。为了说明小规模提取物的效率，我们表明，他们是为大宗核提取物耦合RNA聚合酶II转录/剪接反应积极。为了证明提取协议的效用，我们表明，拼接准备从HeLa细胞提取物取消小号E7107拼接抑制剂药物治疗。小规模的协议应该是普遍适用于任何过程或细胞类型，可在体外用细胞提取物。这些措施包括病人的细胞，只有数量有限，或接触到众多的代理商，如毒品，DNA损伤剂，RNA干扰，或转，这就需要使用小细胞群的细胞。此外，少量的新鲜细胞生长的方便和/或某些应用程序所需的。

Watch this Scientific Journal Video about Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries at JoVE.com

Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries

一个健壮的，小规模的HeLa细胞的核提取物制备的协议。该协议是为需要使用小种群的细胞，如细胞治疗药物或RNAi，有价值的实验。该方法应该适用于各种各样的基因表达分析和其他类型的细胞，包括病人的细胞。

小规模的核提取物，基因表达机具功能分析

A great deal of progress in understanding gene expression has been made using in vitro systems.  For most studies, functional assays are carried out using ...

small-scale-nuclear-extracts-for-functional-assays-gene-expression

Research

JoVE Journal

Biology

25.8K 次观看.  Harvard Medical School. 一个健壮的，小规模的HeLa细胞的核提取物制备的协议。该协议是为需要使用小种群的细胞，如细胞治疗药物或RNAi，有价值的实验。该方法应该适用于各种各样的基因表达分析和其他类型的细胞，包括病人的细胞。

视频: Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries

Bacterial Gene Expression Analysis Using Microarrays

使用微阵列的细菌基因表达分析

科研

生物学

In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

在使用分馏爪蟾卵提取物的体外核大会

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 2

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 2 of 3.

牛痘病毒感染及病毒基因表达的时空分析：第2部分

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells.  Members of ...

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

脊髓电

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

使用自动细胞计数器简化基因表达研究：在Namalwa细胞的IL - 4依赖的基因表达的siRNA击倒

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits1.
This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants)2,3. Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells.
DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers4. The primer extension assay is carried out using the MassARRAY (Sequenom)5 platform according to the manufacturer's specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results.

Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.

等位基因特异性基因表达检测，测试的遗传协会的功能基础

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

石蜡包埋及冷冻的部分果蝇成人肌肉

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...