这项工作是支持的，部分由美国国家科学基金会（IOS-0920417）（ACL）。

1。构建慢病毒荧光素酶记者哺乳动物的昼夜记者的结构通常包含一个表达盒，其中一昼夜的荧光素酶基因启动子的融合。双方结扎和重组基于策略通常用于DNA克隆。作为一个例子，在这里，我们描述了一个重组的Gateway克隆方法产生一个P（Per2基因 ）-D 吕克慢记者，在不稳定的荧光素酶 （D 吕克 ）的小鼠Per2基因启动子的控制下。 <ol><li> Per2基因启动子的克隆。使用PCR扩增的Per2基因的启动子的526 bp的DNA片段，从小鼠Per2基因的BAC克隆9-13的转录起始位点的上游侧，用正向引物（的5&#39;-CTCGAGCGGATTACCGAGGCTGGTCACG TC-3&#39;）和反向引物（5&#39; CTCGAGTCCCTTGCTCGGCCCGTCAC TTGG-3&#39;），并克隆入pENTR5&#39;-TOPO载体（Invitrogen）中，以产生pENTR5&#39;-P（Per2基因 ）。 </li><li>克隆的D 吕克 。的d 吕克含有萤火虫荧光素酶基因的C-末端的病虫害如先前描述的21序列的快速蛋白质降解。使用PCR扩增的d 吕克的DNA片段，并克隆到pENTR / D-TOPO载体（Invitrogen）的生成pENTR / Dd的吕克 。 </li><li>报告载体的构建。混合使用这两种pENTR的质粒，pENTR5&#39;-P（Per2基因 ）和pENTR / Dd的吕克，与慢病毒的目标向量pLV7-BSD的（BSD的 ，杀稻瘟菌素抗性基因），并执行使用Clonase生成pLV7-BSD的 -P的重组反应（Per2基因 ）的-d 吕克记者（ 图1）。 pLV7-BSD是一个修改后的版本（在我们的实验室）pLenti6/R4R2/V5-DEST（Invitrogen公司）的土拨鼠肝炎病毒转录后调节元件（WPRE）序列22个直接插入下游的表达CA<html> ssette以增强基因的表达。 </li></ol> 2。慢病毒颗粒的生产 1。种子293T细胞（1天） <ol><li> 90-100％汇合在定期的DMEM补充有10％FBS和1x青霉素 - 链霉素-谷氨酰胺（PSG）的10厘米培养皿上培养人类胚胎肾（HEK）293T细胞。 （快速生长的细胞与低通道数的高效转染的关键。） </li><li>之前，通过添加0.001％聚-L-赖氨酸在PBS中的1毫升到各孔中，并在室温下孵育20分钟，转染，涂层6孔培养板中接种细胞。吸取溶液，并在使用前用1×PBS冲洗一次。 </li><li>游离293T细胞，用胰蛋白酶和种子0.75×10 6个细胞的各孔上的预涂层钢板用2ml的DMEM定期。旋流板彻底的各孔中的细胞，以获得均匀的分布。在孵化器中于37℃培养过夜的细胞成长。 </li></ol>TEP“&gt; 2。瞬时转染，通过投诉警察课4 / DNA沉淀（第2天） <ol><li>观察从第1天的种子细胞。细胞应达到80％至90％的汇合点。 </li><li>准备质粒转染混合物在1.5 ml离心管中，加入2微克记者质粒DNA的慢病毒（ 例如，pLV7-P（Per2基因 ）-D 吕克·刘实验室）和3个包装载体（1.3微克的加格/波尔，0.5微克牧师和0.7微克VSVG Invitrogen公司）。另外，作为对照转染和随后的感染，我们通常包括一个额外的井慢病毒GFP表达载体的转染，pLV156-CMV-EGFP（ 图1A），窝藏增强型绿色荧光蛋白（EGFP）的CMV启动子的控制下如前所述20。 </li><li>加入100μl0.25 M 氯化钙 （稀释后用DNase / RNase-free的DDH 2 O从2.5 M股票）在第2步中的质粒混合调匀。然后加入100μl的2倍BBS溶液（50 mM的BES，280 mM氯化钠，1.5毫米的Na 2 HPO 4，PH值为6.95），轻轻地混合，但彻底。的DNA混合，在室温下孵育15分钟。 </li><li>在等待过程中，从293T细胞吸出培养基，并更改为2毫升新鲜培养基。板返回到至少10分钟，平衡介质的pH值在转染前的孵化器。 </li><li>染293T细胞一滴一滴从第3步组合。轻轻旋转板，并在显微镜下观察颗粒的形成。孵育，5％CO 2，37℃培养过夜。 （投诉警察课4 / DNA沉淀物细粒子形成的高效转染的关键。） </li></ol> 3。收获病毒颗粒（天3-4） <ol><li>关于转染后16小时（3天），细胞应达到100％汇合，从细胞中吸出培养基，，更换2毫升新鲜定期DMEM。孵育，37℃培养过夜。 </li><li>第4天，通过观察EGFP表达的转录评估转染效率sfection对照组（转染效率90-100％高EGFP表达的是一个病毒准备好一个可靠的预测。） </li><li>收集培养基中分泌，感染性病毒颗粒。离心&gt; 2,000 xg离心5分钟以除去残留的293T细胞，并收集含病毒上清液。可替代地，介质也可以用0.45μm的膜过滤器清零。准备用于在感染的病毒颗粒。 </li></ol> 3。感染的3T3细胞 1。种子3T3细胞（第3天） 第二天分割和种子适当的数字（12000）的3T3细胞在12孔板中获得20％至30％汇合。孵育，37℃培养过夜。 2。感染3T3细胞（第4天） <ol><li>观察种子细胞。合流的20-30％（小于50％）所需的感染。 </li><li>加入聚凝胺（polybrene）至终浓度为5微克/毫升到所收集的培养基中病毒颗粒。移液拌匀。 </li><li>吸液介质从3T3细胞，上述病毒的混合物，每孔加入1毫升。孵育，37℃培养过夜。 （聚凝胺的使用，以提高感染效率，但不是绝对必需的，正如它可能是有毒的一些细胞，建议事先测试）。 </li></ol> 3。选择受感染的细胞（第5天及以后） <ol><li>二十四小时，感染后，吸出含有从受感染的细胞的病毒和聚凝胺（polybrene）的培养基中，用1×PBS洗一次，并改变到新鲜培养基。孵育在37°C过夜复苏和增长。 </li><li>当汇合（一般1-2天），分裂的细胞，在37°C过夜孵育。 </li><li>翌日，吸出培养基，细胞（&lt;50％汇合需要的话）和含10μg/ ml的杀稻瘟菌素选择稳定地用新鲜培养基更换转导的细胞。 （杀稻瘟菌素杀曲线需要凭经验确定为特定的细胞系。） </li><l我切换到新鲜培养基含有稻瘟菌素，每2-3天抗生素耐药细胞表达时钟记者（一般为4-6天）连续选择。 </li></ol> 4。记者细胞的生物发光的记录 1。种子记者细胞传播稻瘟素抗性记者细胞分裂到35毫米的培养皿。在37°C孵育，直至汇合。我们通常准备≥3个菜，每一个记者的细胞在各种条件下昼夜表型。 2。同步和变更记录介质<ol><li>吸出培养基从汇合记者细胞，用PBS洗一次，更换含10μMforskolin的（或200 nm地塞米松）。在37℃下孵育1小时，以同步的细胞。 （或者，细胞可以通过温度循环23或血清休克24同步）。 </li><li>在等待时，作好记录3T3细胞如下：1倍DMEM（HyClone）的含有10％FBS，1×笔/链霉素/谷氨酰胺，1μM福斯克林，1mM的荧光素，25mM的HEPES，pH值7.4的介质。血清和毛喉素浓度可以凭经验确定。对于非常暗淡的细胞，无酚红培养基也可使用。 </li><li>毛喉素治疗结束时，吸出培养基并更换新鲜配制的记录介质。 </li></ol> 3。记者细胞的生物发光记录<ol><li>介质的变化，包括培养皿，用40毫米的无菌盖玻片和密封的地方，真空润滑脂，以防止水分蒸发。 </li><li>加载到的LumiCycle光度计，这是保持在36°C孵化器内无H 2 O或CO 2的菜。 </li><li>启动实时生物发光的记录。我们通常记录的节奏每周1次，其次是介质的变化和第二周的连续记录（见，萨韦利耶夫等人 。详细信息）25。 （96我们的记录会板，协同SL2被用作记录装置，请参阅详细讨论1.1。） </li></ol> 5。数据分析和演示记者细胞促进高清晰度的定量发光记录，确定表型的影响生物钟功能的关键。为了获得昼夜参数包括相位，周期长，节奏振幅，和阻尼率，我们使用的LumiCycle分析的程序（Actimetrics）分析生物发光数据5,14。简言之，原始数据是基线拟合的第一，和基线减去的数据被装配到一个正弦波，从该参数被确定。表明持久性的节奏，善良的拟合&gt;通常达到90％的样品。由于高瞬态生物发光换液时，我们通常不包括数据分析的第一个周期。 有关数据显示，我们通常绘制原始数据（生物发光，计数/秒）对TI我（天）。在必要时，可以绘制基线减去数据用来比较的振幅和相位。 6。代表性的成果 1。特定相位的昼夜记者生物钟是根据上的生化负反馈机制1。核心的反馈环路的转录激活因子BMAL1，CLOCK，和阻遏市盈率及晶体，产生有节奏的基因表达（早晨阶段， 例如 ， 牧师雇员再培训局α）作用于昼夜E / E&#39;箱的增强子元件。的核心循环调节和集成所述至少两个其它的昼夜顺式元素，DBP/E4BP4结合元件（D-盒;天的阶段， 例如，Per3的 ）和ROR / REV-ERB结合元件（RRE;晚相例如，BMAL1）17。规管的多个昼夜元素的组合可以产生新的中间阶段。例如，转录因子CRY1tion是介导的所有三个昼夜的元素（ 即 E / E&#39;框和D-BOX元素中的第一个内含子的CRY1基因的启动子和RRES），从而引发不同的CRY1晚上的时间第13期。 这些基因表达调控的机制的基础上，我们产生了四种不同的报告结构：P（ 肺癌 ）-D 吕克·P（CRY1）-D吕克记者的E / E&#39;框和D-box元件在监管区17， 26,27 P（CRY1） - 内含子 -D 吕克所有三个元素的组合调控（ 即 E / E&#39;箱，D-BOX，RRE）13,17和P（BMAL1）-D吕克·稳压完全由：RRE 9,17,19,21。我们这些记者介绍3T3细胞产生预期的不同阶段的记者表达（ 图2）。 2。通过RNA干扰基因敲除和药理学激活已经化合物当转染效率是高的，合成的siRNA可以瞬时转染到细胞中基因表达击倒。当转染在技术上是困难的，可以是一个shRNA表达载体稳定转导通过慢病毒感染的细胞，这样的shRNA处理由细胞产生的基因敲除（KD）的siRNA。在这里，我们介绍KD CRY1和CRY2基因的siRNA在U2OS细胞（ 图3A）和shRNA使用pLL3.7网关表达载体（ 图3B）在3T3细胞的影响。除了​​3T3细胞，，U2OS模式已成为另一个卓越的细胞时钟模型，主要是因为它符合市售的人siRNA库（ 例如 ，人类的起源，能够产生强大的昼夜节律，验证功能的高通量筛选的关键要求所有已知的时钟基因，可以高效转染和定量的发光录制）。在这两种类型的细胞中，RNA干扰介导KD导致的时钟表型符合先前的小鼠基因敲除（KO）和细胞KD研究5,10,11,28。例如，的CRY1 KD缩短周期长度和降低节奏持久性，而CRY2 KD延长期。此外，选定的小分子可以使用药理学目标和干扰的蛋白质的功能（ 图3C）。 <img alt="图1" src="/files/ftp_upload/4234/4234fig1.jpg" /> 图1。慢病毒载体介导的基因传递系统。 （A）示意图2慢P（ 肺癌 ）-D 吕克记者载体和CMV-EGFP。只有整合到宿主细胞的基因组的区域被示出。在这两个记者的结构，直接控制下的Per2基因启动子转录的D 吕克 。在pLV7-BSD-P（Per2基因 ）-D 吕克载体（重新的组合为基础的克隆），共表达的杀稻瘟菌素抗性基因（bsd）促进感染的细胞的选择。 -D： 吕克向量（基于结扎的克隆）在pLV156-P（Per2基因 ），绿色荧光蛋白翻译是由一个内部核糖体进入位点（IRES）的d 吕克下游介导的，允许的视觉观察和感染细胞的FACS分类。此外，使用SV40启动子/终止子（P / T）作为绝缘体（见讨论1.3）。在CMV-EGFP的对照载体，一个强大的CMV启动子控制下的EGFP表达（B）感染与转染表达GFP的细胞的荧光图像。通常情况下，我们实现了高效率，无论是在瞬时转染293T细胞，在慢病毒感染的细胞株，我们的利益，在这些细胞中的GFP表达。 <a href="http://www.jove.com/files/ftp_upload/4234/4234fig1large.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图2" sRC =“/ files/ftp_upload/4234/4234fig2.jpg”/&gt; 图2。第一阶段特异性表达的3T3细胞中的生物发光记者。记者的慢病毒载体在这个实验中pLV7-BSD-P（Per2基因 ），P-D 吕克（CRY1）-D，P（CRY1） - 吕克内含子 -D 吕克 ，和P（BMAL1）-D吕克 。每个记者表现出明显的振荡相位，如箭头所指示的。虽然肺癌和 cry1发起人驱动器峰值生物发光早上天相和BMAL1子在夜间阶段，组合调整的P（CRY1） - 内含子窝藏的E-box，D-BOX，和RRE元素的赋予晚上阶段的峰值生物发光<a href="http://www.jove.com/files/ftp_upload/4234/4234fig2large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图3" src="/files/ftp_upload/4234/4234fig3.jpg" /> 图3。记者的昼夜生物发光的节奏在细胞遗传学和药理学的扰动。 （A）CRY1和CRY2击倒siRNA对细胞节律的U2OS记者细胞的影响。 LumiCycle光度计用于在35 mm培养皿中的细胞生物发光的记录。图适用 从参考第10，爱思唯尔（2009年）的许可。（B）CRY2击倒的shRNA对细胞节律的3T3记者细胞的影响。 CRY2基因敲除一个pLL3.7 Gateway载体包含一个U6-shRNA组磁带的使用。 shRNA2有一个更好的Western blot分析（数据未示出）所确定的效率比shRNA1击倒。和合光度计用于生物发光记录细胞在96孔板中。记录的设置如下：孵化温度33°C;积分时间，15秒，间隔时间30分钟。（C）的小分子抑制剂的影响CEL的U2OS记者细胞的lular的节奏。 Chir99021和roscovitine处理，分别针对GSK-3和CDK抑制剂。用于生物发光的细胞录音ViewLux的系统（Chir99021测定）和Tecan公司光度计（roscovitine处理测定）在384孔板中。图改编自“参考编号＃19（版权2008年美国国家科学院，美国）， <a href="http://www.jove.com/files/ftp_upload/4234/4234fig3large.jpg" target="_blank">点击这里查看大图</a> 。

没有利益冲突的声明。

1。目前的协议的修改 1.1记录装置和吞吐量的考虑由于其商用中，LumiCycle（Actimetrics）已成为最常用的自动光度计设备实时记录4,5,9,19,29-31的 。 LumiCycle采用光电倍增管（PMT）作为光探测器，它提供了极高的灵敏度和低噪声14，因此特别适合于数据采集极其暗淡的基于荧光素酶发光。其他相似的PMT的设备（ 例如 ，克罗诺斯，阿托公司; POL...

<table border="1"><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> DMEM </td><td>胎牛血清</td><td> SH30243FS </td><td>对于正常细胞生长</td></tr><tr><td> DMEM </td><td> Invitrogen公司</td><td> 12100-046 </td><td>对于luminometry </td></tr><tr><td> FBS </td><td>胎牛血清</td><td> SH3091003 </td><td></td></tr><tr><td>青/链霉素/谷氨酰胺（100X） </td><td>胎牛血清</td><td> SV3008201 </td><td></td></tr><tr><td> B-27 </td><td> Invitrogen公司</td><td> 17504-044 </td><td></td></tr><tr><td> D-荧光素</td><td> Biosynth </td><td> L-8220 </td><td></td></tr><tr><td>聚-L-赖氨酸</td><td>西格玛</td><td> P4707 </td><td></td></tr><tr><td>聚凝胺</td><td>密理博</td><td&gt; TR-1003-G </td><td></td></tr><tr><td>佛司可林</td><td>西格玛</td><td> F6886 </td><td></td></tr><tr><td>所有其他化学品</td><td>西格玛</td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> 设备 </td></tr><tr><td>组织培养箱</td><td></td><td></td><td>在37°C，5％CO2 </td></tr><tr><td>组织培养罩</td><td></td><td></td><td> BSL-2认证</td></tr><tr><td>光与荧光显微镜</td><td></td><td></td><td>相衬可选</td></tr><tr><td> LumiCycle </td><td> Actimetrics </td><td></td><td></td></tr></table>

monitoring cell-autonomous circadian clock rhythms of gene expression using luciferase bioluminescence reporters

在哺乳动物中，许多方面的行为和生理的，如睡眠-觉醒周期和肝脏代谢的调节内源性生物钟（审阅1,2）。时间的昼夜节律系统是一个分层的多振荡器网络，与中央的时钟位于视交叉上核（SCN）同步和协调其他额外的SCN和外设时钟1,2。单个细胞的生成和维护的昼夜节律3,4，这些振荡器在有机体份额显着相似的生化负反馈机制的不同组织类型的功能单元。然而，由于在神经元网络级在SCN和有机体一级通过节奏，系统性的线索的相互作用，在有机体一级的昼夜节律不一定细胞自主5-7。相对于传统的研究在体内的自发活动和SCN外植体离体 ，CELL-总部设在体外实验中可以发现的细胞自主的昼夜缺陷5,8。在战略上，基于细胞的实验模型更易于处理的表型特征和基本时钟机制的快速发现5,8-13。 因为昼夜节律是动态的，纵向的高时间分辨率的测量来评估时钟功能。近年来，实时使用萤火虫荧光素酶的生物发光记录，记者已成为一种常见的技术研究在哺乳动物中的昼夜节律14,15，因为它允许分子节奏的持久性和动态检查。细胞的自我监控的昼夜节律基因表达，荧光素酶记者可以被引入到细胞通过瞬时转染13,16,17或稳定转导5,10,18,19。在这里，我们描述了一个稳定传导协议的慢病毒介导的基因传递。 Ţ他慢病毒载体系统是优于传统的方法，如瞬时转染和生殖传输，因为它的效率和多功能性：它可以有效地提供稳定整合到宿主基因组的分裂和非分裂细胞20。记者细胞系建立之后，时钟功能的动态可以检查通过生物发光记录。我们首先介绍了新一代的P（Per2基因 ）-D 吕克记者的线条，然后和其他昼夜记者从目前的数据。在这些实验中，3T3小鼠成纤维细胞和U2OS人骨肉瘤细胞被用作细胞模型。我们还讨论了不同的方式使用这些时钟模型在昼夜节律的研究。这里描述的方法可以应用到多种类型的细胞，研究细胞和分子基础的生物钟，并在处理其他生物系统的问题可能会被证明是有用的。

Watch this Scientific Journal Video about Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters at JoVE.com

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

生物钟单个细胞内发挥作用，也就是说，它们是细胞的自我。在这里，我们描述了使用非侵入性，实时荧光素酶为基础的生物发光技术产生的细胞自主的时钟模型的方法。记者细胞提供听话的，功能性的昼夜节律生物学研究的模型系统。

监测细胞自主的生物钟节律基因表达，利用荧光素酶生物发光记者

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

monitoring-cell-autonomous-circadian-clock-rhythms-gene-expression

Research

JoVE Journal

Biology

22.1K 次观看.  The University of Memphis. 生物钟单个细胞内发挥作用，也就是说，它们是细胞的自我。在这里，我们描述了使用非侵入性，实时荧光素酶为基础的生物发光技术产生的细胞自主的时钟模型的方法。记者细胞提供听话的，功能性的昼夜节律生物学研究的模型系统。

视频: Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

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

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 ...

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 ...

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 ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

全球合成的RNA在单细胞水平以下的低氧分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

代谢率的测量<em&gt;果蝇</em&gt;使用呼吸运动

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic levels. Tight control over ER calcium is imperative for protein folding, modification and trafficking. Perturbations to ER calcium can result in the activation of the unfolded protein response, a three-prong ER stress response mechanism, and contribute to pathogenesis in a variety of diseases. The ability to monitor ER calcium alterations during disease onset and progression is important in principle, yet challenging in practice. Currently available methods for monitoring ER calcium, such as calcium-dependent fluorescent dyes and proteins, have provided insight into ER calcium dynamics in cells, however these tools are not well suited for in vivo studies. Our lab has demonstrated that a modification to the carboxy-terminus of Gaussia luciferase confers secretion of the reporter in response to ER calcium depletion. The methods for using a luciferase based, secreted ER calcium monitoring protein (SERCaMP) for in vitro and in vivo applications are described herein. This video highlights hepatic injections, pharmacological manipulation of GLuc-SERCaMP, blood collection and processing, and assay parameters for longitudinal monitoring of ER calcium.

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies. A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store. This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.

监测内质网钙稳态使用<em&gt; Gaussia</em&gt;荧光素酶SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

利用荧光标记的纳米粒子检测的小鼠，人与猪源活细胞内的基因表达

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable biotic and abiotic stress. Conversely, disruption of circadian timekeeping severely compromises plant health and reduces agricultural crop yields. It is therefore imperative that we understand the intricate regulation of circadian rhythms in plants, including the factors that affect motion of the transcriptional clockwork itself.
Testing circadian defects in the model plant Arabidopsis thaliana (Arabidopsis) traditionally involves crossing specific mutant lines to a line rhythmically expressing firefly luciferase from a circadian clock gene promoter. This approach is laborious, time-consuming, and could be fruitless if a mutant has no circadian phenotype. The methodology presented here allows a rapid initial assessment of circadian phenotypes. Protoplasts derived from mutant and wild-type Arabidopsis are isolated, transfected with a rhythmically expressed luminescent reporter, and imaged under constant light conditions for 5 days. Luminescent traces will directly reveal whether the free-running period of mutant plants is different from wild-type plants. The advantage of the method is that any Arabidopsis line can efficiently be screened, without the need for generating a stably transgenic luminescent clock marker line in that mutant background.

The circadian clock regulates about a third of the Arabidopsis transcriptome, but the percentage of genes that feed back into timekeeping remains unknown. Here we visualize a method to rapidly assess circadian phenotypes in any mutant line of Arabidopsis using luminescent imaging of a circadian reporter transiently expressed in protoplasts.

在拟南芥原生质体转染昼夜的表型有荧光时钟记者的快速分析

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable ...