这项工作得到了优异评论奖道格拉斯D.罗斯和从退伍军人事务部的阿里夫·侯赛因的支持。

1. 在 BCRP1的替代外显子第一硅片预测上使用鼠标EST数据库的BLAST分析注:本协议描述了如何搜索小鼠表达序列标签（EST）数据库与序列相似性的无害环境技术外显子BCRP1（包含翻译起始位点）2，再怎么匹配的EST序列对准基因组序列，以确定他们的位置相对于BCRP1外显子2的5&#39;末端的小鼠BCRP1基因英寸<ol><li>通过输入mRNA的参考序列ID（NM_011920.3）进入ENSEMBL基因组浏览器17的搜索窗口获得鼠标BCRP1外显子序列，点击"GO"。在接下来的屏幕中，选择全长序列（包括外显子16）: <ol><li>在接下来的屏幕（"基于抄本 - 显示"），选择"16外显子。"这将显示含有BCRP1的所有外显子的顺序的画面。 BCRP1的外显子2将改为"59; -AAAGGC ... TATCAA-3&#39;"获得将类似于在参照图18中所示的那些结果。 </li><li>点击"下载序列"选项。在接下来的屏幕上，选择FASTA格式，然后点击"预览"。在下一屏，复制外显子2的预览序列到剪贴板。 </li></ol></li><li>导航到国家生物技术信息中心（NCBI）网站20 BLAST主页19。 <ol><li>选择"鼠标"的基因组。从剪贴板粘贴到查询框中的外显子2序列。 </li><li>从数据库下拉列表中选择"无害环境技术"，优化"高度相似的序列"，然后选择"BLAST"。运行时会需要几分钟。当BLAST运行完成后，将显示"结果"页面。 </li><li>在"结果"页面上的"说明"的副标题，选择"ALL"，然后选择"下载"，然后"FASTA（完整序）"中的下拉菜单，然后选择"继续"。txt文件就会出现;开放，整个文件复制到剪贴板中的.txt文件包含了所有的EST用鼠标BCRP1外显子高的序列相似的序列。但不是他们的有关位置，以在BCRP1基因外显子2。 注:2015年4月15日进行的分析发现，与外显子BCRP1对准2.这些在表1中列出14个EST小鼠。 </li></ol></li><li>识别的EST序列是5&#39;在BCRP1基因外显子2的位置。鼠标BCRP1基因位于6号染色体重叠群NC_000072.6（GI:372099104）。 <ol><li>下的"专业化爆炸"的副标题BLAST主页，选择"使用BLAST（使用bl2seq）对准两个（或更多）的序列。" </li><li>从剪贴板文本文件粘贴到查询框，并在主题序列框中输入372099104。优化方案下的"高度相似的序列"选择和运行BLAST。 </li><li>一旦出现结果窗口，通过图形化的"路线"窗口中单击"图形"查看路线。使用左，右箭头和放大专注于BCRP1 / ABCG2和序列比对。 </li><li>保存序列比​​对:在"说明"框中选择"ALL"，然后在"下载"下拉列表中选择"打表（CSV）"，然后点击"继续"。此文件包含BCRP1外显子2的序列比对，并相对于该核苷酸在小鼠染色体6重叠群的编号与序列相似的所有EST序列以BCRP1外显子2的取向。每个完整的EST序列可能产生多重比对跨越的区域的5&#39;和3&#39;外显子2包括与BCRP1外显子2重叠序列。 </li><li>作为外显子2的5&#39;端的位置将对应于染色体6重叠群的核苷酸58655638，指定这为+ 1，然后计算每个EST 5&#39;的部分序列的位置58655638。为14个EST的结果在表1中给出。 注意:要小心分析是潜在的外显子第5&#39;+1（ 即有负面核苷酸值）EST序列。例如，在两块与表1（AI647825和AI664571）所示BCRP1外显子2对准的EST序列的剩余序列是3&#39;外显子2。 </li></ol></li></ol> 2. BCRP1评价替代的启动子功能<ol><li> 记者设计构建了BCRP1 E1U，E1A，E1B和E1C推动者 <ol><li>使用从搜索EST数据库中得到的E1U的序列，指定E1U的第一5&#39;核苷酸为+1。 </li><li>得到的细菌人工染色体（BAC），它包含了BCRP1 / ABCG2基因序列和序列的至少100 kb的上游小鼠染色体6的克隆BCRP1 / ABCG2基因（见材料和​​设备清单）。 <ol><li>识别如萤光素酶报道质粒的pGL3-Basic中的合适的报告基因载体。 </li><li>确定在报告载体的多克隆位点。的KpnI，SacI位，Mlu1，NheI位，SmaⅠ位，XhoI位，BglⅡ和HindⅢ位处于pGL3-基本载体的多克隆位点&#39;至3&#39;方向的限制性内切酶位点，在5。 注意:酶切位点的序列是得自产生的限制酶公司的产品目录。 </li><li>确定在E1U所有外显子酶切位点和2 kb的5&#39;区域使用E1U软件程序，如DNA5的。确定未在E1U或2 kb的上游区域发现了pGL3-基本多克隆位点的至少两个消化位点。 注意:一定要选择正向引物是选择了反向引物之一上游的多克隆位点酶切位点，以确保插入WILl条方向正确。 </li></ol></li><li>制备正向和反向包含用于使用商业基因/引物合成服务或引物的选择软件（见材料和​​设备的列表）选择的pGL3碱性多克隆位点的限制性内切酶位点的序列用于PCR的引物。选择位于〜2kb的上游从E1U序列和E1U序列内的反向引物正向引物。在作者使用的引物"以前的工作引入的正向引物一个SacI位点，而在反向引物的NheI位点，并与第一5放大从约-1,906的基因组区域至+64&#39;E1U的指定为核苷酸+ 1（见上文步骤2.1.1），并在参考16被提供。 <ol><li> PCR用这些引物，所述的BAC 0.01至1微克扩增E1U基因组区，并含有高保真的Taq聚合酶（参见材料和设备的列表）PCR主混合物与变性95℃30秒，然后PCR的35至40个循环，以延伸72℃（2分钟），和退火并基于所使用的引物的熔化特性熔融温度。 注:使用高保真的Taq聚合酶的是具有高GC含量测序启动子区域至关重要。当使用大基因组DNA片段，如BAC为模板，基因组DNA的二级结构可能使其难以用于PCR的引物结合。如果这个问题出现了，如果长基因组DNA模板轻轻通过超声处理PCR反应之前剪切可以得到更好的PCR结果。 </li><li>通过使用0.8％TAE琼脂糖凝胶电泳对一个1 kb的DNA梯比较验证扩增的PCR产物的长度。 </li></ol></li><li>消化获得的PCR产物和pGL3-基本矢量，使用限制性内切核酸酶特异于引入向前的限制酶切位点和反向引物为每项用限制性消化酶供给tructions。 <ol><li>净化使用商业SV凝胶和PCR清理系统套件（参见材料和设备清单），继包协议21消化反应。 <ol><li>通过比较其对利用琼脂糖凝胶电泳的未切割的载体验证向量的消化线性化，然后切割和纯化消化的载体DNA带。测量纯化的PCR产物的浓度和使用UV光谱法（见材料和​​设备的列表）的纯化载体。 </li></ol></li></ol></li><li>通过结扎纯化制备E1U报道构建，限制性内切酶消化的PCR产物和pGL3-基本萤火虫萤光素酶报告基因载体（包含在报告测定试剂盒;见材料清单和设备）在插入:11，21和31的矢量比，使用"T4 DNA连接酶快速连接套件"按试剂盒说明书22，从而产生E1Uřeporter结构，命名为pGL3-启动E1U。 <ol><li>使用的pGL3-E1U质粒转化细菌克隆扩大在TA克隆试剂盒的pGL3-E1U下面的说明（见材料和​​设备清单）。 </li><li>确认通过序列分析的插入方向和保真度。 </li></ol></li><li>记者准备使用类似方法为E1U的E1A，E1B和E1C构建。确认通过测序插入件的保真度与第2.1.5.2。 注意:E1A，E1B和E1C启动子插入物应为约-1,875 / + 10，-1,847 / + 60，和-1,904 / + 83相对于E1A，E1B的第一5&#39;核苷酸（表示为+ +1），或E1C，分别为。 </li></ol></li><li> 记者测定方法 注意:报告分析的一般策略:一个基因的推定的启动子（5&#39;上游区域）被插入到包含"reporte一个空的"记者"载体的多克隆位点抗病基因"（ 例如 ，萤火虫萤光素酶）的下游的多克隆位点，然后将载体转染到表达目的基因的真核细胞中。促进感兴趣的这些细胞中的基因的表达应该激活的反式作用因子启动子在转染报告基因载体，导致萤火虫萤光素酶，可与发光测定法来容易量化的表达。 <ol><li>选择合适的细胞系以用于报告测定。 注意:为了评价E1U备选启动子报告构建体的活性，有必要来转染该构造成，根据E1U启动子控制表达BCRP1细胞。所述TM4鼠塞尔托利细胞系（见材料清单与设备）表达BCRP1蛋白和含有E1U，以及E1A，E1B和E1C 5 BCRP1的mRNA。作为阴性对照，请考虑使用不表达BCRP1蛋白或mRNA的细胞系。 </li><li>文化TM4 CE在24孔板LLS以200,000细胞的初始密度/孔，在1:火腿的F12培养基和Dulbecco氏改性Eagle氏1.2 g / L的碳酸氢钠介质的1:1混合物，15毫米的HEPES补充有5％马血清，2.5％牛胎儿血清，青霉素（100IU / ml）和链霉素（100微克/毫升），在37℃，5％的CO 2，如先前5所述。 </li><li>放置细胞的培养后六小时，转染空的pGL3-basic载体的0.2微克或0.2微克含有-1,906 / + 64 E1U或-1,875 / + 10 E1A或-1,847 / + 60 E1B的pGL3载体的细胞或-1,904 / + 83 E1C BCRP 1缺失构建连同4纳克PRL-TK（一海肾荧光素酶表达载体）的内部控制，使用商业DNA转染试剂盒和制造商的协议23（见材料和设备清单） 。 </li><li> 30小时转染后，从文化性除去生长培养基编辑转染细胞。用200微升PBS的一次洗涤细胞，然后取出洗涤液。 <ol><li>测量萤火虫和使用商业试剂盒根据制造商的协议24 Renilla荧光素酶的活性。 </li></ol></li><li>表示每个管作为萤火虫荧光素酶活性由内部（Renilla荧光素酶）的控制划分的比率的活性;总的结果通常被表示为每个报告的活性构造相对于与空的pGL3基本载体，其被赋予值1转染的细胞。 </li></ol></li></ol>

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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+controls:+the+role+of+alternative+promoters+in+mammalian+genomes.">Complex controls: the role of alternative promoters in mammalian genomes.</a> Trends Genet. (11), 640-648 (2009).</li><li>Park, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChIP-seq:+advantages+and+challenges+of+a+maturing+technology.">ChIP-seq: advantages and challenges of a maturing technology.</a> Nat Rev Genet. 10 (10), 669-680 (2009).</li><li>Bulun, S. E., 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=Regulation+of+aromatase+expression+in+breast+cancer+tissue.">Regulation of aromatase expression in breast cancer tissue.</a> Ann N Y Acad Sci. 1155, 121-131 (2009).</li></ol>

道格拉斯D.罗斯和马里兰大学巴尔的摩持有专利权人的BCRP。

大多数提出的方法和代表性的结果在以前的题目这是发表在2013年5"在小鼠睾丸乙 CRP1转录由启动子上游通过类固醇生成因子-1调控的小说第一外显子（E1U），可控"的工作介绍除了这里所描述的代表性的结果，先前的纸张提供了一种使用5&#39;-RACE PCR和RT-PCR检测方法替代第一外显子的利用率的估计。此外，在硅片在启动子上游E1U推定SF-1应答元件的鉴定来完成，和染色质免疫沉淀测定法表明SF-1结合于推定的SF-1应答元件。功能研究表明，在小鼠Sertoli细胞，BCRP 1转录由SF-1经由E1U启动子SF-1应答元件控制。这些功能性研究中TM4细胞TRA包括SF-1的上调nsfection或通过使用组蛋白脱乙酰酶抑制剂（伏立诺他），这导致在BCRP 1 E1U mRNA的表达增强，并且在BCRP 1蛋白在报道实验的增加以及BCRP 1 E1U启动子的活性。最后，这些研究提供的证据表明，在成年睾丸支持细胞特异性SF-1基因敲除小鼠的睾丸，BCRP 1 E1U mRNA表达，总BCRP 1 mRNA 的表达，并BCRP1蛋白显着减少5。同样的工作也报道在各种鼠组织中，包括肾，肝，睾丸，脑，心脏，肺，肌肉和脾5 BCRP1的mRNA同种型的表达模式。 使用BCRP 1的组织特异性调控作为模型- -这里所描述的协议提供一个浅显试验框架解开任何基因的转录复杂，以确定其差在各种组织或细胞亚型的表达。必须强调的是，从在硅片评价描述的协议的步骤中获得的结果可能是依赖于时间的，因为这容纳软件和使用可能会受到改变的数据库的各种网站。这里提出的在硅片方法利用搜索如以前29日报道的EST数据库（dbEST数据库），它估计，在EST数据集中的所有人类基因约有18％采用替代推动者。搜索dbEST中可能会低估替代第一外显子，因为其他研究者 - 使用"低聚帽"方法从许多人体组织产生的cDNA的180万的5&#39;末端序列 - 能够识别为人类基因和估计推定的转录起始位点研究人类的RefSeq基因有52％的可能是通过促 ​​进替代上调1。有趣的是，后者的研究发现，灵活运用公认的替代促销员最多的是睾丸和脑组织;此外，他们发现，编码信号转导途径相关的蛋白质的基因更可能采用组织特异性替代的启动子1。尽管所提到的缺点，dbEST中分析了在用最小的努力多种组织的特定基因提供5&#39;端非编码区的使用的一个粗略的概述。 虽然dbEST中分析提供了在替代第一外显子的使用一个浅显的惊鸿一瞥，我们强烈建议执行5&#39;cDNA末端（5&#39;-RACE）分析的快速扩增，以验证正在调查一个组织或细胞系第一外显子的用法。商业套件可用于5&#39;-RACE，由制造商提供详细和直接的程序（见材料和​​设备的列表）。虽然耗费的时间稍长，5&#39;-RACE研究提供存在实际的估计以及替代第一八佰伴的序列组件在感兴趣的基因;此外，多个转录起始位点的存在，也可检测出13。为了保证足够的代表性，需要至少15至25个克隆的测序。测序之前，重要的是从5&#39;RACE产物除去污染的载体序列。如果不这样做，BLAST分析可能会完全无法检测与小鼠基因组的序列比对。一旦第一外显子的使用被建立，5&#39;-RACE发现可以用于验证替代第一外显子随后定量RT-PCR测定。在一般情况下，作者的以前的工作中发现，有由5&#39;RACE恢复的第一外显子的mRNA的克隆的百分比和PCR产物的从相同的组织或细胞系5回收对于一个给定的替代第一外显子的百分比之间的良好的相关性。此外，良好的相关性萤光素酶启动子构建活动之间找到替代BCRP1促销员次对应的替代第一外显子的在特定细胞系5的表达。 在寻 ​​找组织特异性启动子的替代使用，如果使用了整个器官，人们必须意识到，找到替代促销员/第一外显子将反映器官组织的异质性，如腺元素，血管，间质等 。例如，睾丸是体细胞（间质，塞尔托利，肌样，和内皮）细胞和生发（单倍体）细胞，以及脉管的混合。探测组织特异性第一外显子的表达，这将是必要的，以隔离来自器官特定组织。 其他方法已被报道用于确定替代的启动子的使用和管理;然而，这些要求新一代测序和生物信息学计算能够分析大量产生的数据。这些方法包括整个transcriptosome测序（RNA测序）以及芯片起组蛋白如H3K4me3的，其优先结合促销员30。虽然这些方法可以进行脱从头 ，当焦点上的几个特定的基因和组织中的表达昂贵，现有数据挖掘可能是一个更实际的方法。 用于估计这里提出的启动子活性的测定法采用pGL3-基本向量，其中包含一个多克隆区上游的萤火虫荧光素酶基因。启动子活性通过生产萤火虫萤光素酶，这是很容易量化的在商业光度计（见材料和​​设备的列表）测得，除辅因子的和发光底物后，通过发光的测量。相关pGL4载体可制成含有可选择标记（ 例如 ，新霉素，潮霉素，嘌呤霉素），从而使生产稳定转染的细胞。本文给出的协议利用瞬时转染。通常情况下，记者对检测promot尔活性使用共转染与组成性表达的Renilla（海上三色堇）荧光素酶（PRL-TK）的载体，以控制在细胞数量的变型和转染的效率完成。在对于给定的基因建立的启动子测定法的一个关键步骤是，以确保感兴趣的基因在被转染的报道构建的细胞系中被表达。其次，当可变启动子的用法是本，它使用对应于转染的细胞系中表达的替代第一外显子的启动子构建是重要的。例如，不希望看到的发光为转染入表达BCRP1纯粹的E1B启动子控制下的细胞E1U子结构。可替代地，作为阴性对照，记者构建体转染到不表达该基因或感兴趣的mRNA同种型应当导致没有生产萤火虫荧光素酶的细胞系。 的C替代子用法onsequences包括生产相同的蛋白质，生产不同的N端，或不同的蛋白质29的蛋白质的生产。在BCRP1 / BCRP，多个的情况下（组织或细胞类型特异性）启动子控制生产相同的蛋白质。类似的模式存在的CYP19（芳香化酶）基因29,31。这里介绍的协议提供了人们可以使用在组织或细胞系破译的目的蛋白质的转录控制的复杂机制的基本步骤。

超过人类基因中有一半是通过促 ​​进替代上调1。每个备选启动子可以含有调控元件，可能是从那些在其他替代的启动子不同。在一个组织中使用的启动子（多个）可能与另一组织中使用不同。例如，它可能是一个给定的信号传导途径的激活可以触发用于在一个组织利用的基因替代的启动子，但有没有影响或抑制对于被利用在另一组织相同基因的单独的替代型启动子。 所述BCRP1基因的表达是通过替代的启动子支配。 BCRP1是人类乳腺癌耐药蛋白（BCRP）基因的小鼠同源基因。 BCRP是ATP结合盒（ABC）转运，正式指定ABCG2 2，3。作为一个顶质膜蛋白质，BCRP / BCRP1功能外排多种天然和异生底物<suP&gt; 3，4。在人类和小鼠中，BCRP / BCRP1高度在药理学相关机关表示如肝（胆小管），肠和肾，以及器官血液-组织屏障如胎盘，脑和睾丸2，5 - 12。 BCRP / BCRP1的造血和其它干细胞包括癌干细胞，表达可以赋予这些细胞的抗外源物和癌症化疗药物3。 在早期的工作，了解BCRP表达在正常和肿瘤细胞的调节，进行5&#39;cDNA末端（5&#39;-RACE）BCRP mRNA的分析，快速扩增，以确定其确切的转录起始位点13。不仅是找到多个转录起始位点;也遇到是第一外显子，这是BCRP翻译三种替代形式。这些替代第一外显子 - 指定中E1a，E1B，E1C - 在各种人体组织中表达不同。两个附加第一外显子的变体在BLAST搜索用BCRP 13的第二外显子的人的EST数据库的被发现。四场比赛显露上游从被指定E1u外显子2第一外显子&gt; 70 kb的;其他四场比赛显露BCRP表达，缺乏完全是第一外显子，这被指定E1- 13。 替代领导人外显子的存在被认为是替代的启动子的使用14的体现。 在小鼠中，BCRP1四个替代第一外显子中描述了可以对应于管理在不同鼠组织BCRP 1转录替代的启动子;这些外显子/启动子指定E1U，E1A，E1B和E1C，并位于约70，从BCRP1外显子2 5,15 58，15和5 kb的上游。在E1A的mRNA同种型被认为是在murin主要È造血干细胞，心脏，肺，脾和脑，而E1B亚型在小鼠小肠，胎肝细胞和红细胞前体细胞在骨髓5,15表示。上游E1B启动子被证明是管辖在小鼠肠BCRP1转录的主要替代子，至少部分调节的，由磷酸环-AMP反应元件结合蛋白（对CREB）和独特的，替代一个CREB响应元件BCRP1子16。该E1C表达亚型在成年小鼠肝脏和肾脏5中表达。该E1U亚型是在除了鼠睾丸，它是表示5的主要同种型测试大多数组织中检测不到。 BCRP1在大鼠睾丸表达的在两个躯体（内皮细胞紧密连接，管周肌样细胞和支持细胞）细胞和生殖细胞中发现（生精内皮细胞，它可以保护晚期精子4）。该注册离子上游E1U包含类固醇生成因子1（SF-1）5的官能响应元素。 BCRP1 mRNA和蛋白在塞尔托利细胞特异性SF-1敲除小鼠的睾丸被显着降低，这表明在小鼠Sertoli细胞BCRP1表达通过SF-1 5控制。 详细介绍方法的协议来检测BCRP1替代第一外显子在硅片上 ，并从上游确定的替代外显子第一次为建立促进推测基于荧光素酶报告基因分析。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>COMMERCIAL BIOLOGIC MATERIALS AND KITS:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>First Choice RLM-RACE kit</td>
			<td>Ambion Inc., Austin, TX, currently available through Life Technologies</td>
			<td>AM1700</td>
			<td>5&#39;-RACE PCR kit</td>
		</tr>
		<tr>
			<td>TOPO TA cloning kit&#160;</td>
			<td>Life Technologies</td>
			<td>K450001</td>
			<td>This kit contains the TOP10 chemically competent E. coli bacteria.</td>
		</tr>
		<tr>
			<td>T4 DNA ligase quick ligation kit</td>
			<td>New England Biolabs</td>
			<td>M2200</td>
			<td></td>
		</tr>
		<tr>
			<td>Faststart high-fidelity Taq- DNA polymerase</td>
			<td>Sigma-Aldrich/Roche</td>
			<td>3553400001/RMB-4738284001&#160;</td>
			<td>Contains a high-fidelity Taq polymerase enzyme blend</td>
		</tr>
		<tr>
			<td>XtremeGENE HP DNA transfection reagent</td>
			<td>Roche</td>
			<td>6366236001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual-luciferase reporter assay kit</td>
			<td>Promega</td>
			<td>E1910</td>
			<td>Includes the pGL3-basic empty reporter vector (firefly luciferase), pRLTK renilla luciferase expressing vector, and other control vectors.</td>
		</tr>
		<tr>
			<td>QIAprep</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td>For plasmid miniprep - isolation and purification of plasmid DNA from bacterial colonies</td>
		</tr>
		<tr>
			<td>pCR2.1 vector</td>
			<td>part of the TOPO TA cloning kit</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pGL3-basic luciferase containing vector</td>
			<td>Promega</td>
			<td>E1751</td>
			<td></td>
		</tr>
		<tr>
			<td>pRL-TK Renilla luciferase expressing vector</td>
			<td>Promega</td>
			<td>E2241</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacterial artificial chromosome</td>
			<td>BACPAC Resources Center, Children&#8217;s Hospital Oakland Research Institute, Oakland, CA</td>
			<td>RP23-285A12 and RP24-314E24</td>
			<td>http://bacpac.chori.org/clones.htm</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company&#160;</td>
			<td>Catalog Number&#160;</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>REAGENTS/CHEMICALS/MEDIA/SUPPLIES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol</td>
			<td>Life Technologies</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr>
			<td>Wizard&#174; SV Gel and PCR Clean-Up System&#160;</td>
			<td>Promega</td>
			<td>A9281</td>
			<td>Useful for purifying PCR products following digestion with restriction endonucleases</td>
		</tr>
		<tr>
			<td>CRYOVIALS</td>
			<td>Denville Scientific</td>
			<td>V9012</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company&#160;</td>
			<td>Catalog Number&#160;</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>SOFTWARE:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ensembl software</td>
			<td>Ensembl project</td>
			<td>http://Ensembl.org&#160;</td>
			<td>The Ensembl project produces genome databases for vertebrates and other eukaryotic species, and makes this information freely available online.</td>
		</tr>
		<tr>
			<td>Blast software</td>
			<td>NCBI</td>
			<td>http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD= 
			Web&#38;PAGE_TYPE=BlastHome</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer 3 software</td>
			<td>Simgene</td>
			<td>http://simgene.com/Primer3</td>
			<td>Useful for designing primers for 5&#39;-RACE PCR</td>
		</tr>
		<tr>
			<td>NCBI Nucleotide database</td>
			<td>NCBI</td>
			<td>http://www.ncbi.nlm.nih.gov/nucleotide/</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company&#160;</td>
			<td>Catalog Number&#160;</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>CELL LINES:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TM4 murine Sertoli cells</td>
			<td>ATCC, Manassas, Virginia</td>
			<td>CRL-1715&#8482;</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company&#160;</td>
			<td>Catalog Number&#160;</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>INSTRUMENTS:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Luminometer</td>
			<td>Turner Biosystems</td>
			<td>TD-20/20</td>
			<td>This is a relatively inexpensive, manually operated luminometer. Automated systems are also available from the same manufacturer that utilize 96 well plates.</td>
		</tr>
		<tr>
			<td>NanoDrop spectrophotometers</td>
			<td>Thermo Scientific, Inc.</td>
			<td>NanoDrop 2000</td>
			<td>Spectrophotometer can use very small quantities of sample</td>
		</tr>
		<tr>
			<td>DU 800 UV/VIS spectrophotometer and Nucleic Acid Analysis II software</td>
			<td>BeckmanCoulter Inc, Fullerton, CA</td>
			<td>DU 800</td>
			<td></td>
		</tr>
	</tbody>
</table>

methods to discover alternative promoter usage and transcriptional regulation of murine bcrp1

通常非翻译 - - 第一外显子在不同组织中的基因表达通常通过导致mRNA的合成具有独特的备选启动子控制。 BCRP1（ABCG2），ABC转运乳腺癌耐药蛋白（BCRP，ABCG2）的小鼠同源基因，具有由生产相应的四个备选外显子先指定至少有四种可供选择的发起人:E1U，E1A，E1B和E1C。这里， 在计算机芯片上的协议被提供给预测备选启动子用法BCRP1。此外，被描述报告基因检测方法，以产生替代的启动子报道构建并确定推定的启动子上游的所确定的替代第一外显子的功能。

BCRP 1 替代促进利用 的鉴定 在小鼠睾丸被领导者的外显子的分析 当在NCBI的EST数据库，使用在方案1中列出的步骤进行探测（2015年4月）中，EST序列发现，分别邻接在BCRP的5&#39;外显子2的端部1 mRNA 的示于表1，与它们在基因组的位置沿相对于外显子2，从C57BL / 6J小鼠睾丸即邻接外显子2中BCRP 1的mRNA具有从外显子2中的基因组DNA 70 kb的上游序列中，对应于E1U（ 表1）衍生的一个EST的起始的DNA。同样地，检测到对应于E1A，E1B和E1C的EST。说明的是，对应于E1C两个EST序列也含有E1B拼接到E1C的5&#39;端。这些预测的第一外显子的位置的关系到小鼠染色体6上BCRP1外显子2中示出在图1中。 BCRP 1 可变启动子功能 的评价 对应于E1U，E1A典型萤光素酶测定的结果，转染到TM4小鼠塞尔托利细胞（参见第2.2节）E1B和E1C启动子-荧光素酶报道构建体示于图2A。 在以前的工作中，SF-1反应元件被预测为是在E1U启动子5。如果此推定SF-1反应元件是参与BCRP1转录在小鼠睾丸的调节，那么在E1U启动子报道构建该反应元件（如在一个先前的纸5所述）突变会降低由该构建体产生的荧光素酶活性当转染入TM4细胞。一个典型实验的结果示于图2B。突变的构建体显示出比未突变构建体下部的荧光素酶的活性，用活性媲美的空的pGL3-basic载体的，即使是在与SF-1为强制表达（在先前的纸5所描述）细胞。 SF-1的表达强制增加了非突变结构的活性，但不是突变之一。 <img alt="图1" src="/files/ftp_upload/53827/53827fig1.jpg" /> 图 1。与序列同一性BCRP1外显子2与鼠染色体6.这些比4月份进行的dbEST中BLAST搜索被认定无害环境技术的基因组比对图 ，发现2015年四个不同路线，相当于替代BCRP1第一外显子E1U，E1A， E1B和E1C。这个数字从先前公布5转载。 <aHREF ="htt​​ps://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53827/53827fig1large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/53827/53827fig2highres.jpg" width="500" /> 图 2。 （A）记者试验的发起人BCRP1 E1U，E1A，E1B和E1C在BALB / c小鼠睾丸支持细胞衍生TM4细胞。数据表示为萤火虫萤光素酶的归一化到Renilla荧光素酶的发光，利用在第2所示的数据中描述的方法进行的平均值和三种不同的实验的标准偏差，在不同的日子进行。星号表示，从使用t检验（P &lt;0.01），空pGL3载体控制显著差异。该图是从以 ​​前的出版物5再现。在BCRP1 E1U重新对SF-1应答元件的突变的（B）的影响。波特在构建荧光素酶活性。 BCRP1记者随推定SF-1结合区构建（突变或未突变）转染TM4细胞（SF-1转），无（转矢量）显示SF-1，数据的强制表达为平均值的3个不同的实验中，在不同的日子进行;误差棒代表标准偏差。对于每个实验，从个人测定以一式两份进行。在图中，（A）表示从空载体的pGL3对照组（P &lt;0.05），使用t检验一个显著的差异;（B）表示相比，使用t检验的E1U子构建一个统计学上显著差异（P &lt; 0.05）。这个数字从先前公布5转载。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53827/53827fig2highres.jpg" target="_blank">请点击此处查看本图的放大版本。</a> 表 1。 高炉搜索对BCRP1 25 -28的第二个外显子序列鼠标EST数据库， <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53827/53827table1highres.jpg" target="_blank">请点击这里查看此表的放大版本。</a>

Watch this Scientific Journal Video about Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1 at JoVE.com

Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1

方法发现小鼠BCRP1的另类用法子和转录调控

与鼠ABC转运BCRP1（ABCG2）作为一个例子， 在硅片呈现协议，以检测在小鼠组织中表达的基因的备选启动子的使用，并评价使用报告基因分析鉴定的备选启动子的功能。

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually ...

methods-to-discover-alternative-promoter-usage-transcriptional

Nanyang Technological University

Research

JoVE Journal

Biology

8.0K 次观看.  University of Maryland School of Medicine. 与鼠ABC转运BCRP1（ABCG2）作为一个例子， 在硅片呈现协议，以检测在小鼠组织中表达的基因的备选启动子的使用，并评价使用报告基因分析鉴定的备选启动子的功能。 

视频: Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1

Preparation of 2-dGuo-Treated Thymus Organ Cultures

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo can limit analysis of individual cellular components and particular stages of development. In vitro culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

This video demonstrates the dissection and removal of the fetal thymus as well the preparation of ex vivo cultures of 2-dGuo-treated thymus.

2 dGuo治疗胸腺器官培养的制备

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play ...

科研

生物学

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

夹层和染色果蝇幼虫卵巢

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

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 ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

成年小鼠心肌细胞的单细胞转录谱

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

核糖体束缚新生多肽的分离<em&gt;在体外</em&gt;确定沿mRNA的翻译暂停网站

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

作为一种工具来隔离核糖体的束缚多肽SECM逮捕序列

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, and multiwell-based assays are used to measure transcription factor activity. However, these assays are nonquantitative, lack specificity, may involve the use of radiolabeled oligonucleotides, and may not be adaptable for the screening of inhibitors of DNA binding. On the other hand, using a quantitative DNA-binding enzyme-linked immunosorbent assay (D-ELISA) assay, we demonstrate nuclear protein interactions with DNA using the RUNX2 transcription factor that depend on specific association with consensus DNA-binding sequences present on biotin-labeled oligonucleotides. Preparation of cells, extraction of nuclear protein, and design of double stranded oligonucleotides are described. Avidin-coated 96-well plates are fixed with alkaline buffer and incubated with nuclear proteins in nucleotide blocking buffer. Following extensive washing of the plates, specific primary antibody and secondary antibody incubations are followed by the addition of horseradish peroxidase substrate and development of the colorimetric reaction. Stop reaction mode or continuous kinetic monitoring were used to quantitatively measure protein interaction with DNA. We discuss appropriate specificity controls, including treatment with non-specific IgG or without protein or primary antibody. Applications of the assay are described including its utility in drug screening and representative positive and negative results are discussed.

We developed a quantitative DNA-binding, ELISA-based assay to measure transcription factor interactions with DNA. High specificity for the RUNX2 protein was achieved with a consensus DNA-recognition oligonucleotide and specific monoclonal antibody. Colorimetric detection with an enzyme-coupled antibody substrate reaction was monitored in real time.

定量检测，以研究蛋白质:DNA的相互作用，发现转录基因表达的调节，并确定新的抗肿瘤剂

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

siRNA的筛选识别泛素和泛素样系统压力表生物学途径在培养的哺乳动物细胞

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&#945; Promoter Using Gibson Assembly

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized plasmids for expression of exogenous genes in mouse embryonic stem cells (mESCs). Expression of exogenous genes under the control of the SV40 or human cytomegalovirus promoters diminishes quickly after transfection into mESCs. A remedy for this diminished expression is to use the human elongation factor-1 alpha (hEF1&#945;) promoter to drive gene expression. Plasmid vectors containing hEF1&#945; are not as widely available as SV40- or CMV-containing plasmids, especially those also containing N-terminal 3xFLAG-tags. The protocol described here is a rapid method to create plasmids expressing FLAG-tagged CstF-64 and CstF-64 mutant under the expressional regulation of the hEF1&#945; promoter. GA uses a blend of DNA exonuclease, DNA polymerase and DNA ligase to make cloning of overlapping ends of DNA fragments possible. Based on the template DNAs we had available, we designed our constructs to be assembled into a single sequence. Our design used four DNA fragments: pcDNA 3.1 vector backbone, hEF1&#945; promoter part 1, hEF1&#945; promoter part 2 (which contained 3xFLAG-tag purchased as a double-stranded synthetic DNA fragment), and either CstF-64 or specific CstF-64 mutant. The sequences of these fragments were uploaded to a primer generation tool to design appropriate PCR primers for generating the DNA fragments. After PCR, DNA fragments were mixed with the vector containing the selective marker and the GA cloning reaction was assembled. Plasmids from individual transformed bacterial colonies were isolated. Initial screen of the plasmids was done by restriction digestion, followed by sequencing. In conclusion, GA allowed us to create customized plasmids for gene expression in 5 days, including construct screens and verification.

Synthesis of custom plasmids is labor and time consuming. This protocol describes the use of Gibson assembly cloning to reduce the work and duration of custom DNA cloning procedure. The protocol described also produces reliable tagged protein constructs for mammalian expression at similar cost to the traditional cut-and-paste DNA cloning.

质粒载体产生表达FLAG标记的蛋白质在人延伸因子1α启动子的使用吉布森大会规则

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized ...

Plant Promoter Analysis: Identification and Characterization of Root Nodule Specific Promoter in the Common Bean

The upstream sequences of gene coding sequences are termed as promoter sequences. Studying the expression patterns of promoters are very significant in understanding the gene regulation and spatiotemporal expression patterns of target genes. On the other hand, it is also critical to establish promoter evaluation tools and genetic transformation techniques that are fast, efficient, and reproducible. In this study, we investigated the spatiotemporal expression pattern of the rhizobial symbiosis-specific nodule inception (NIN) promoter of Phaseolus vulgaris in the transgenic hairy roots. Using plant genome databases and analysis tools we identified, isolated, and cloned the P. vulgaris NIN promoter in a transcriptional fusion to the chimeric reporter &#946;-glucuronidase (GUS) GUS-enhanced::GFP. Further, this protocol describes a rapid and versatile system of genetic transformation in the P. vulgaris using Agrobacterium rhizogenes induced hairy roots. This system generates &#8805;2 cm hairy roots at 10 to 12 days after transformation. Next, we assessed the spatiotemporal expression of NIN promoter in Rhizobium inoculated hairy roots at periodic intervals of post-inoculation. Our results depicted by GUS activity show that the NIN promoter was active during the process of nodulation. Together, the present protocol demonstrates how to identify, isolate, clone, and characterize a plant promoter in the common bean hairy roots. Moreover, this protocol is easy to use in non-specialized laboratories.

Promoter expression analyses are crucial to improving the understanding of gene regulation and the spatiotemporal expression of target genes. Herein we present a protocol to identify, isolate, and clone a plant promoter. Further, we describe the characterization of the nodule-specific promoter in the common bean hairy roots.

植物启动子分析: 菜豆根瘤特异启动子的鉴定与表征

The upstream sequences of gene coding sequences are termed as promoter sequences. Studying the expression patterns of promoters are very significant in ...