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?...

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><tbody><tr><td colspan="4">&lt;strong&gt;COMMERCIAL BIOLOGIC MATERIALS AND KITS:&lt;/strong&gt;</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&#039;-RACE PCR kit</td></tr><tr><td>TOPO TA cloning kit</td><td>Life Technologies</td><td>K450001</td><td>This kit contains the TOP10 chemically competent &lt;em&gt;E. coli&lt;/em&gt; 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&amp;nbsp;</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&amp;rsquo;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 colspan="4">&lt;strong&gt;REAGENTS/CHEMICALS/MEDIA/SUPPLIES&lt;/strong&gt;</td></tr><tr><td>TRIzol</td><td>Life Technologies</td><td>15596026</td><td></td></tr><tr><td>Wizard&amp;reg; SV Gel and PCR Clean-Up System&amp;nbsp;</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 colspan="4">&lt;strong&gt;SOFTWARE:&lt;/strong&gt;</td></tr><tr><td>Ensembl software</td><td>Ensembl project</td><td>http://Ensembl.org&amp;nbsp;</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=&lt;br/&gt; Web&amp;amp;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&#039;-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 colspan="4">&lt;strong&gt;CELL LINES:&lt;/strong&gt;</td></tr><tr><td>TM4 murine Sertoli cells</td><td>ATCC, Manassas, Virginia</td><td>CRL-1715&amp;trade;</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;INSTRUMENTS:&lt;/strong&gt;</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?...

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

Universidad de Cartagena UDEC

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

Promoter Capture Hi-C: High-resolution, Genome-wide Profiling of Promoter Interactions

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the spatio-temporal expression of their target genes through physical contact, often bridging considerable (in some cases hundreds of kilobases) genomic distances and bypassing nearby genes. The human genome harbors an estimated one million enhancers, the vast majority of which have unknown gene targets. Assigning distal regulatory regions to their target genes is thus crucial to understand gene expression control. We developed Promoter Capture Hi-C (PCHi-C) to enable the genome-wide detection of distal promoter-interacting regions (PIRs), for all promoters in a single experiment. In PCHi-C, highly complex Hi-C libraries are specifically enriched for promoter sequences through in-solution hybrid selection with thousands of biotinylated RNA baits complementary to the ends of all promoter-containing restriction fragments. The aim is to then pull-down promoter sequences and their frequent interaction partners such as enhancers and other potential regulatory elements. After high-throughput paired-end sequencing, a statistical test is applied to each promoter-ligated restriction fragment to identify significant PIRs at the restriction fragment level. We have used PCHi-C to generate an atlas of long-range promoter interactions in dozens of human and mouse cell types. These promoter interactome maps have contributed to a greater understanding of mammalian gene expression control by assigning putative regulatory regions to their target genes and revealing preferential spatial promoter-promoter interaction networks. This information also has high relevance to understanding human genetic disease and the identification of potential disease genes, by linking non-coding disease-associated sequence variants in or near control sequences to their target genes.

DNA regulatory elements, such as enhancers, control gene expression by physically contacting target gene promoters, often through long-range chromosomal interactions spanning large genomic distances. Promoter Capture Hi-C (PCHi-C) identifies significant interactions between promoters and distal regions, enabling the assignment of potential regulatory sequences to their target genes.

启动子捕获高分辨率, 全基因组的启动子交互分析

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the ...

科研

遗传学

Inducible and Reversible Dominant-negative (DN) Protein Inhibition

Dominant-negative (DN) protein inhibition is a powerful method to manipulate protein function and offers several advantages over other genome-based approaches. For example, although chimeric and Cre-LoxP targeting strategies have been widely used, the intrinsic limitations of these strategies (i.e., leaky promoter activity, mosaic Cre expression, etc.) have significantly restricted their application. Moreover, a complete deletion of many endogenous genes is embryonically lethal, making it impossible to study gene function in postnatal life. To address these challenges, we have made significant changes to an early genetic engineering protocol and combined a short (transgenic) version of the Rb1 gene with a lysosomal protease procathepsin B (CB), to generate a DN mouse model of Rb1 (CBRb). Due to the presence of a lysosomal protease, the entire CB-RB1 fusion protein and its interacting complex are routed for proteasome-mediated degradation. Moreover, the presence of a tetracycline inducer (rtTA) element in the transgenic construct enables an inducible and reversible regulation of the RB1 protein. The presence of a ubiquitous ROSA-CAG promoter in the CBRb mouse model makes it a useful tool to carry out transient and reversible Rb1 gene ablation and provide researchers a resource for understanding its activity in virtually any cell type where RB1 is expressed.

Inducible and Reversible Dominant-negative DN Protein Inhibition

Here we present a protocol to develop a dominant-negative inducible system, in which any protein can be conditionally inactivated by reversibly overexpressing a dominant-negative mutant version of it.

诱导和可逆显性阴性 (dn) 蛋白抑制

Dominant-negative (DN) protein inhibition is a powerful method to manipulate protein function and offers several advantages over other genome-based ...

In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for reversible and tunable expression control of an endogenous gene of interest in living model systems. The REMOTE-control system employs enhanced lac repression and tet activation systems to achieve down- or upregulation of a target gene within a single biological system. Tight repression can be achieved from repressor binding sites flexibly located far downstream of a transcription start site by inhibiting transcription elongation. Robust upregulation can be attained by enhancing the transcription of an endogenous gene by targeting tet transcriptional activators to the cognate promoter. This reversible and tunable expression control can be applied and withdrawn repeatedly in organisms. The potency and versatility of the system, as demonstrated for endogenous Dnmt1 here, will allow more precise in vivo functional analyses by enabling investigation of gene function at various expression levels and by testing the reversibility of a phenotype.

This protocol outlines the steps needed to generate a model system in which the transcription of an endogenous gene of interest can be conditionally controlled in live animals or cells using enhanced lac repressor and/or tet activator systems.

remtoe 控制系统在内源基因表达调控中的应用

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for ...

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding sites for transcriptional regulators to modulate gene expression. Alterations of CREs have been implicated in various diseases including cancer. While promoters and enhancers have been the primary CREs for studying gene regulation, very little is known about the role of silencer, which is another type of CRE that mediates gene repression. Originally identified as an adaptive immunity system in prokaryotes, CRISPR/Cas9 has been exploited to be a powerful tool for eukaryotic genome editing. Here, we present the use of this technique to delete an intronic silencer in the human RUNX1 gene and investigate the impacts on gene expression in OCI-AML3 leukemic cells. Our approach relies on electroporation-mediated delivery of two preassembled Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complexes to create two double-strand breaks (DSBs) that flank the silencer. Deletions can be readily screened by fragment analysis. Expression analyses of different mRNAs transcribed from alternative promoters help evaluate promoter-dependent effects. This strategy can be used to study other CREs and is particularly suitable for hematopoietic cells, which are often difficult to transfect with plasmid-based methods. The use of a plasmid- and virus-free strategy allows simple and fast assessments of gene regulatory functions.

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

Direct delivery of preassembled Cas9/guide RNA ribonucleoprotein complexes is a fast and efficient means for genome editing in hematopoietic cells. Here, we utilize this approach to delete a RUNX1 intronic silencer and examine the transcriptional responses in OCI-AML3 leukemic cells.

CRISPR/Cas9核糖核酸蛋白在急性骨髓性白血病细胞中的RUNX1电子消光器的转录作用研究

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding ...

研究稀缺细胞中启动子相互作用组的综合方法

An Integrated Workflow to Study the Promoter-Centric Spatio-Temporal Genome Architecture in Scarce Cell Populations

Spatiotemporal gene transcription is tightly regulated by distal regulatory elements, such as enhancers and silencers, which rely on physical proximity with their target gene promoters to control transcription. Although these regulatory elements are easy to identify, their target genes are difficult to predict, since most of them are cell-type specific and may be separated by hundreds of kilobases in the linear genome sequence, skipping over other non-target genes. For several years, Promoter Capture Hi-C (PCHi-C) has been the gold standard for the association of distal regulatory elements to their target genes. However, PCHi-C relies on the availability of millions of cells, prohibiting the study of rare cell populations such as those commonly obtained from primary tissues. To overcome this limitation, low input Capture Hi-C (liCHi-C), a cost-effective and customizable method to identify the repertoire of distal regulatory elements controlling each gene of the genome, has been developed. liCHi-C relies on a similar experimental and computational framework as PCHi-C, but by employing minimal tube changes, modifying&#160;the reagent concentration and volumes, and swapping or eliminating steps, it accounts&#160;for minimal material loss during library construction. Collectively, liCHi-C enables the study of gene regulation and spatiotemporal genome organization in the context of developmental biology and cellular function.

作者聚焦:研究稀缺细胞群中以启动子为中心的时空基因组结构的集成工作流程  

Gene expression is regulated by interactions of gene promoters with distal regulatory elements. Here, we descirbe how low input Capture Hi-C (liCHi-C) allows the identification of these interactions in rare cell types, which were previously unmeasurable.

研究稀缺细胞群中以启动子为中心的时空基因组结构的集成工作流程

Spatiotemporal gene transcription is tightly regulated by distal regulatory elements, such as enhancers and silencers, which rely on physical proximity ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

施加诱导表达系统研究与细胞内信号细菌毒力因子的干扰

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

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Retroviral Overexpression of CXCR4 on Murine B-1a Cells and Adoptive Transfer for Targeted B-1a Cell Migration to the Bone Marrow and IgM Production

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide further insight on cell function. B-1a cells are a unique B cell subset in mice that produce protective natural IgM antibodies against oxidation-specific epitopes that arise during health and disease. B-1a cell IgM production differs depending on B-1a cell location, and therefore it becomes useful from a therapeutic standpoint to target B-1a localization to niches supportive of high antibody production. Here we describe a method to target B-1a cell migration to the bone marrow by retroviral-mediated overexpression of the C-X-C motif chemokine receptor 4 (CXCR4). Gene induction in primary murine B cells can be challenging and typically yields low transfection efficiencies of 10-20% depending on technique. Here we demonstrate that retroviral transduction of primary murine B-1a cells results in 30-40% transduction efficiency. This method utilizes adoptive cell transfer of transduced B-1a cells into B cell-deficient recipient mice so that donor B-1a cell migration and localization can be visualized. This protocol can be modified for other retroviral constructs and can be used in diverse functional assays post-adoptive transfer, including analysis of donor cell or host cell phenotype and function, or analysis of soluble factors secreted post B-1a cell transfer. The use of distinct donor and recipient mice differentiated by CD45.1 and CD45.2 allotype and the presence of a GFP reporter within the retroviral plasmid could also enable detection of donor cells in other, immune-sufficient mouse models containing endogenous B cell populations.

Here we describe a method for retroviral overexpression and adoptive transfer of murine B-1a cells to examine in vivo B-1a cell migration and localization. This protocol can be extended for diverse downstream functional assays including quantification of donor B-1a cell localization or analysis of donor cell-derived secreted factors post-adoptive transfer.

CXCR4在Murine B-1a细胞上的逆转录病毒过度表达和靶向B-1a细胞向骨髓和IgM生产转移的采用转移

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide ...

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.

方法发现小鼠BCRP1的另类用法子和转录调控

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