Name: associated chromosome trap for identifying long-range dna interactions
Uploaded: 2011-04-23T14:00:00
Duration: 14 min 49 s
Description: Watch this Scientific Journal Video about Associated Chromosome Trap for Identifying Long-range DNA Interactions at JoVE.com

我们非常感谢阿德尔Murell和沃尔夫Reik分享他们的3C协议。这项工作是由国防部和退伍军人事务部的研究服务部的支持。

1。甲醛固定远距离的染色质相互作用<ol><li>文化人类细胞株与15％FBS和1个孵化器青霉素/链霉素80-90％汇合的RPMI1640培养基中的HL - 60提供5％的CO 2在37 ° C。 </li><li> 50毫升NUNC管，离心收集到的细胞在15分钟1200转，然后取出介质的吸</li><li>添加5毫升培养液中悬浮细胞沉淀，使用血球计数细胞。到40毫升的RPMI1640 / 10％FBS量取约1 × 10 7细胞，再加入1.7毫升37％的甲醛，修复淡化染色。 </li><li>在室温下孵育10分钟，轻轻摇动，然后用2.4毫升2M甘氨酸解渴。 15分钟1200转的离心机在4 ° C，并去除上清。用40 mL冰冷的PBS洗涤沉淀一次，然后降速沉淀和删除的PBS。 </li></ol> 2。隔离细胞核的细胞裂解<ol><li>悬浮细胞40毫升冰冷的裂解液（10毫米的Tris - HCl，pH8.0，10 mM氯化钠，0.2％NP - 40）与刚加入的蛋白酶抑制剂（1:500稀释）和0.1 mm PMSF。在一个旋转的冷室孵育90分钟，15分钟2500转离心，去除上清。 </li></ol> 3。 用 Bgl II限制性内切酶消化<ol><li>重悬在0.5毫升1 × NEB缓冲区3的原子核，并添加15μlof10％SDS。用颤抖的在37 ° C孵育1小时，然后加入45μl20％的Triton X - 100的的封存SDS。在37℃，1小时用颤抖的孵育。 </li><li>使用1 × 10 6核酶切（约15微克，原始细胞的十分之一）等分。取出55μl核解决方案的步骤3.1和1个，NEB 缓冲的 Bgl II（50U/ul）3和12μL与433μL到500μL 。在37℃过夜孵育。 </li></ol> 4。结扎相互作用的DNA片段<ol><li>限制性内切酶失活，加入95μL10％SDS和变性加热65 ° C时在水浴20分钟。 </li><li>添加7毫升1 ×连接缓冲液（30毫米的Tris - HCl，pH值8.0，MgCl 2的 10毫米，10毫米数码地面电视，1毫米ATP）和20％的Triton X - 100的360μL，在37 ° C，1小时。 </li><li>温度降低到16℃，添加400 U /μLT4 DNA连接酶50μL。样品在16℃孵育4小时，然后在室温下30分钟。 </li></ol> 5。 DNA纯化<ol><li>添加300微克的蛋白酶K，65℃过夜孵育。 </li><li>添加一个，在37 ° C孵育30分钟的5微克的RNase </li><li>净化酚/氯仿抽提，异丙醇沉淀DNA的DNA。在150μL无菌蒸馏水溶解的DNA。 </li></ol> 6。 MSP我的消化和结扎寡核苷酸连接器<ol><li>纯化DNA与5个单位 MSP我为4-6小时，在37 ° C孵育2μg 。灭活MSP我在65 ° C 10分钟，然后在乙醇沉淀的DNA与1μL5 mg / ml的糖原。在50μL无菌水溶解DNA沉淀。 </li><li>混合2μL20微米的连接寡核苷酸大号50μLMSP我处理过的DNA（5&#39; - gctgaccctgaattcgcacgtgcctgtcgttagcggacacagggcgattcac - 3&#39;），1μl的20微米寡核苷酸S（5&#39; - cggtgaatc - 3&#39;），1 μL无菌蒸馏水和6 10 × T4 DNA连接酶缓冲液。盖蜡液的混合物。 </li><li>变性寡核苷酸在50℃，1分钟，并允许逐步降温至10℃，0.5℃/分钟的梯度在热循环仪。 </li><li>添加1 400 U /μLT4 DNA连接酶液，并在15℃过夜孵育。净化的连接器结扎DNA，使用QIAquick PCR纯化试剂盒，并在50μL无菌蒸馏水洗脱。 </li></ol> 7。 PCR扩增和序列分析<ol><li>选择一个特定的DNA，要进行检查长程相互作用（即“诱饵”）地区的引物。在这个例子中，我们使用ABL - 1。 </li><li>取1μL纯化的DNA进行的第一轮PCR使用1μL20微米-1 ABL的具体底漆＃4656（5&#39; - gttcaagcgattctcctgcctcga - 3&#39;），1μL20微米的连接器的特异性引物＃2963（ 5&#39; - gctgaccctgaattcgcacgtgcct - 3&#39;），3μl3 × Klen的Taq DNA聚合酶我鸡尾酒会和3μL无菌蒸馏水。 32个P - dCTP液加入到100μL3 × Klen的Taq DNA聚合酶PCR扩增前我鸡尾酒。热启动PCR的热循环72 ° C为2分钟，95℃保温2分钟; 18周期为95 ° C，20秒，65 ° C下40秒，和72℃1分钟;最后延伸72℃5分钟。 </li><li>净化第一轮PCR产物使用无菌蒸馏水在30μL一个QIAquick PCR纯化试剂盒，和洗脱DNA。 </li><li>取1μL稀释后的第一次PCR产物加入1μl20微米的 ABL - 1＃4626特异性引物（5&#39; - ggagaatttttatctgcctctgtga - 3&#39;）（图1A），1μL，以执行第二轮的使用嵌套引物PCR的100 ×连接器的具体底漆＃2961（5&#39; - gtcgttagcggacacagggcgattc - 3&#39;），3 × Klen的Taq DNA聚合酶我鸡尾酒会和3μL无菌蒸馏水3μL20微米。热循环时间表是25个周期为95 ° C，20秒，67 ° C下40秒，和72℃1分钟，随后在72℃延伸5分钟。 </li><li>可视化运行5％的尿素- PAGE凝胶扫描暴露在PhosphoImager屏幕（图1b）的PCR产物。每个PCR波段可循环使用的凝胶的Eppendorf管含60μL无菌蒸馏水溶解的凝胶带，并培育在95℃5分钟。短暂离心收集的所有样本的10秒10,000 RPM。取出1μLDNA为模板进行PCR引物，在相同条件下，如上所述4626分之2961。可以使用QIAquick PCR纯化试剂盒纯化后进行序列分析。 </li><li> DNA序列分析，使用了一个在线工具，以确定其染色体定位的网站：http://genome.ucsc.edu（图1c）。点击“BLAT”进入了一个新的窗口，它允许粘贴DNA序列，点击“提交”后，出现一个新窗口，并显示“BLAT”搜索结果。点击“浏览器”命中100％的身份进入下一个窗口，显示的DNA序列的位置。 </li></ol> 8。代表性的成果 1。使检测ABL - 1区域为诱饵，以确定其远距离DNA的相互作用正如图1a中，两个经Bgl II网站，并用BamH I网站所示的ACT检测选择。在第二轮的PCR引物2961分之4​​626是用来放大ABL - M1，二千九百六十一分之四千六百三十零是用于ABL - M2的，2961分之4​​636ABL - M3。一个典型的凝胶模式显示几个乐队之一（图1b）。每个片段从行为分析包括两个结合的DNA片段：一个部分是来自诱饵DNA区域，而第二部分是从相关的合作伙伴，这是第一限制性内切酶识别序列诱饵地区分部加入。第二种酶识别序列会出现在相关的合作伙伴序列（图1c）结束。 ABL - M1“克隆片段中包含ABL1的区域位于染色体9q32.4从+133,592,306-133,592,399的DNA，和相关的合作伙伴是位于染色体3p13从+71,869,882-71,870,107。相关的合作伙伴的身份，发现爆破使用序列UCSC基因组浏览器（GRCh7/hg19在2月份发布，2009年）。PROK2 chr3p13轨迹相关的合作伙伴。 同样，ABL - M2的相关合作伙伴的本地化，以chr5q21.1，而克隆ABL - M3附近ABL - 1位点的染色体内协会确定。 2。确定普遍的远距离交互使用的ACT检测基于对3C的其他化验报告更互动，比我们在图1和IGF2 / H19轨迹12所示的许多合作伙伴。我们提出的方法，将选择最流行的长程相互作用。但是，通过增加PCR循环数，以及确定额外的，那么频繁协会（图2）。 3。与正常组织相比，在癌细胞中的长程相互作用的差异。 ACT的检测也可以被用来确定在核架构之间的差异和正常细胞和癌细胞（图3）长程相互作用。这些差异可能反映了核结构的变化，细胞转化过程中出现的，因此这个实验最终可能会被用于诊断目的。在正常和癌组织中出现类似凝胶的模式表明，该法的检测是可靠的和可重复的。虽然ACT检测可以识别长距离DNA的合作伙伴，进一步分析，如鱼和芯片检测，需要验证之间遥远的基因位点的确定协会的存在。遗传，生理生化研究，然后可以进行澄清这些远距离DNA协会的生物影响。 <img src="/files/ftp_upload/2621/2621fig1.jpg" alt="图1" /> 图1法检测使用ABL - 1区域在HL - 60细胞DNA为诱饵 。 A. DNA的结构茜在ABL - 1地区。第一限制性内切酶，使检测中使用的是经Bgl II。第二轮的PCR引物上，也有箭头和相应的引物号码。 B.凝胶格局在5％的尿素页的ACT检测。二千九百六十一分之四千六百三十零，对引物2961分之4​​626的巢式PCR扩增克隆ABL - M1“，是用于克隆ABL - M2的，2961分之4​​636克隆ABL - M3。 C. DNA序列的克隆ABL - M1。 ABL - 1诱饵DNA片段的DNA标记为红色，相关的DNA标记的合作伙伴是青色。，经 Bgl II网站（AGACTC）绿色标记， 和 MSP我的网站（CCGG）是在紫色的标记。 <img src="/files/ftp_upload/2621/2621fig2.jpg" alt="图2" /> 图2 PCR循环影响ACT检测结果。Igf2/H19为诱饵，在小鼠成纤维细胞的DNA位点的印记控制区（ICR），不同的循环方案被应用在PCR的第一和第二轮的ACT检测。 18日至20日在第一轮的PCR周期没有足够的信号进行可视化放大。二十五个周期在第二次PCR结果清晰的条带，而第二轮的PCR循环次数增加导致涂抹除了更多的波段模式。 <img src="/files/ftp_upload/2621/2621fig3.jpg" alt="图3" /> 图3核架构之间的差异使检测正常结肠组织和结肠癌组织的检测。 A. IGF2/H19轨迹和IGF2基因的DNA结构的ICR地区。DPN II ACT的检测网站的标记。在第二轮PCR引物标记由不同颜色的箭头和数字，对应于每个面板图的b巷。 B.凝胶格局在5％的尿素页的ACT检测。正常结肠组织MAD03 - 1423和结肠癌组织MAD04 - 149获得合作人体组织网络（CHTN）西部分部。每个结肠组织匀浆后，使测定按照本文所述的程序执行。巷1代表使用引物＃​​2961and＃4161（5&#39; - tctgcgccatcagggcagtgagac - 3&#39;）在粉红色标记面板的PCR结果;巷2表示使用引物＃​​2961和＃4163（5&#39; - gccgcgcggccacttccgattcc - 3的PCR结果“）在面板上标有橙色的; 3巷代表的PCR结果用引物对＃2961和＃5145（5&#39; - gccatgcaggtaggatttgagctg - 3”）在蓝色标记面板; 4巷代表的PCR结果用引物对＃2961 ＃5151（5&#39; - gtctcaaataggggccagctagcttgg - 3&#39;）在绿色标记面板A.黄色箭头标记出现在正常结肠组织中唯一的乐队，和那些只出现在结肠癌组织的乐队，是在红色箭头标记。 ICR的，印记控制区; DMR的，不同的甲基化区域。

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德克尔等人发展的染色体构象捕获（3C）的方法来检测两个1的基因位点之间的互动频率，并已广泛用于3C 调查 2哺乳动物细胞中的两个已知的DNA区域内的染色体和染色体间协会-9。虽然新开发的高- C的方法可用于对全基因组DNA协会的研究应用，使检测位点特异性DNA相互作用10-11的研究仍然是一个有效的技术。我们已经修改了这种方法，识别未知的DNA，在培养的小鼠和人类细胞（图1）已知的DNA区域关联区域。我们命名这个方法相关的染色体陷阱（ACT）的检测，因为它为我们提供了一个可靠和可重复性的方法来识别的一种新的未知的DNA伙伴，与已知的目标DNA区域的12副。一个成功的3C检测与适当的控制是在执行小说方面的ACT检测13之前执行。 tofind为了尽可能多的相关的DNA区域，它是需要使用的各种组合的第一个和第二个限制性内切酶。尤其重要的是使用限制性内切酶，CpG甲基化不敏感执行的第一个3C结扎步骤。蛋白结合和DNA甲基化也可以影响酶切效率，并可能导致对某些限制性内切酶消化相关的DNA区域结扎失败。发生染色体内或跨结扎取决于蛋白质- DNA交联和相关的DNA区域的适当的物理图谱。因此，一些初步的实验，都必须建立在ACT检测一个切实可行的有效的甲醛浓度和处理时间。终浓度的甲醛（from1.5％至2％）的范围已被用于在几个实验室在实验 7,9 3C部分。另外，作为连接器所使用的寡核苷酸，可设计相匹配的第二个限制性内切酶切割的凝聚力结束。虽然我们发现，18日至20日在第一轮PCR和20-25周期，在第二轮的PCR循环，可以提供清晰的条带，它是要建立每个实验（图2）最佳的PCR条件，内部分子之间的5&#39; -端和3&#39; -末端的DNA链可能会发生在PCR的互补适配器序列退火，它能够抑制适配器特异性引物退火的DNA分子，扩增效率低得多，导致前几个周期。循环的扩增目标DNA后，其数额可能远远比这些非特异性反应，并可能有利于引物退火的目标DNA分子的竞争。这也是为什么我们可以看到背景放大，为何第一轮PCR产物进行稀释，以减少背景amplification.It重要的是从PCR反应中删除多余的引物，以减少在第二轮PCR的背景。在所有基于PCR的实验，这是至关重要的，设计引物中不重复序列的区域，这构成了大多数人类和小鼠的DNA位于。虽然新开发的高- C的方法可用于对全基因组DNA协会的研究应用，使检测位点特异性的DNA相互作用的研究仍然是一个有效的技术。

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>RPMI1640 medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>22400-105</td>
<td>&nbsp;</td>
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<td>acrylamide</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15512-023</td>
<td>&nbsp;</td>
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<td>ATP solution, 10mM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>AM8110G</td>
<td>&nbsp;</td>
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<td>fetal bovine serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16000-044</td>
<td>&nbsp;</td>
<tr>
<td>penicillin-streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>1M Tris pH8.0</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>AM9856</td>
<td>&nbsp;</td>
<tr>
<td>RNase A</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12091-039</td>
<td>&nbsp;</td>
<tr>
<td>SDS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15525-017</td>
<td>&nbsp;</td>
<tr>
<td>urea</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15505-035</td>
<td>&nbsp;</td>
<tr>
<td>BamH I</td>
<td>&nbsp;</td>
<td>NEB Biolabs</td>
<td>R0136T</td>
<td>&nbsp;</td>
<tr>
<td>Bgl II</td>
<td>&nbsp;</td>
<td>NEB Biolabs</td>
<td>R0144M</td>
<td>&nbsp;</td>
<tr>
<td>Dpn II</td>
<td>&nbsp;</td>
<td>NEB Biolabs</td>
<td>R0543T</td>
<td>&nbsp;</td>
<tr>
<td>Msp I</td>
<td>&nbsp;</td>
<td>NEB Biolabs</td>
<td>R0106S</td>
<td>&nbsp;</td>
<tr>
<td>dNTPs</td>
<td>&nbsp;</td>
<td>NEB Biolabs</td>
<td>N0447L</td>
<td>&nbsp;</td>
<tr>
<td>proteinase K</td>
<td>&nbsp;</td>
<td>NEB Biolabs</td>
<td>P8102S</td>
<td>&nbsp;</td>
<tr>
<td>T4 DNA ligase</td>
<td>&nbsp;</td>
<td>NEB Biolabs</td>
<td>M0202T</td>
<td>&nbsp;</td>
<tr>
<td>37% formaldehyde</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>F8775</td>
<td>&nbsp;</td>
<tr>
<td>Bis-acrylamide</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>146072</td>
<td>&nbsp;</td>
<tr>
<td>dithiothreitol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>43815</td>
<td>&nbsp;</td>
<tr>
<td>glycine</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>50046</td>
<td>&nbsp;</td>
<tr>
<td>PMSF</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>93482</td>
<td>&nbsp;</td>
<tr>
<td>proteinase inhibitor</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S8830</td>
<td>&nbsp;</td>
<tr>
<td>Nonidet P-40</td>
<td>&nbsp;</td>
<td>Roche Applied Science</td>
<td>11754599001</td>
<td>&nbsp;</td>
<tr>
<td>KlenTaq1</td>
<td>&nbsp;</td>
<td>Ab peptides</td>
<td>1001</td>
<td>&nbsp;</td>
<tr>
<td>dCTP alpha P32</td>
<td>&nbsp;</td>
<td>PerkinElmer</td>
<td>BLU513H250UC</td>
<td>&nbsp;</td>
<tr>
<td>PTC-100 Thermal Cycler</td>
<td>&nbsp;</td>
<td>MJ Research</td>
<td>mjptc100</td>
<td>&nbsp;</td>
<tr>
<td>Power Supply</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>164-5056</td>
<td>&nbsp;</td>
<tr>
<td>OmniPAGE Maxi</td>
<td>&nbsp;</td>
<td>Aurogene Life Science</td>
<td>VS20D</td>
<td>&nbsp;</td>
<tr>
<td>Typhoon 9400</td>
<td>&nbsp;</td>
<td>GE Healthcare </td>
<td>63-0055-78</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

associated chromosome trap for identifying long-range dna interactions

由DNA编码的遗传信息是在一个复杂和高度管制的染色质结构的组织。每个染色体上占据一个特定的领土，这可能会改变的发展阶段或细胞周期。基因表达可出现在专门的转录工厂染色段可能从不同的染色体领土循环，导致合作本地化的DNA片段上可能存在不同的染色体或同一染色体上相距甚远。相关的染色体陷阱（ACT）的检测提供了一种有效的方法来识别这些偏见地远射DNA协会通过扩展和修改的染色体构象捕获技术。 ACT检测使得我们有可能调查的反转录调控机制，可以帮助解释核架构的关系，在正常的生理和在疾病状态下基因表达。

Watch this Scientific Journal Video about Associated Chromosome Trap for Identifying Long-range DNA Interactions at JoVE.com

Associated Chromosome Trap for Identifying Long-range DNA Interactions

相关的染色体陷阱（ACT）的分析是用于识别远射DNA相互作用的一种新的不带偏见的方法。远距离DNA相互作用的特性，将使我们在正常的生理和患病状态，以确定核架构的关系，基因的表达。

确定远距离的DNA相互作用的相关染色体陷阱

Genetic information encoded by DNA is organized in a complex and highly regulated chromatin structure.  Each chromosome occupies a specific territory, ...

associated-chromosome-trap-for-identifying-long-range-dna-interactions

Research

JoVE Journal

Biology

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

基于分体式泛素膜酵母双杂交（神话）系统：一个强大的工具用于识别蛋白质 - 蛋白质相互作用

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

科研

生物学

Preparation of Drosophila Polytene Chromosome Squashes for Antibody Labeling

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological advantages provided by the giant salivary gland polytene chromosomes of third instar larvae.  In this tissue the chromosomes undergo many rounds of replication in the absence of cell division giving rise to approximately 1000 copies.  The DNA remains aligned after each replicative cycle resulting in greatly enlarged chromosomes that provide a unique opportunity to correlate chromatin morphology with the localization of specific proteins.  Consequently, there has been a high level of interest in defining the epigenetic modifications present at different genes and at different stages of the transcription process. An important tool for such studies is the labeling of polytene chromosomes with antibodies to the enzyme, transcription factor, or histone modification of interest. This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

果蝇聚乙烯南瓜抗体标记染色体的制备

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological ...

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.

We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.

测量细菌细胞的抗弯刚度，使用光学陷阱

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue1,2. GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (Gi and Go) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K+ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate.
GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders3. However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations3.
Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K+ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K+ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry4 or radiotracer analysis5, the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.

A real-time screening procedure for identifying drugs that interact with G protein-gated inward rectifier K+ (GIRK) channels is described. The assay utilizes membrane potential-sensitive fluorescent dyes to measure GIRK channel activity. This technique is adaptable for use on a number of cell lines.

一个荧光识别GIRK通道调制器的筛选试验

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

结合单分子操纵和成像蛋白-DNA相互作用的研究

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) allows the detection of nucleic acids in their native environment within cells. We here describe a protocol for the combined, simultaneous detection of RNA and DNA by means of FISH, which can be used to study X chromosome inactivation in mouse embryonic stem cells.

结合DNA-RNA荧光<em&gt;原位</em&gt;杂交（FISH）研究X染色体失活的分化雌​​性小鼠胚胎干细胞

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

染色质免疫沉淀拟南芥蛋白-DNA相互作用的鉴定<em&gt;在体内</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is an alternative approach to chromatin immunoprecipitation (ChIP). An expressed fusion protein consisting of the protein of interest and the E. coli DNA adenine methyltransferase can methylate the adenine base in GATC motifs near the sites of protein-DNA interactions. Adenine-methylated DNA fragments can then be specifically amplified and detected. The original DamID assay detects the genomic locations of methylated DNA fragments by hybridization to DNA microarrays, which is limited by the availability of microarrays and the density of predetermined probes. In this paper, we report the detailed protocol of integrating high throughput DNA sequencing into DamID (DamID-seq). The large number of short reads generated from DamID-seq enables detecting and localizing protein-DNA interactions genome-wide with high precision and sensitivity. We have used the DamID-seq assay to study genome-nuclear lamina (NL) interactions in mammalian cells, and have noticed that DamID-seq provides a high resolution and a wide dynamic range in detecting genome-NL interactions. The DamID-seq approach enables probing NL associations within gene structures and allows comparing genome-NL interaction maps with other functional genomic data, such as ChIP-seq and RNA-seq.

We describe herein an assay by coupling DNA adenine methyltransferase identification (DamID) to high throughput sequencing (DamID-seq). This improved method provides a higher resolution and a wider dynamic range, and allows analyzing DamID-seq data in conjunction with other high throughput sequencing data such as ChIP-seq, RNA-seq, etc.

DamID-SEQ：蛋白质-DNA相互作用的全基因组映射腺嘌呤甲基化的DNA片段通过高通量测序

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

逆转录病毒整合位点的扩增，新一代测序和基因组DNA制图

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...