技术援助和马里兰大学Greenebaum癌症中心转化核心设施，尤其是博士的仪器。瑞纳拉皮迪和MARIOLA Sadowska，表示诚挚的谢意。负责该实验的发展工作是由美国国立卫生研究院RO1CA108846，AHA格兰特在急救GRNT2130014，一个VA优异奖美联社报道，由马里兰州卷烟归还资金提供给马琳和斯图尔特大学（CRF）的部分资助Greenebaum癌症中心。

的时间被执行几个步骤前进，几个试剂制备和储存的过程之前：（1）细胞培养和蛋白质分离，（2）制备的寡核苷酸，（3）制备的96孔板，（4）核提取孵育过夜。程序要求，因为核蛋白质与DNA寡核苷酸孵育过夜2天。 1。缓冲液的制备 1.1核蛋白分离<ol><li>低渗缓冲液：要制备100ml的低渗缓冲液中，加入1 ml（1M的HEPES，pH值7.9），150微升（1μM的MgCl 2）中，333微升（3M KCl）中，以98.3毫升H 2 O。终浓度：10毫米肝素钠，1.5毫米氯化镁2，10毫米氯化钾。对于低渗缓冲液+ NP40（在步骤2.11使用）：加入0.5毫升（10％NP40），以9.5毫升低渗缓冲液进行最后的0.5％NP40。 </li><li>低盐缓冲液：要制备100ml低盐缓冲液（在步骤2.15使用），加入1 ml（1M的HEPES，pH值7.9），25米升甘油，加入150μl（1μM的MgCl 2）中，666微升（3M KCl）中，加入40μl（0.5M EDTA）至73毫升水2 O。终浓度：10毫米的HEPES，25％甘油，1.5毫米氯化镁2，20毫米氯化钾，0.2毫米EDTA。 </li><li>高盐缓冲液：要制备100ml高盐缓冲液（用于在步骤2.15使用），加入1 ml（1M的HEPES，pH值7.9），将25ml甘油，加入150μl（1μM的MgCl 2）中，26.7毫升（3M KCl）中，加入40μl（0.5M EDTA）的47毫升H 2 O。终浓度：10毫米的HEPES，25％甘油，1.5毫米氯化镁2，800毫米氯化钾，0.2毫米EDTA。 </li><li> 10％NP40洗涤剂：10毫升NP40〜90毫升水2 O </li><li>蛋白酶和磷酸酶抑制剂[加入低渗缓冲液+ NP40（步骤2.11），以低盐缓冲液（步骤2.15），并以高盐缓冲液（步骤2.15），在使用前才]：添加2.5微升（1 M DTT）和50微升蛋白酶抑制剂（100倍）的至5ml每个缓冲区为0.5mM的DTT和1x蛋白酶抑制剂的最终浓度。蛋白酶抑制剂100X库存混合物包括：氟化钠（丝氨酸/苏氨酸，酸性磷酸酶），原钒酸钠（酪氨酸，碱性磷酸酯酶），β-磷酸甘油（丝氨酸/苏氨酸磷酸酶），焦磷酸钠（丝氨酸/苏氨酸磷酸酶），抑酶肽（丝氨酸蛋白酶），苯丁抑制素（氨基肽酶），E64（半胱氨酸蛋白酶），亮抑酶肽（丝氨酸/半胱氨酸蛋白酶），EDTA（金属蛋白酶）。 </li></ol> 1.2制备测定板的<ol><li>板固定的解决方案：要准备500ml溶液，加5.25克碳酸钠至500ml H 2 O，拌匀，调节pH值至9.7为0.1M的碳酸钠的终浓度。 </li><li>板封闭液：制备聚股票的dI / DC寡核苷酸，重悬1单位（〜50毫克），聚DI / DC在1毫升10毫米的Tris /盐酸，含1毫米EDTA的股票浓度为50微克pH值7.9 /微升。股票稀释到1微克/使用微升前。 </li></ol> 1.3洗涤缓冲液和抗体稀释液注：链霉亲和洗涤缓冲液是<html>用于在添加之间洗涤板并以稀释初级和次级抗体。 <ol><li>以制备1升的链霉亲和洗涤缓冲液中，添加2.4克的Tris /盐酸，37.5毫升（4M NaCl）中，将10毫升（10％BSA）中，加入5ml（10％的Tween-20），以1升Millipore公司的无菌过滤的水，pH值为7.18。贮存于4℃。终浓度：20毫摩尔Tris /盐酸，150 mM氯化钠，0.1％BSA，0.05％Tween-20的。 </li></ol> 1.4 DNA结合缓冲液注意：这个缓冲区存放于-20℃。 <ol><li>为了制备将15ml DNA结合缓冲液中，添加180微升（1M的HEPES，pH值7.9），300微升（3M KCl）中，12微升（0.5M EDTA，pH值8.0），7.5微升（1 M DTT），3.0毫升（60％甘油）中，加入300μl（50微克/微升聚的dI / DC）稀释用的去离子/蒸馏水H 2 O的（12毫升）中。终浓度：12mM的HEPES / pH值7.9，60mM的氯化钾，0.4mM的EDTA，0.5mM的DTT，12％甘油，1微克/微升聚的dI / DC。 </li></ol> 2。细胞培养和核蛋白质分​​离T“&gt;注：编制，需要约3小时细胞的刺激将取决于实验中使用的每一个实验的细胞数量会有所不同，所有卷反映了一个典型的实验中使用3×10 7细胞/点调整量，以适应。在实验6中实际使用的细胞数目。 <ol><li>为了准备核蛋白质的DNA结合实验，培养人细胞表达RUNX2（内皮细胞，骨肉瘤，乳腺癌细胞）在合适的介质6。 </li><li>治疗会合细胞与噻氨酯哒唑（0.2微克/毫升16小时）逮捕细胞在G2 / M期细胞周期边界6。在这些条件下，本RUNX2蛋白是稳定的和最大的DNA结合得以实现。以这种方式，为多个测定和抑制剂可从同一制剂相比足够核蛋白是可用的。 </li><li>对于每个步骤，预冷却离心机4℃，制备缓冲液和细胞收获前放松20分钟，在4℃下。</li><li>寒冷细胞（4℃）和在冰上进行的程序。 </li><li>离心细胞以1,000 rpm离心5分钟在15毫升聚丙烯圆底试管中。 </li><li>洗1X与冰冷的PBS（的Ca 2 +和Mg 2 +免费）。 </li><li>再次离心，弃PBS上清液。 </li><li>加入500μl低渗缓冲的无蛋白酶抑制剂，小心翼翼地完全重悬细胞沉淀。 </li><li>传送内容到1.5ml的Eppendorf管中。 </li><li>立时离心机在5,500转5.5分钟，弃去上清液，并保持细胞沉淀。 </li><li>重悬细胞沉淀于500μl低渗缓冲液中含有0.5％NP40的。添加蛋白酶和磷酸酶抑制剂和0.5mM DTT（如在步骤1.1.5中描述）。 </li><li>使样品休息在冰上30分钟。 </li><li>离心机以13,000 rpm离心10分钟。 </li><li>弃上清（含有胞质蛋白）。 </li><li>重悬细胞核沉淀在每个低SAL的60微升吨缓冲液（向其中蛋白酶抑制剂和0.5mM DTT已经加入从步骤1.1.5）和高盐缓冲液（向其中蛋白酶抑制剂和0.5mM DTT已经加入从步骤1.1.5），总共120微升。加60微升低盐缓冲液中的沉淀物的第一和重悬，然后加入60微升的高盐缓冲液中。 VORTEX颗粒的悬浮帮助。 </li></ol>注：此步骤有助于摆脱一些核膜蛋白和设立裂解步骤：低盐第一（重悬沉淀，涡旋），然后高盐（涡流）优化这一步骤。保持提取物置于冰上30分钟，在第一10分钟后涡旋。 <ol start="16"><li>离心机以12,000 rpm离心15分钟;保持含有核蛋白质的上清液。 </li><li>测量蛋白质浓度（尽量保持样品在2-5毫克/毫升），分装在合适的量（10μL/管），并储存在-80°C。 </li></ol>注：预计产量：30×10 6 CELLS将产生5微克/微升蛋白120微升。 5×10 6个细胞将产生2.2微克/微升的蛋白质在40微升。 3。的双链寡核苷酸的制备<ol><li>设计互补的寡核苷酸与兼容的半位点上的两端。为RUNX2这些是：5&#39;-CGTATT AACCACA ATACTCGCGTATT AACCACA ATACTCGCGTATT AACCACA ATAC TCG-3&#39;-生物素（有义）和5&#39;CGAGTAT TGTGGTT AATACGCGAGTAT TGTGGTT AATACGCGAGTATŦGTGGTT AATACG-3&#39;-生物素（反义）。 </li></ol>注：确定有三种RUNX2结合位点和单端标记生物素产生的最重复性的结果试点实验。 <ol start="2"><li>准备5X退火缓冲液：加入300微升（1米的Tris-HCl，pH值为7.6），30微升（1米氯化镁2），10微升（1 M DTT），以260微升H 2 O的（最终600微升）。 </li><li>到200皮摩尔各单链OLIG的onucleotide（3微升），加入12微升的5倍退火缓冲液和45微升H 2 O的共60微升（200 pmol/60微升）。 </li><li>热在加热块95℃下5-10分钟。 </li><li>冷却至65℃，缓慢地（通过温度设定为65℃时，需要15分钟），然后冷却至室温缓慢地通过关闭加热块（或通过放置在50毫升的65℃的水的动力在冰的烧杯中，这需要15-20分钟）。 </li></ol> 4。 96孔板准备注意：不要用牛奶蛋白质的洗涤缓冲液。 <ol><li>固定板与固定溶液（0.1M碳酸钠）：加300微升/孔。 </li><li>通过摇摆在室温（RT），或在4°C（O / N）无摇动孵育2小时。 </li><li>与链霉亲和洗涤缓冲液（WB）洗板3次：每次加300微升/孔。 </li><li>添加生物素标记的双链寡核苷酸（每3个核苷酸共有结合位点），2小时瓦特第i个摆动（1.25纳摩尔/孔;100μl/孔）。 </li><li>洗涤3次用冷WB：加300微升/孔，然后立即颠倒板放入一个水槽和印迹在纸巾上的边缘每次加入后。 </li></ol>注意：不要使用移液器吸头从板块中删除流体，因为这可能会刮抗生物素蛋白：生物素：从井DNA复合物。这样做对所有后续的洗涤步骤。 5。孵化与核提取物注意：请不要用鲑鱼或鲱鱼精DNA的聚DI / DC阻断缓冲区。避免阻塞板与牛奶蛋白质。这两种封闭剂的导致高背景值。 <ol><li>孵育核提取物（90微升/孔）制备特异性转录因子。制备含核提取物的母液（DNA结合蛋白; 3-9微克/孔）+聚DI / DC在1x的DNA（1微克/微升从50微克/微升股票）结合缓冲液。体积根据需要为总数所需的井（90微升/孔）。 </li><li>任何潜在的抑制剂，如维生素D 3（ 图2）或CADD化合物5221975（ 图3），或者适当的稀释溶剂对照，如乙醇，在此步骤中加入。 </li><li>在摇摆平台，O / N在4°C的地方板</li><li>洗3次与巴菲特：加入300微升/孔</li></ol> 6。另外一抗注：一抗稀释液应新鲜配制 - 存储是不推荐。 <ol><li>稀RUNX2特异性单克隆抗体（股票1微克/微升）的1/5，在链霉亲和000洗缓冲区。准备足够的用于添加到96孔平板（90微升/孔）以一式三份的每个孔中。 </li><li>孵育1小时，在室温下在摇动平台上。 </li><li>洗3次与世行，加300微升/孔。 </li></ol> 7。此外二级抗体注：二级抗体稀释液可以是申通快递红色，在4℃过夜，如果需要的第二天。 <ol><li>稀F AB-特异性亲和纯化，HRP标记抗体（7.1 mg / ml的股票），以1:1,400的链霉亲和清洗缓冲液：3.5微升antibody/5.0毫升洗涤缓冲液。准备足够的用于添加到96孔平板（90微升/孔）以一式三份的每个孔中。 </li><li>孵育在室温下在摇动平台上30分钟。 </li><li>洗涤6次以WB由反相板放入水槽（300微升/孔）。 </li></ol> 8。 HRP基质及产品开发<ol><li>从股票瓶加入50微升TMB底物，每孔直接。 </li><li>在黑暗中孵育10-20分钟，在室温（停止反应法）或直接测量吸光度（连续动力学监测）。 </li></ol> 9。反应测量<ol><li>停止反应的方法：对于Visual停止反应，检查从清晰的彩色变为蓝色，并停止与50微升硫酸反应。 </li><li>测量450nm处的吸光度与该Biotrak二可见酶标仪分光光度计（Amersham Biosciences社制; GE医疗集团生物科技股份有限公司皮斯卡塔韦NJ，USA）或类似的仪器。 </li><li>连续动力学监测方法：在最后一次洗涤后，加入底物，只是在此之前放置在分光光度计（不停止反应）。 </li><li>测量吸光度在635 nm处的生物康协同HT多反应酶标仪分光光度计（BIO-Tek仪器，公司，威努斯基，VT，USA）或类似的仪器。 </li></ol>

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我们什么都没有透露。

DNA结合测定法来测量的转录因子与DNA的相互作用的能力。检测DNA结合包括电泳迁移率（EMSA）1和染色质immuneprecipitation（芯片）为基础的分析3以及检测采用96孔格式4，如化学发光检测2。该EMSA是非定量和使用放射性标记（32 P）的寡核苷酸。这些孵育核蛋白质和结合复合体上分离的琼脂糖或聚丙烯酰胺凝胶。与此相反，定量的D-ELISA是能够测量对应于在RUNX2靶?...

DNA结合分析在测量转录因子与DNA相互作用的能力效用。检测DNA结合包括电泳迁移率变动分析（EMSA）依赖于放射性标记的寡核苷酸1或化学发光检测2。染色质immuneprecipitation（芯片）为基础的分析3以及测定使用96孔格式4也被描述。然而，EMSA是一个非定量测定要求使用放射性标记的寡核苷酸。当核的蛋白质相关联的特定核苷酸序列的启动子，结合复合体是滞后于聚丙烯酰胺凝胶和特定的转录因子可与抗体“超迁移”进行验证。我们已经开发出利用酶联免疫格式（D-ELISA），它能够测量与对应于所定义的启动子元件的DNA结合序列，RUNX2的相互作用的定量DNA结合测定法我Ñ​​RUNX2靶基因。使用防RUNX2抗体提供了特异性的检测和缺乏放射性标记的从传统的凝胶迁移实验5分辨这个实验。检测结合复合体是可能的使用结合到辣根过氧化物酶（HRP），它的HRP底物四甲基联苯胺（TMB）转化成有色产物进行分光光度分析的二次抗体。本文报道的测定可以合并使用突变的DNA的寡核苷酸作为对照，并可以用于检测的DNA的竞争性或非竞争性抑制剂结合。新颖的抗肿瘤化合物的筛选，也可以用此试验。

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		<tr>
			<td>Name of the Reagent</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Poly dI/dC</td>
			<td>GE Healthcare, Piscataway, NJ</td>
			<td>US20539-5UN</td>
			<td>1 U ~50mg</td>
		</tr>
		<tr>
			<td>RUNX2 antibody</td>
			<td>MBL International Corp., Woburn, MA</td>
			<td>D130-3</td>
			<td>1 mg/ml</td>
		</tr>
		<tr>
			<td>Fab-specific peroxidase conjugated antibody</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>A9917</td>
			<td>7.1 mg/ml</td>
		</tr>
		<tr>
			<td>TMB Substrate (tetramethyl benzidine)</td>
			<td>EXALPHA Biologicals, Shirley, MA</td>
			<td>X1189S</td>
			<td>100 ml</td>
		</tr>
		<tr>
			<td>Sodium carbonate</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>57995</td>
			<td>Plate-fixing</td>
		</tr>
		<tr>
			<td>Sulfuric Acid</td>
			<td>VWR, West Chester, PA</td>
			<td>BDH-39922-1</td>
			<td>Stop solution</td>
		</tr>
		<tr>
			<td>Multi-well plates</td>
			<td>Greiner Bio-One, Basel, Switzerland</td>
			<td>655996</td>
			<td>Avidin-coated, black sides</td>
		</tr>
		<tr>
			<td>HALT</td>
			<td>Thermo-Scientific/Pierce, Rockford, IL</td>
			<td>78440</td>
			<td>Protease and phosphatase inhibitors</td>
		</tr>
		<tr>
			<td>Chemicals</td>
			<td>Various manufacturers</td>
			<td>Laboratory grade</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Table 1. Reagents</td>
		</tr>
		<tr>
			<td>Spectrophotometer: Biotrak II Visible plate reader</td>
			<td>Amersham Biosciences</td>
			<td>&nbsp;</td>
			<td>For use with stop reaction method</td>
		</tr>
		<tr>
			<td>Spectrophotometer: Bio-Tek Synergy HT Multi-reaction microplate reader</td>
			<td>Bio-Tek Instruments, Inc.</td>
			<td>&nbsp;</td>
			<td>For use with continuous kinetic monitoring</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Table 2. Equipment</td>
		</tr>
	</tbody>
</table>

a quantitative assay to study protein:dna interactions, discover transcriptional regulators of gene expression, and identify novel anti-tumor agents

许多DNA结合测定法，如电泳迁移率变动分析（EMSA），化学发光测定法，染色质免疫沉淀（ChIP）为基础的检测，和多孔基测定法用于测量转录因子的活性。然而，这些测定是非定量，缺乏特异性，可能涉及使用放射性标记的寡核苷酸，并且可能并不适用于DNA结合的抑制剂的筛选。另一方面，用一个定量的DNA结合的酶联免疫吸附测定法（D-ELISA）测定中，我们展示了核蛋白质相互作用与DNA的使用依赖于特定的关联与存在于生物素共有DNA结合序列的RUNX2转录因子标记的寡核苷酸。细胞的制备，提取核蛋白，和双链寡核苷酸的设计进行说明。抗生物素蛋白包被的96孔板中是固定的碱性缓冲液，温育在核苷酸封闭缓冲液中的核蛋白质。傅杰仕由于板的彻底清洗，特定的一抗和二抗孵育，随后加入比色反应的辣根过氧化物酶底物和发展。停止反应模式或连续动力学监测被用来定量测量与DNA蛋白相互作用。我们讨论了相应的特异性控制，包括与非特异性IgG或无蛋白质或初级抗体治疗。该测定的申请中描述，包括其在药物筛选程序和代表正和负的结果进行了讨论。

对D-ELISA法是高度特异性的针对指定的DNA结合蛋白，只要使用含有共识RUNX2结合位点（ACACCA）的三个拷贝的序列特异性双链寡核苷酸。第一抗体识别的蛋白因子也提高了特异性。二次抗体包含共价连接的辣根过氧化物酶（HRP），一个透明基底（四甲基联苯胺）转换为有色产物，便于检测（ 图1）。出于这些原因，一些重要的背景控制需要被包括在每个测定板，以确保特定的DNA结合。这些...

Watch this Scientific Journal Video about A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents at JoVE.c

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

我们开发了一种定量的DNA结合，基于ELISA的测定法来测量与DNA的转录因子相互作用。高特异性的RUNX2蛋白与共识的DNA寡核苷酸识别和特异性单克隆抗体来实现。比色检测用酶偶联抗体底物反应的实时监测。

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, ...

a-quantitative-assay-to-study-proteindna-interactions-discover

Research

JoVE Journal

Biology

18.5K 次观看.  University of Maryland School of Medicine. 我们开发了一种定量的DNA结合，基于ELISA的测定法来测量与DNA的转录因子相互作用。高特异性的RUNX2蛋白与共识的DNA寡核苷酸识别和特异性单克隆抗体来实现。比色检测用酶偶联抗体底物反应的实时监测。 

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

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

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

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

主电穿孔的小鼠骨髓造血细胞培养制备

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

科研

生物学

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

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

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

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

嗨 - C：一个方法研究基因组的三维架构。

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

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

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

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

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

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

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

在激活人类细胞中的基因表达的动态检测组蛋白修饰染色质免疫沉淀（ChIP）

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

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 ...

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 ...

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

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

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

全球合成的RNA在单细胞水平以下的低氧分析

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

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 ...

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1

DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression.