这项工作是由弗吉尼亚大学，弗吉尼亚大学癌症中心和托马斯F和凯特米勒Jeffress纪念信托基金的支持。我们感谢詹姆斯E.达内尔实验室（美国洛克菲勒大学的）罗伯特罗斯实验室（Fordham大学）细胞株。

规划前一天，一个芯片实验计划，使足够数量的，应该是培养细胞。假设每个芯片检测将需要〜1-1.5 × 10 7细胞。始终做负球蛋白（IgG）和积极的（潘组蛋白抗体）控制和重复实验。 提示 -对于2FTGH细胞，每个芯片的检测要求在80％汇合约15 cm组织培养皿中。 第1部分：准备染色质和执行系统<ol><li>取出培养基，诱导细胞20毫升10％的宇宙小牛血清（CCS）/ 5毫微克/毫升含有γ-干扰素的DMEM所需的时间。替换20毫升10％CCS / DMEM培养液，以及对非诱导板的媒体。 </li><li>在通风橱中，添加540μL37％的甲醛20毫升媒体15 cm培养皿中（终浓度1％）。 提示 -倾斜板，然后添加甲醛的介质池-岩石板扩散均匀。 </li><li>允许继续在室温下10分钟的交联。 </li><li>添加甘氨酸0.125米决赛浓度停止交联。 </li><li>真空吸媒体和洗细胞一次用10 mL冰冷的磷酸盐缓冲液（PBS）。 </li><li>收集细胞与细胞刮刀〜7毫升PBS，并结合细胞（6板），共收治50毫升锥形管冰相同。 </li><li>离心机（美国贝克曼Coutler Allegra的12 - R离心机）在1800 RPM在4 ° C和5分钟吸的PBS。 提示 -捕捉冻结的细胞沉淀，如果停在这里，并储存于-80 ° C 。 </li><li>重悬细胞沉淀在4毫升膨胀的缓冲液（25毫米HEPES pH值7.8，1.5毫米氯化镁 2，氯化钾10毫米，0.1％NP40。添加1毫米DTT，完整的蛋白酶抑制剂和寒意上冰前使用），并转移至15 mL锥形管。 提示 -这种膨胀的缓冲液是适合6板池（〜6-9 x 10 7细胞） 。或多或少的缓冲区可能改变douncing效率。 </li><li>孵育10分钟，在冰上的细胞悬液。 </li><li>转让暂停一个预冷15毫升douncer（惠顿）和dounce杵答：20杆</li><li>转让dounced细胞恢复到15 mL锥形管上冰。 </li><li> 5分钟2500转的离心机在4 ° C，弃上清。 </li><li>重悬在3毫升的核颗粒（约200μL6板）超声缓冲液（15颗粒卷）（的50 mM HEPES pH值7.9，氯化钠140毫米，1毫米EDTA，1％TRITON X - 100，0.1％去氧胆酸钠， 0.1％SDS在冰上添加完整的蛋白酶抑制剂和寒冷，在使用前）。 提示 -此超声缓冲液是适合6板池（〜6-9 × 10 7细胞）。或多或少的缓冲区可能会改变的超声效率。 </li><li>超声对冰核停牌10个周期（20秒on/40 8秒/关闭幅度设置）使用一个Misonix Sonicator 3000配备的微尖。这通常会导致染色质片段，平均200-1000个基点大小。 提示 -由于sonicators会有所不同，超声效率，必须凭经验确定。要做到这一点，前超声和超声循环后，每个分装20μL。在65 ° C加热4小时，这些样品。添加DNA上样缓冲液，运行一个标准的1％琼脂糖/ TBE凝胶，以可视化的DNA片段。） </li><li>转让超声20分钟的材料SS34离心管，离心4 °彗星在SS34转子13000 RPM（Sorvall RC6加离心机）颗粒通常是少量的细胞碎片。 </li><li>小心将上清转移至15 mL锥形管上冰。 </li><li>加入360μL的鲑鱼精子DNA /蛋白质A或G琼脂糖珠浆，在4 ° C摇晃至少30分钟的预清除。 提示 -这一步降低噪音试验从非特异性结合事件发生。 </li><li>在4 ° C离心5分钟沉淀琼脂糖珠在1000 rpm。 </li><li>传送前清除上清另一个15毫升锥形管冰。 </li><li>测量前清除染色的A260单位和超声缓冲区的音量调整到四（4）（0.2 mg / mL的假设1单位= 0.050毫克/毫升A260 A260单位） 提示 -要衡量A260单位，稀释，在190μLDDW 10μL预先清除染色;空白分光光度计10μL超声缓冲液190μL，DDW。 </li><li>保存75μL的染色质（4 A260单位）作为输入采样并存储在20 ° C，直到准备反向交联。 提示 -这一步是进行数据分析的关键。不要忽视输入等分。 </li><li>分装1.7 ml离心管上我准备染色CE为1毫升等分。 提示 -如果停在这里，单元冻结干冰和存储的准备染色，在-80 ° C。注：存储染色，可降解，生产不到最佳的效果，这样就可以避免如​​果可能的话。 </li><li>添加的芯片级抗体诱导和未诱导的样品重复设置。加入IgG抗体阴性和阳性对照，以诱导和未诱导的样品集（2微克）和泛组蛋白H3抗体（15μL）。 提示 -使用的抗体量，必须凭经验确定或建议金额的供应商的产品负债表。一个芯片级的抗体，通常为1-2微克效果很好。必须确定适当的音量经验，提供血清抗体。 </li><li>岩石在一夜之间所有的管子在4 ° C。 </li></ol>第2部分：COLLECT免疫复合物<ol><li>加入60μL鲑鱼精DNA /蛋白A或蛋白G琼脂糖珠浆所有的管子。 提示使用小鼠单克隆抗体，多克隆抗体和G蛋白的蛋白质。 </li><li>在4 ° C的岩石，至少60分钟。 </li><li>颗粒的免疫复合物（琼脂糖珠）microfuging在2000转，轻轻地在4 ° C时3分钟。 </li><li>吸出上清液。 </li><li>洗净的免疫复合物，用1毫升每10分钟以下洗净缓冲器摇摆在4 ° C。在4℃离心3分钟在2000转每洗，吸上清彗星之间。让冷冻样品在冰上。加入PMSF在使用前。 <ul><li>低盐清洗 - 0.1％SDS，1％TRITON X - 100，2毫米EDTA，20毫米，150毫米氯化钠，pH值8.0的Tris - CL，1毫米PMSF </li><li>高盐液 - 0.1％SDS，1％TRITON X - 100，2毫米EDTA，20毫米，pH值8.0的Tris - CL 500毫米氯化钠，1毫米PMSF </li><li>氯化锂洗净 - 氯化锂0.25米，1％NP - 40，1％去氧胆酸钠，1 mM的EDTA，10毫米的Tris - CL，pH值8.0，1毫米PMSF </li><li>特洗衣 - 两个TE缓冲液-10毫米的Tris - CL，pH值8.0，1 mM的EDTA，PMSF 1毫米清洗</li></ul></li><li>删除最后的TE缓冲液洗珠，加入500μl洗脱缓冲液（50毫米的Tris - CL，pH值8.0，10 mM的EDTA，1％SDS）。 提示 -洗脱缓冲液应新鲜准备。 </li><li>涡旋混合30分钟琼脂糖珠，洗脱缓冲液和发热量，在65 ° C。 提示 -涡每管至少在这30分钟内每10分钟。 </li><li>离心30秒，在2000转和洗脱液转移到一个新的离心管。 提示 -在洗脱，解冻您的输入样本，并加入500μl洗脱缓冲液，以每一个。这些样本包括前进中的所有步骤。 </li><li> 5 M氯化钠加入终浓度为200毫米的所有样品和涡。 </li><li>在65 ° C孵育过夜（或至少4个小时）。 </li></ol>第3部分：净化ChIP&#39;d和输入的DNA <ol><li>所有样品离心30秒收集凝结盖。 </li><li> 1μL核糖核酸酶A（10 U /μL）添加到每个管，室温孵育15分钟。 </li><li>加入10μL，0.5 M EDTA，40μL0.5 M Tris - CL的pH值6.5，2μL蛋白酶K（10毫克/毫升）和孵育在45 ° C为60分钟。提示准备掌握了这些试剂混合，并添加52μL，每管。 </li><li>酚/氯仿/异戊醇提取物和氯仿/异戊醇提取样品。 </li><li>加入2μL的糖原（25微克/μL）和0.1卷3M钠醋酸pH值5.2和旋涡。 </li><li>添加2.5 100％的乙醇和旋涡卷。 </li><li>孵育在20 ° C过夜。 </li></ol>第4部分：净化ChIP&#39;d及输入的DNA续<ol><li>离心样品最低为20分钟，在13000转4 ° C至沉淀的DNA /糖原颗粒。 </li><li> 1毫升70％的乙醇清洗DNA /糖原颗粒。 </li><li>空气干燥颗粒在DNA SAVANT高速真空离心机或快速干燥。 提示 -不要过度干燥的DNA。 </li><li>重悬在200μLTE缓冲液中的DNA /糖原颗粒。 </li><li> DNA（1μL）通过实时PCR定量。商店剩余的DNA在20 ° C。 提示 -使用超过1μL的DNA能抑制实时PCR反应，如此规模的谨慎。 </li></ol>第5部分：代表性的结果： 芯片和输入的DNA样本进行量化实时PCR 7,8使用特定利益的基因位点的引物（应用生物系统公司7500快速实时PCR系统）。目标基因组序列已知的富集或缺乏在被检测的组蛋白修饰的引物可用于以及作为阳性和阴性对照。数据作为输入％。芯片程序应完全řepeated与三个生物复制，以确保报道组蛋白修饰的变化有统计学意义。 使用实时PCR定量的DNA，包括引物设计，PCR的效率，并适当的方法来计算％的输入和规范化程序时，有几个重要的问题需要考虑。在分析跨基因组蛋白修饰入住，各引物对PCR效率必须一致。实时PCR系统制造商可以提供必要的信息和培训。 图1显示了代表染色质免疫沉淀在STAT1的IRF1基因的激活，引发由干扰素-γ 。 <img src="/files/ftp_upload/2053/2053fig1.jpg" alt="图1" /> 图1。 IRF1基因在STAT1的激活STAT1，RNA聚合酶II和H3K36me3占用是动态的。 （A，B，C）的芯片与α- H3K36me3，α- STAT1和α-波尔染色质II 2FTGH细胞收集诱导IFN -γ为30分钟（红色方块），1.5小时（蓝色圆圈），5小时（绿色三角形）或非诱导（黑钻石）。抗体是阴性对照组（灰色跨越），（四 ）泛H3是积极的控制和显示横跨在诱导（蓝色圆圈）位点的组蛋白占用和非诱导条件（黑钻石）。周围-5 BP下跌是由于核小体耗尽是在转录起始位点的发现。

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	<li>Levy, D., Darnell, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signalling:+Stats:+transcriptional+control+and+biological+impact.">Signalling: Stats: transcriptional control and biological impact.</a> Nature Reviews Molecular Cell Biology. 3, 651-662 (2002).</li><li>Bromberg, J., Darnell, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+STATs+in+transcriptional+control+and+their+impact+on+cellular+function.">The role of STATs in transcriptional control and their impact on cellular function.</a> Oncogene. 19, 2468-2473 (2000).</li><li>Hu, X., Ivashkiv, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-regulation+of+signaling+pathways+by+interferon-gamma:+implications+for+immune+responses+and+autoimmune+diseases.">Cross-regulation of signaling pathways by interferon-gamma: implications for immune responses and autoimmune diseases.</a> Immunity. 31, 539-550 (2009).</li><li>Campos, E. I., Reinberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histones:+annotating+chromatin.">Histones: annotating chromatin.</a> Annu Rev Genet. 43, 559-599 (2009).</li><li>Kouzarides, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+Modifications+and+Their+Function.">Chromatin Modifications and Their Function.</a> Cell. 128, 693-705 (2007).</li><li>Jenuwein, T., Allis, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translating+the+histone+code.">Translating the histone code.</a> Science. 293, 1074-1080 (2001).</li><li>Wittwer, C. T., Herrmann, M. G., Moss, A. A., Rasmussen, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+fluorescence+monitoring+of+rapid+cycle+DNA+amplification.">Continuous fluorescence monitoring of rapid cycle DNA amplification.</a> Biotechniques. 22 (130-131), 134-138 (1997).</li><li>Higuchi, R., Dollinger, G., Walsh, P. S., Griffith, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+amplification+and+detection+of+specific+DNA+sequences.">Simultaneous amplification and detection of specific DNA sequences.</a> Biotechnology (N Y). 10, 413-417 (1992).</li><li>Kroger, A., Koster, M., Schroeder, K., Hauser, H., Mueller, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activities+of+IRF-1.">Activities of IRF-1.</a> J Interferon Cytokine Res. 22, 5-14 (2002).</li><li>Ross, R. A., Spengler, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+neuroblastoma+stem+cells.">Human neuroblastoma stem cells.</a> Seminars in cancer biology. 17, 241-247 (2007).</li></ol>

芯片协议概述已成功地应用于实验室检测超过20种不同的组蛋白修饰。此外，我们有特点的转录因子的入住变化，组蛋白修饰酶，蛋白质识别组蛋白修饰和RNA聚合酶II的转录机器，几个干扰素γ诱导的基因。我们也使用的过程，刻画在一个癌症干细胞的分化10组蛋白修饰的变化。 芯片的数据的质量最终取决于所使用的抗体的有效性和抗体之间有很大差别。故障芯片的修?...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>37% Formaldehyde</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP531-500</td>
<td>&nbsp;</td>
<tr>
<td>Chloroform/Isoamyl alcohol</td>
<td>&nbsp;</td>
<td>Acros</td>
<td>AC32715-5000</td>
<td>&nbsp;</td>
<tr>
<td>Complete Mini Protease Inhibitors</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>1836153</td>
<td>&nbsp;</td>
<tr>
<td>Cosmic Calf Serum</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30087.04N</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30243</td>
<td>&nbsp;</td>
<tr>
<td>DTT</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D0632</td>
<td>&nbsp;</td>
<tr>
<td>EDTA</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP118-500</td>
<td>&nbsp;</td>
<tr>
<td>Ethanol</td>
<td>&nbsp;</td>
<td>Pharmco</td>
<td>zp1110EP</td>
<td>&nbsp;</td>
<tr>
<td>Glycine</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>100149</td>
<td>&nbsp;</td>
<tr>
<td>Glycogen</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>901393</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3784</td>
<td>&nbsp;</td>
<tr>
<td>IFN-gamma</td>
<td>&nbsp;</td>
<td>R&amp;D Systems</td>
<td>285-IF-100</td>
<td>&nbsp;</td>
<tr>
<td>IgG</td>
<td>&nbsp;</td>
<td>Jackson Immunoresearch</td>
<td> 109-005-003</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>&nbsp;</td>
<td>MP Biologicals</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>LiCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L4408</td>
<td>&nbsp;</td>
<tr>
<td>MgCl2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M8266</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Acetate</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP333-500</td>
<td>&nbsp;</td>
<tr>
<td>Deoxycholic Acid Sodium Salt</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP349-100</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>7647-14-5</td>
<td>&nbsp;</td>
<tr>
<td>NP40</td>
<td>&nbsp;</td>
<td>US Biological</td>
<td>N3500</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Histone H3, CT, pan</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>07-690</td>
<td>&nbsp;</td>
<tr>
<td>Phenol/chloroform/isoamyl</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>108-95-2</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate Buffered Saline</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>7647-14-5</td>
<td>&nbsp;</td>
<tr>
<td>PMSF</td>
<td>&nbsp;</td>
<td>EMD</td>
<td>50-230-0316</td>
<td>&nbsp;</td>
<tr>
<td>Proteinase K</td>
<td>&nbsp;</td>
<td>5-Prime</td>
<td>2500140</td>
<td>&nbsp;</td>
<tr>
<td>RNAse A</td>
<td>&nbsp;</td>
<td>5-Prime</td>
<td>2500130</td>
<td>&nbsp;</td>
<tr>
<td>Salmon Sperm DNA/Protein A Agarose</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>16-157</td>
<td>&nbsp;</td>
<tr>
<td>Salmon Sperm DNA/Protein G Agaorse</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>16-201</td>
<td>&nbsp;</td>
<tr>
<td>SDS</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP166-500</td>
<td>&nbsp;</td>
<tr>
<td>Tris-Cl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T5941</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Acros</td>
<td>327372500</td>
<td>&nbsp;</td>
<tr>
<td>Anti-K36me3</td>
<td>&nbsp;</td>
<td>Abcam</td>
<td>ab9050</td>
<td>&nbsp;</td>
<tr>
<td>Anti-STAT1</td>
<td>&nbsp;</td>
<td>Santa Cruz</td>
<td>sc-345X</td>
<td>&nbsp;</td>
<tr>
<td>Anti-RNA Polymerase II</td>
<td>&nbsp;</td>
<td>Santa Cruz</td>
<td>sc-899</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

chromatin immunoprecipitation (chip) to assay dynamic histone modification in activated gene expression in human cells

在应对各种胞外配体时，STAT（信号转导和转录激活因子）转录因子迅速招募来，他们是由在一个单一的酪氨酸残基 1的磷酸化激活的细胞表面受体在细胞质中的潜伏状态。然后，他们二聚体，并转运到细胞核驱动的靶基因的转录，影响生长，分化，动态平衡和免疫反应。毫不奇怪，因为他们在正常细胞过程中的广泛参与，STAT的功能失调导致人类疾病， 特别是2和3的癌症自身免疫性疾病。 它是公认的，是通过改变转录调节染色质模板 4,5 。这些变化包括ATP依赖的复合物，以及共价组蛋白修饰和DNA甲基化6的活动。由于基因表达激活的STAT是迅速而短暂的，它需要在STAT相关的基因位点的调节染色质模板的具体机制。要定义这些机制中，我们描述的组蛋白修饰和酶活动产生响应STAT信号的基因位点。此协议介绍，STAT信号激活基因表达染色的研究提供宝贵的一个方法，是染色质免疫沉淀。

Watch this Scientific Journal Video about Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells at JoVE.com

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

本协议描述了如何染色质免疫沉淀（ChIP）是用来研究的动态改变，调节信号转导通路诱导转录的染色质模板。

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 ...

chromatin-immunoprecipitation-chip-to-assay-dynamic-histone

Research

JoVE Journal

Biology

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

28.5K 次观看.  University of Virginia. 本协议描述了如何染色质免疫沉淀（ChIP）是用来研究的动态改变，调节信号转导通路诱导转录的染色质模板。

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

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

从人类胚胎干细胞的染色质免疫沉淀

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

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

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

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

机器人技术和植物组织的基因表达研究的动态图像分析

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

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 ...

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits1.
This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants)2,3. Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells.
DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers4. The primer extension assay is carried out using the MassARRAY (Sequenom)5 platform according to the manufacturer's specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results.

Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.

等位基因特异性基因表达检测，测试的遗传协会的功能基础

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

克隆形成含量：贴壁细胞

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

使用早期的小鼠胚胎组织特异性基因的染色质免疫沉淀检测

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

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 ...

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at different developmental stages. Epigenetic regulation contributes to a broad spectrum of biological processes, including cellular differentiation during embryonic development and homeostasis in adulthood. A critical strategy in epigenetic studies is to examine how various histone modifications and chromatin factors regulate gene expression. To address this, Chromatin Immunoprecipitation (ChIP) is used widely to obtain a snapshot of the association of particular factors with DNA in the cells of interest.
ChIP technique commonly uses cultured cells as starting material, which can be obtained in abundance and homogeneity to generate reproducible data. However, there are several caveats: First, the environment to grow cells in Petri dish is different from that in vivo, thus may not reflect the endogenous chromatin state of cells in a living organism. Second, not all types of cells can be cultured ex vivo. There are only a limited number of cell lines, from which people can obtain enough material for ChIP assay.
Here we describe a method to do ChIP experiment using Drosophila tissues. The starting material is dissected tissue from a living animal, thus can accurately reflect the endogenous chromatin state. The adaptability of this method with many different types of tissue will allow researchers to address a lot more biologically relevant questions regarding epigenetic regulation in vivo1, 2. Combining this method with high-throughput sequencing (ChIP-seq) will further allow researchers to obtain an epigenomic landscape.

Chromatin Immunoprecipitation ChIP using Drosophila tissue

Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system.

染色质免疫沉淀（ChIP）使用果蝇组织

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at ...

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 ...