这项工作是由美国国立卫生研究院R01 GM56244安仁，其中包括通过美国复苏与再投资法“，2009年授予的资金支持，由美国国立卫生研究院R01 GM87130 JARP

1。胚胎隔离 注 ：所有涉及小鼠的操作，应当按照相应的动物护理和使用的政策和协议执行<ol><li>检查雌性小鼠交配塞存在交配后的上午，他们放置在不同的笼子分开根根男性的交配女性。交配塞观察的一天中午，被认为是发展的0.5天（E0.5）胚胎。 </li><li>在E8.5，（或所需的阶段，如果有不同的），牺牲鼠标使用经批准的协议。 </li><li>湿安乐死动物用70％乙醇的腹部，打开腹腔。植入网站，说明胚胎发育的存在被看作是沿每个子宫角（图1A）的长度“上一个珠子串”安排。用剪刀，删除这两个子宫角，削减每个着床部位单独分开（图1B），并放置在100毫米培养皿含有室温（RT）菜夹层培养基（DMEM培养液，10％胎牛血清，20毫米的HEPES pH值7.4 60微克/ ml青霉素，链霉素2毫米）。 </li><li>使用镊子，取出每个胚胎从子宫组织（图1C）和周围的膜，并放置在新鲜剥离液。 </li><li>在解剖显微镜使用镊子下分离单个胚胎。在这个发展阶段，胚胎被包围在壁层卵黄囊，其中接触deciduum组织，卵黄囊和羊膜。羊膜是一种透明膜，在胚胎的直接接触。位于之间的羊膜和顶叶卵黄囊卵黄囊和存在突出的血管，培育胚胎在E8.5 - E9.5胚胎很容易分辨。删除从每个胚胎及其周围的膜和含有夹层的媒体，以除去多余的血液进入一个新的板块单独转让。发育阶段，可以从胚胎到胚胎和窝之间不同;代表E8.5胚胎和一个代表E9.5胚胎图1D所示。 </li><li>每个胚胎转移到1.5 ml的Eppendorf管中含有200μL的夹层媒体（上述步骤4中所描述）。 </li></ol> 2。同质化隔离胚胎<ol><li>胶原酶II型20个单位在一个体积为200 UL（Dubbecco 1X的磷酸盐缓冲盐水; DPBS）添加到每个Eppendorf管。 </li><li>轻轻摇动20分钟100转的样品在37℃摇床。 </li><li>重悬以及吹打扰乱细胞的团块。 </li><li>应用细胞悬液，在无菌细胞过滤器放置在1.5 mL Eppendorf管（网目尺寸40微米）的顶部。 </li><li>立即申请600μL室温1X细胞过滤器的顶部DPBS，以完成分离。丢弃的过滤器。 </li><li>离心样品在4 ° C在4000 XG为5分钟。 </li><li>上清液。 </li><li> 1毫升RT 1X DPBS重悬沉淀。 </li><li>离心机的样品在室温下1分钟在4000 XG。 </li><li>上清液。 </li><li> 200μLRT清扫媒体的重悬沉淀。 </li><li>胚胎干细胞的计数。平均3-5 × 10 6 cells/E8.5胚胎。 </li></ol> 3。交叉连结染色质<ol><li>添加5.6μL，37％的甲醛终浓度为1％甲醛200μL样品。 </li><li>孵育样品在室温下10分钟。 </li><li>在4 ° C在4000 XG 3分钟离心样品。 </li><li>上清液。 </li><li>重悬的细胞用冷1X含有80μL/毫升的蛋白酶抑制剂的鸡尾酒（PIC）的DPBS。 </li><li>离心样品在4 ° C在4000 XG为3分钟。 </li><li>上清液。在这一步，样品可冷冻于-80 ° C。数个月的冻结，交联样品是稳定的。 </li></ol> 4。超声<ol><li>细胞沉淀重悬于100μL的RT SDS裂解液（50毫米的Tris - HCl（pH值8.1），10毫米EDTA和1％SDS刚加入1μLPIC/800μL总体积PIC）。当重悬，添加额外的100 UL RT SDS裂解液促进再悬浮。吹打和反演混合。 </li><li>孵育细胞悬液，冰浴10分钟。 </li><li>超声裂解细胞剪切DNA之间的200和500碱基对使用Diagenode Bioruptor™UCD200（5分钟× 8倍幅度设置为30秒on/30秒高（320瓦）功率）。样品应被包围的冰浆。不要让样品温4 ° C以上或冻结。 sonicators提供有多种类型。超声条件下，应优化每个sonicator导致交联的DNA剪切长度约500 bp的。 </li><li>离心机超声样品在4 ° C时10分钟XG 14000。 </li><li> Transfer上清液到一个新的Eppendorf管中，在冰上预冷。 </li><li>超声样品分成5等份的最大。我们有经验确定一个E8.5胚胎可分为5等份。较小或较大的样本量可以相应地调整。在这一步，样品可存放于-80 ° C，直到进一步的使用。 </li><li>保留一个等份作为输入，并储存在-20 ° C时，直到第6步交叉链接将得到扭转。淡化其他等分芯片缓冲液稀释（16.7毫米的Tris - HCl（pH值8.1），1.2毫米EDTA，1.1％Triton X - 100的的，167毫米NaCl和0.01％SDS）的10倍，含有1μl/800刚添加的PIC μL缓冲。 </li></ol> 5。预结算和免疫<ol><li>预清除鲑鱼精子DNA /蛋白A或G琼脂糖- 50％的浆料在4 ° C，旋转，1小时与75μL稀释上清。任应确定要使用的芯片的抗体A或蛋白G珠蛋白使用。例如，建议兔抗体蛋白A珠，而G蛋白珠山羊抗体或鼠IgG抗体的建议。如需详细资讯，请参阅珠制造商提出的建议。 </li><li>在4 ° C离心1分钟，2500 XG。 </li><li>将上清转移至一个新的，预冷的Eppendorf管。 </li><li>加入抗体（4微克/样品）前清除等分，并在4℃孵育过夜旋转彗星。 </li></ol> 6。洗衣机的染色质蛋白珠复合物<ol><li>添加到每个小瓶和孵化在4 ° C旋转1小时60μL鲑鱼精DNA /蛋白质A或G琼脂糖浆。使用相同的珠型，用于在上述步骤3预结算。 </li><li>在4 ° C离心1分钟1000 XG。 </li><li>小心取出上清液并丢弃。置于冰上的DNA -蛋白质珠复合物。 </li><li>重悬和洗的颗粒直接与下面的洗缓冲区1，2，3，和4。洗缓冲区1-3应冷却到4 ° C和这些缓冲区清洗应表现在4 ° C。洗缓冲液4，应在室温和与此缓冲区清洗应在室温下进行。每次清洗是在适当的温度为5分钟旋转。每次洗，在4 ° C在1000 XG 1分钟（室温缓冲液洗4清洗）离心，然后小心地吸去或去除上清。 </li></ol> 缓冲器1：低盐缓冲液洗（20毫米的Tris - HCl（pH值8.1），2毫米EDTA，1％的Triton X - 100， 150毫米NaCl和0.1％SDS），1毫升× 2洗。 缓冲区2：高盐缓冲液洗（20毫米的Tris - HCl（pH值8.1），2毫米EDTA，1％的Triton X - 100， 500毫米NaCl和0.1％SDS），1毫升× 2洗。 缓冲区3：氯化锂盐缓冲液洗（10毫米的Tris - HCl（pH值8.1），1 mM的EDTA，1％IGEPAL - CA630， 0.25米LiCl和1％去氧胆酸（钠）），1毫升x 1洗。 缓冲区4：TE缓冲液（10毫米的Tris - HCl（pH值8.1），1毫米EDTA），1毫升x 2洗。 7。染色质抗体复合物的洗脱<ol><li>重悬于250μL新鲜配制洗脱缓冲液（1％SDS，0.1 M的碳酸氢钠3）洗过的染色质蛋白珠复杂。大力涡样本为5秒，然后在室温下孵育15分钟旋转。 </li><li>离心机在2500 XG在室温下1分钟，并转移到一个新的Eppendorf管中的洗脱液。 </li><li>重复步骤1和2，并结合在同一个Eppendorf管中的洗脱液。洗脱液应在室温下保存。这一步完成后，洗脱液可以储存在-20 ° C，直到进一步利用。 </li></ol> 8。反向交叉链接和恢复DNA <ol><li> 500μL洗脱液（40μL/毫升输入控制），加入20μL5 M氯化钠。 </li><li>热洗脱液在65℃为4小时至过夜水浴。 </li><li> 10μL每个样本（包括输入控制），可以运行在2％琼脂糖凝胶跨越0.1至1 KBP检查的DNA片段大小的尺寸标记。 DNA会出现一抹黑，而不是一个尖锐的乐队。其余的样本，可以留在65 ° C，而凝胶运行。 </li><li>恢复使用QIAquick凝胶提取试剂盒（QIAGEN）从每个样品中的DNA。加入600μLQG缓冲液（试剂盒中提供），以优化的DNA吸附在QIAquick离心柱和200μL到500μL样品异丙醇的硅膜。请注意该资历组别的缓冲区中包含允许容易测定样品的pH值pH值指标。如果样品的颜色从黄色到紫色圈（pH值&gt; 7.5），混匀后，加10μL3米NaAcetate（pH值5.2）的样品和混合。这降低了混合物的pH值，以最大限度地列珠的DNA结合。 </li><li>装货前列，检查，以确定是否存在任何SDS沉淀的混合物。如果SDS的降水发生，样品应在回暖42℃的热水洗澡，直到沉淀消失。 </li><li> QIAquick自旋列（工具包中的紫色列）装入700μl和混合物在室温下离心1分钟，在14000 XG。通过从列在出发前的流量丢弃，其余样品重复此步骤。 </li><li>加入500μlQG缓冲液洗柱。在室温下离心2分钟，在14000 XG。丢弃的流量通过从列在出发前。 </li><li> PE洗涤缓冲液700μL（工具包中的提供），已加入乙醇制造商的指示清洗之列。在室温下离心1分钟，在14000 XG。丢弃的流量通过从列在出发前。 </li><li>重复离心步骤完全删除列的PE缓冲区的其余部分。放弃通过流量和收集管。 </li><li>洗脱的DNA从列到一个新的Eppendorf管中，加入60μL的EB缓冲液（洗脱缓冲液试剂盒中提供）。 </li><li>在室温下离心1分钟，在14000 XG。洗脱下的DNA可以存储在-20 ° C，直到检测。回收的材料260读数通常表明浓度为90-120毫微克/ UL，总回收率为〜6-7微克每等份。然而，一个260/280这些样本的比例通常&gt; 1.8，表明RNA样品中。因此，回收DNA的确切数额可能会更低。 </li></ol> 9。回收DNA分析<ol><li> SDS会干扰PCR Taq DNA聚合酶的活性。 SDS应完全去除，然后才进行PCR扩增。孵化在冰上的DNA沉淀1分钟，在14000 XG 4任何残留的SDS和离心机° C。强烈建议将上清转移至一个新的Eppendorf tube.This一步。 </li><li>通过常规PCR和实时定量PCR（Q - PCR）与具体每一个要分析的序列的引物可检测的DNA洗脱液。 5-10μL总DNA洗脱液可用于PCR和Q - PCR反应。 PCR与输入控制可以进行5-10倍稀释的DNA与其他样本量相比。 PCR和Q - PCR条件会有所不同，但是，起始原料的数量有限，需要更多的PCR循环。对于常规的PCR检测，我们建议开始，40个循环，调整需要。 </li></ol> 10。代表性的成果我们已经用这个协议来执行E8.5和E9.5胚胎（图2）芯片。结果表明，肌细胞生成素是在E8.5和E9.5胚胎肌细胞生成素推动者。 CHIP纯化的DNA进行了分析常规PCR（图2A）和实时定量PCR（图2B）。与此相反，有没有卵黄囊，不表达肌细胞生成素在肌细胞生成素促进肌细胞生成素的指示结合。干扰素-γ（IFNγ）发起人，其中包含了肌细胞生成素的结合位点匹配的序列，是作为一个负序控制使用。正如预期的那样，肌细胞生成素，不一定要在任何组织测试样本IFNγ的发起人。在E8.5和9.5的胚胎中，肌细胞生成素是具体 ​​表现在体节（图3，6,10），这是骨骼肌的前体。因此，结果表明，肌细胞生成素是必然要在体节肌细胞生成素在E8.5和E9.5推动者。 <img src="/files/ftp_upload/2677/2677fig1.jpg" alt="图1" /> 图1。E8.5胚胎清扫。 （一）离体子宫角（顶部面板在下部面板上显示更高的放大倍率）。 （二）子宫角用剪刀剪开，以单独的个体包含单个胚胎植入网站。 （三）从子宫组织在清扫胚胎突起。箭头标志仍然受到额外的胚胎组织覆盖的胚胎。 （d）两个E8.5胚胎来自同一窝。左边的胚胎还没有开始转动的过程中。在右边的胚胎正处于转折点。极右 - 代表E9.5胚胎。 <img src="/files/ftp_upload/2677/2677fig2.jpg" alt="图2" /> 图2的ChIP实验证明E8.5和E9.5胚胎肌细胞生成素促进肌细胞生成素具有约束力。 （上）常规5 UL使用一个肌细胞生成抗体的非特异性IgG抗体放大肌细胞生成素启动子的部分，从-79到69相对的转录起始位点的引物进行芯片实验的DNA纯化，PCR分析包含了肌细胞生成素的结合位点，位于-12或引物扩增IFNγ的发起人，其中包含一个序列匹配的肌细胞生成素的结合位点，位于〜1075 bp的转录起始位点上游的部分。 IFNγ的引物序列5&#39; - GCT GAC TCA AGA CCC认证CGA GGC - 3&#39;和5&#39; - TGA GGA TGG GGC AGG AGG的CC - 3&#39;。 （下）实时定量PCR分析（一）使用相同的样本。 “数据绘制成输入+ / - 标准差％。 <img src="/files/ftp_upload/2677/2677fig3.jpg" alt="图3" /> 图3。肌细胞生成素，特别是在E8.5和E9.5胚胎的体节表示。肌细胞生成素原位杂交整装显示特定的mRNA的表达在体节。 E8.5形象 - 200微米的大小酒吧。 E9.5形象 - 500微米的大小酒吧。

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E., Arnold, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baculovirus-expressed+myogenic+determination+factors+require+E12+complex+formation+for+binding+to+the+myosin-light-chain+enhancer.">Baculovirus-expressed myogenic determination factors require E12 complex formation for binding to the myosin-light-chain enhancer.</a> Eur J Biochem. 198, 187-193 (1991).</li><li>Chakraborty, T., Brennan, T., Olson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+trans-activation+of+a+muscle-specific+enhancer+by+myogenic+helix-loop-helix+proteins+is+separable+from+DNA+binding.">Differential trans-activation of a muscle-specific enhancer by myogenic helix-loop-helix proteins is separable from DNA binding.</a> J Biol Chem. 266, 2878-2882 (1991).</li><li>French, B. A., Chow, K. L., Olson, E. N., Schwartz, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterodimers+of+myogenic+helix-loop-helix+regulatory+factors+and+E12+bind+a+complex+element+governing+myogenic+induction+of+the+avian+cardiac+alpha-actin+promoter.">Heterodimers of myogenic helix-loop-helix regulatory factors and E12 bind a complex element governing myogenic induction of the avian cardiac alpha-actin promoter.</a> Mol Cell Biol. 11, 2439-2450 (1991).</li><li>Brennan, T. J., Chakraborty, T., Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutagenesis+of+the+myogenin+basic+region+identifies+an+ancient+protein+motif+critical+for+activation+of+myogenesis.">Mutagenesis of the myogenin basic region identifies an ancient protein motif critical for activation of myogenesis.</a> Proc Natl Acad Sci U S A. 88, 5675-5679 (1991).</li><li>Lassar, A. B., Davis, R. L., Wright, W. E., Kadesch, T., Murre, C., Voronova, A., Baltimore, D., Weintraub, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+activity+of+myogenic+HLH+proteins+requires+hetero-oligomerization+with+E12/E47-like+proteins+in+vivo.">Functional activity of myogenic HLH proteins requires hetero-oligomerization with E12/E47-like proteins in vivo.</a> Cell. 66, 305-315 (1991).</li><li>Chakraborty, T., Brennan, T. J., Li, L., Edmondson, D., Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inefficient+homooligomerization+contributes+to+the+dependence+of+myogenin+on+E2A+products+for+efficient+DNA+binding.">Inefficient homooligomerization contributes to the dependence of myogenin on E2A products for efficient DNA binding.</a> Mol Cell Biol. 11, 3633-3641 (1991).</li><li>Cserjesi, P., Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myogenin+induces+the+myocyte-specific+enhancer+binding+factor+MEF-2+independently+of+other+muscle-specific+gene+products.">Myogenin induces the myocyte-specific enhancer binding factor MEF-2 independently of other muscle-specific gene products.</a> Mol Cell Biol. 11, 4854-4862 (1991).</li><li>Braun, T., Arnold, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+four+human+muscle+regulatory+helix-loop-helix+proteins+Myf3-Myf6+exhibit+similar+hetero-dimerization+and+DNA+binding+properties.">The four human muscle regulatory helix-loop-helix proteins Myf3-Myf6 exhibit similar hetero-dimerization and DNA binding properties.</a> Nucleic Acids Res. 19, 5645-5651 (1991).</li><li>Serna, d. e. l. a., L, I., Ohkawa, Y., Berkes, C. A., Bergstrom, D. A., Dacwag, C. S., Tapscott, S. J., Imbalzano, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MyoD+targets+chromatin+remodeling+complexes+to+the+myogenin+locus+prior+to+forming+a+stable+DNA-bound+complex.">MyoD targets chromatin remodeling complexes to the myogenin locus prior to forming a stable DNA-bound complex.</a> Mol Cell Biol. 25, 3997-4009 (2005).</li><li>Blais, A., Tsikitis, M., Acosta-Alvear, D., Sharan, R., Kluger, Y., Dynlacht, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+initial+blueprint+for+myogenic+differentiation.">An initial blueprint for myogenic differentiation.</a> Genes Dev. 19, 553-569 (2005).</li><li>Cao, Y., Kumar, R. M., Penn, B. H., Berkes, C. A., Kooperberg, C., Boyer, L. A., Young, R. A., Tapscott, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+and+gene-specific+analyses+show+distinct+roles+for+Myod+and+Myog+at+a+common+set+of+promoters.">Global and gene-specific analyses show distinct roles for Myod and Myog at a common set of promoters.</a> EMBO J. 25, 502-511 (2006).</li><li>Ohkawa, Y., Yoshimura, S., Higashi, C., Marfella, C. G., Dacwag, C. S., Tachibana, T., Imbalzano, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myogenin+and+the+SWI/SNF+ATPase+Brg1+maintain+myogenic+gene+expression+at+different+stages+of+skeletal+myogenesis.">Myogenin and the SWI/SNF ATPase Brg1 maintain myogenic gene expression at different stages of skeletal myogenesis.</a> J Biol Chem. 282, 6564-6570 (2007).</li><li>Davie, J. K., Cho, J. H., Meadows, E., Flynn, J. M., Knapp, J. R., Klein, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target+gene+selectivity+of+the+myogenic+basic+helix-loop-helix+transcription+factor+myogenin+in+embryonic+muscle.">Target gene selectivity of the myogenic basic helix-loop-helix transcription factor myogenin in embryonic muscle.</a> Dev Biol. 311, 650-664 (2007).</li><li>Metivier, R., Penot, G., Hubner, M. R., Reid, G., Brand, H., Kos, M., Gannon, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+receptor-alpha+directs+ordered,+cyclical,+and+combinatorial+recruitment+of+cofactors+on+a+natural+target+promoter.">Estrogen receptor-alpha directs ordered, cyclical, and combinatorial recruitment of cofactors on a natural target promoter.</a> Cell. 115, 751-763 (2003).</li><li>Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., Struhl, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Current Protocols in Molecular Biology. , (2010).</li></ol>

在马萨诸塞州医学院IACUC大学规定的准则和法规的规定进行动物实验。

在所描述的芯片协议，我们将展示生肌调节肌细胞生成素是在骨骼肌易制毒化学组织目前在单E8.5和E9.5胚胎肌细胞生成素推动者。此前的研究已经广泛的特点肌细胞生成素E盒含序列的结合，从最初在体外凝胶迁移实验利用体外翻译或细菌产生的肌细胞生成素和放射性标记的DNA编码的靶基因序列11-20监管的相关部分。传统的芯片研究已经证明了肌细胞生成素具有约束力的组织文...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>ChIP Assay Kit</td>
<td>&nbsp;</td>
<td>Upstate Cell Signaling Solutions, Millipore</td>
<td>17-295</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase Type II</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17101015</td>
<td>Dilution by 1 x PBS</td>
</tr>
<tr>
<td>Dulbecco&#x2019;s modified eagle medium (DMEM) </td>
<td>&nbsp;</td>
<td>Gibco Labs, Invitrogen</td>
<td>12100-061</td>
<td>High glucose content</td>
</tr>
<tr>
<td>Dulbecco&#x2019;s phosphate buffered saline 1X (DPBS)</td>
<td>&nbsp;</td>
<td>Gibco Labs, Invitrogen</td>
<td>14190-144</td>
<td>Calcium chloride free, Magnesium chloride free</td>
</tr>
<tr>
<td>Fetal bovine serum (FBS)</td>
<td>&nbsp;</td>
<td>Mediatech, Inc.</td>
<td>35-010-CV</td>
<td>&nbsp;</td>
<tr>
<td>Gel extraction kit</td>
<td>&nbsp;</td>
<td>QIAquick</td>
<td>28704</td>
<td>50 reaction kit</td>
</tr>
<tr>
<td>Penicillin/streptomycin stock solution</td>
<td>&nbsp;</td>
<td>Gibco Labs, Invitrogen</td>
<td>&nbsp;</td>
<td>5000 &mu;g/ml concentration</td>
</tr>
<tr>
<td>Protease Inhibitor Cocktail</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P8340</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>myogenin antibody</td>
<td>&nbsp;</td>
<td>Santa Cruz Biotechnology, Inc.</td>
<td>sc-576</td>
<td>&nbsp;</td>
<tr>
<td>Normal rabbit IgG</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>12-370</td>
<td>&nbsp;</td>
<tr>
<td>Platinum PCR Supermix</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11306-016</td>
<td>&nbsp;</td>
<tr>
<td>GoTaq Q-PCR master mix</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>A6001</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

chromatin immunoprecipitation assay for tissue-specific genes using early-stage mouse embryos

染色质免疫沉淀（ChIP）是一个强大的工具，以确定蛋白质：发生在活细胞的情况下1-3的染色质相互作用。这项技术已被广泛利用组织培养细胞中，并在较小程度上，在基层组织。啮齿类动物的胚胎组织，特别是在发展的早期，芯片的应用，是由组织的数量有限，异质性的胚胎细胞和组织类型复杂。在这里，我们提出了一种方法来执行芯片采用一个分离的胚胎每天8.5（E8.5）胚胎。剪切从单一E8.5胚胎的染色质可分为五个等份，它允许为控制和特定蛋白质的调查研究者足够的材料：染色质相互作用。 我们已经利用这种技术来开始：在组织特异性基因表达程序的规范文件蛋白质染色质相互作用。类型的细胞在胚胎的异质性必然限制了这项技术的应用，因为其结果是不区分的相互作用是否发生在所有检测的蛋白质：染色质相互作用的一个子集，或一个单一的细胞类型（S）。然而，考试期间或之后的组织特异性基因的组织特异性的基因表达发病原因有两个可行的。首先，一定免疫组织特定的因素隔离开来表达的细胞类型，其中的因素是染色质。其次，含有基因激活相关的翻译后修改的共激活因子和组蛋白的免疫应该只被发现在基因的细胞类型的基因和基因调控序列，或已被激活。这项技术应该适用于大多数组织特异性基因的激活事件的研究。 在这个例子中所述，我们利用E8.5和E9.5小鼠胚胎的研究在骨骼肌特定基因的启动子具有约束力的因素。体节，躯干和四肢骨骼肌肉就会形成易制毒化学组织，目前在E8.5 - 9.5 4,5。肌细胞生成素是一种调节因子骨骼肌分化6-9所需。数据表明，肌细胞生成素是与它自己的启动子在E8.5和E9.5胚胎。由于肌细胞生成素只在体节在这个发展的6,10阶段表示，数据表明，肌细胞生成素的相互作用与它自己的启动已经发生E8.5胚胎骨骼肌中的前体细胞。

Watch this Scientific Journal Video about Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos at JoVE.com

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

我们展示了染色质免疫沉淀（ChIP）的方法来识别组织特异性基因表达的组织特异性基因的小鼠胚胎组织中的发病因素相互作用期间或之后。此协议应广泛适用的组织特异性基因的激活的研究，因为它在正常胚胎发育过程中发生。

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 ...

chromatin-immunoprecipitation-assay-for-tissue-specific-genes-using

Research

JoVE Journal

Biology

18.0K 次观看.  University of Massachusetts Medical School. 我们展示了染色质免疫沉淀（ChIP）的方法来识别组织特异性基因表达的组织特异性基因的小鼠胚胎组织中的发病因素相互作用期间或之后。此协议应广泛适用的组织特异性基因的激活的研究，因为它在正常胚胎发育过程中发生。

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

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

The neural tube expresses many proteins in specific spatiotemporal patterns during development. These proteins have been shown to be critical for cell fate determination, cell migration, and formation of neural circuits. Neuronal induction and patterning involve bone morphogenetic protein (BMP), sonic hedgehog (SHH), fibroblast growth factor (FGF), among others. In particular, the expression pattern of Fgf8 is in close proximity to regions expressing BMP4 and SHH. This expression pattern is consistent with developmental interactions that facilitate patterning in the telencephalon.

Here we provide a visual demonstration of a method in which an in ovo preparation can be used to test the effects of Fgfs in the formation of the forebrain. Beads are coated with protein and placed in the developing neural tube to provide sustained exposure. Because the procedure uses small, carefully placed beads, it is minimally invasive and allows several beads to be placed within a single neural tube. Moreover, the method allows for continued development so that embryos can be analyzed at a more mature stage to detect changes in anatomy and in neural patterning. This simple but useful protocol allows for real time imaging. It provides a means to make spatially and temporally limited changes to endogenous protein levels.

A variety of growth factors and proteins interact to induce cells to take on different cell fates during development. Here we demonstrate the use of an in ovo preparation to address possible interactions between different proteins in development by placing beads on E2.5 chick embryos.

配售早期鸡胚生长因子包被的磁珠

The neural tube expresses many proteins in specific spatiotemporal patterns during development.   These proteins have been shown to be critical for cell ...

科研

生物学

A Chromatin Assay for Human Brain Tissue

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental factors that might result in epigenetic alterations of gene expression and other molecular pathology. Traditionally, however, expression studies in postmortem brain were confined to quantification of mRNA or protein. The limitations encountered in postmortem brain research   such as variabilities in autolysis time and tissue integrities   are also likely to impact any studies of higher order chromatin structures. However, the nucleosomal organization of genomic DNA   including DNA:core histone binding - appears to be largely preserved in representative samples provided by various brain banks. Therefore, it is possible to study the methylation pattern and other covalent modifications of the core histones at defined genomic loci in postmortem brain. Here, we present a simplified native chromatin immunoprecipitation (NChIP) protocol for frozen (never-fixed) human brain specimens. Starting with micrococcal nuclease digestion of brain homogenates, NChIP followed by qPCR can be completed within three days. The methodology presented here should be useful to elucidate epigenetic mechanisms of gene expression in normal and diseased human brain.

Until recently, expression studies on human brain were limited to quantification of RNA or protein. With the chromatin immunoprecipitation techniques described in this paper, it will be possible to map histone methylation and other epigenetic regulators of gene expression in postmortem brain.

一个人脑组织中的染色质含量

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental ...

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 ...

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 ...

Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell nuclei1. The technique uses DNA probes with unique sequences that hybridize to whole chromosomes or specific chromosomal regions, and serves as a powerful adjunct to classic cytogenetics. For instance, many earlier studies reported the frequent detection of increased chromosome aberrations in leukemia patients related with benzene exposure, benzene-poisoning patients, and healthy workers exposed to benzene, using classic cytogenetic analysis2. Using FISH, leukemia-specific chromosomal alterations have been observed to be elevated in apparently healthy workers exposed to benzene3-6, indicating the critical roles of cytogentic changes in benzene-induced leukemogenesis. 
Generally, a single FISH assay examines only one or a few whole chromosomes or specific loci per slide, so multiple hybridizations need to be conducted on multiple slides to cover all of the human chromosomes. Spectral karyotyping (SKY) allows visualization of the whole genome simultaneously, but the requirement for special software and equipment limits its application7. Here, we describe a novel FISH assay, OctoChrome-FISH, which can be applied for Chromosomics, which we define here as the simultaneous analysis of all 24 human chromosomes on one slide in human studies, such as chromosome-wide aneuploidy study (CWAS)8. The basis of the method, marketed by Cytocell as the Chromoprobe Multiprobe System, is an OctoChrome device that is divided into 8 squares, each of which carries three different whole chromosome painting probes (Figure 1). Each of the three probes is directly labeled with a different colored fluorophore, green (FITC), red (Texas Red), and blue (Coumarin). The arrangement of chromosome combinations on the OctoChrome device has been designed to facilitate the identification of the non-random structural chromosome alterations (translocations) found in the most common leukemias and lymphomas, for instance t(9;22), t(15;17), t(8;21), t(14;18)9. Moreover, numerical changes (aneuploidy) in chromosomes can be detected concurrently. The corresponding template slide is also divided into 8 squares onto which metaphase spreads are bound (Figure 2), and is positioned over the OctoChrome device. The probes and target DNA are denatured at high-temperature and hybridized in a humid chamber, and then all 24 human chromosomes can be visualized simultaneously. 
OctoChrome FISH is a promising technique for the clinical diagnosis of leukemia and lymphoma and for detection of aneuploidies in all chromosomes. We have applied this new Chromosomic approach in a CWAS study of benzene-exposed Chinese workers8,10.

A novel fluorescence in situ hybridization (FISH) method that simultaneously examines both numerical and structural chromosome alterations, particularly the specific chromosomal translocations associated with leukemia and lymphoma, of all 24 human chromosomes on a single device in one hybridization, is described.

在所有人类染色体24 chromosomics：检测的数值模拟和结构上的改动同时使用一种新型OctoChrome鱼含量

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell ...

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 ...

Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass

Chromatin immunoprecipitation (ChIP) is a widely-used method for determining the interactions of different proteins with DNA in chromatin of living cells. Examples include sequence-specific DNA binding transcription factors, histones and their different modification states, enzymes such as RNA polymerases and ancillary factors, and DNA repair components. Despite its ubiquity, there is a lack of up-to-date, detailed methodologies for both bench preparation of material and for accurate analysis allowing quantitative metrics of interaction. Due to this lack of information, and also because, like any immunoprecipitation, conditions must be re-optimized for new sets of experimental conditions, the ChIP assay is susceptible to inaccurate or poorly quantitative results.
Our protocol is ultimately derived from seminal work on transcription factor:DNA interactions1,2 , but incorporates a number of improvements to sensitivity and reproducibility for difficult-to-obtain cell types. The protocol has been used successfully3,4 , both using qPCR to quantify DNA enrichment, or using a semi-quantitative variant of the below protocol.
This quantitative analysis of PCR-amplified material is performed computationally, and represents a limiting factor in the assay. Important controls and other considerations include the use of an isotype-matched antibody, as well as evaluation of a control region of genomic DNA, such as an intergenic region predicted not to be bound by the protein under study (or anticipated not to show changes under the experimental conditions). In addition, a standard curve of input material for every ChIP sample is used to derive absolute levels of enrichment in the experimental material. Use of standard curves helps to take into account differences between primer sets, regardless of how carefully they are designed, and also efficiency differences throughout the range of template concentrations for a single primer set. Our protocol is different from others that are available5-8 in that we extensively cover the later, analysis phase.

We describe a robust method for chromatin immunoprecipitation using primary T cells. The method is founded on standard approaches, but uses a specific set of conditions and reagents that improve efficiency for limited a quantities of cells. Importantly, a detailed description of the data analysis phase is presented.

生物质的使用限制金额高效的染色质免疫沉淀

Chromatin immunoprecipitation (ChIP) is a  widely-used method for determining the interactions of different proteins with  DNA in chromatin of living ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

代高通量测序技术（ChIP-seq的高品质的染色质免疫沉淀DNA模板）

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

高效，快速的早期胚胎分离<em&gt;拟南芥</em&gt;种子

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

组织特异性荧光的斑马鱼胚胎半自动成像

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...