从加州再生医学研究所（CIRM）的赠款（RS1 - 00477 - 1）财政司司长提出的工作是支持

解冻胚胎干细胞（不视频特色） ES细胞中含有10％DMSO介质被冻结。由于DMSO可诱导ES细胞的分化，它可以解冻的细胞在一天中较晚等，以改变介质的次日早晨，以尽量减少残留的DMSO的影响。 <ol><li>外套6以及组织文化与0.1％，为至少15分钟的明胶板和吸立即关闭前板细胞。 </li><li>在37℃水浴解冻胚胎干细胞（约5 × 10 6细胞，相当于一个汇合6井），并稀释成10毫升预热ES细胞培养基。 </li><li>颗粒细胞10分钟在1000转的旋转，在台式临床离心机。 </li><li>吸了10毫升37℃预热介质中轻轻悬浮细胞。 </li><li>细胞悬液转移到6孔板，生长在37 °饱和湿度5％的CO 2培养箱中的彗星。 </li><li>中等翌日更改，去除死细胞和残余二甲基亚砜。 </li></ol>旅费和扩大胚胎干细胞培养 ES细胞常规传代，每2 / 3天，中期隔日改变。因此，胚胎干细胞需要每天关注。根据我们的经验，馈线独立的ES细胞生长迅速，很快酸化媒介，把它发黄。并使细胞酸化中（不改变媒体每天或通过传代太低稀释细胞），将导致细胞经过危机，引发多余的分化和细胞死亡，之后，他们的全能性无法得到保证。在密度太低，细胞通过分散在不足，或不均匀电镀电镀细胞可引起类似的问题，细胞达到汇合之前，将形成大团块，这些团块内的细胞会分化或死亡。生殖系的传输是在受到虐待，甚至当他们出现在注射时的健康细胞明显减少。 <ol><li>对于一个融合的6孔细胞板吸介质，并用2-3毫升37℃预热PBS，吹打细胞。 </li><li>盖细胞与1毫升1 × 3-4分钟或直至细胞是均匀分散成小团块的胰蛋白酶的解决方案。 </li><li>加入1毫升的中型灭活胰蛋白酶。 </li><li>胰酶消化的细胞和板细胞（通常是良好的2 / 5）收集新鲜糊化的6孔板。 </li></ol>冷冻胚胎干细胞<ol><li> Trypsinize一个汇合6孔板（约1 × 10 7细胞）如上所述。 </li><li>收集9毫升，5分钟1000转的介质和颗粒胰酶消化细胞。 </li><li>吸1毫升新鲜配制的冻结介质中和悬浮细胞沉淀。分装成两个cryotubes 0.5毫升的细胞。 </li><li>小瓶冷冻在-80 ° C过夜，并转移到长期贮存的液氮。 </li></ol>芯片上的芯片程序免疫沉淀<ol><li>甲醛交联细胞（悬浮细胞） <ol><li>使用5 × 10 7 - 1 × 10 8各免疫细胞。 </li><li>添加新鲜的甲醛的浓度为1％的细胞悬液。 </li><li>孵化与甲醛溶液在室温下10分钟的细胞。 </li><li>添加量1 / 10 1.25米甘氨酸解渴甲醛。 </li><li> 10毫升的1X PBS冲洗细胞两次。 </li><li>池细胞在50 ml锥形管和自旋700克为5分钟，4 ° C。丢弃在1.5毫升细胞裂解液上清，重悬沉淀。转入1.5 ml离心管中的细胞。 </li></ol></li><li>闪存冻结三次在液氮中的细胞和使用组织磨床，打破细胞。一旦细胞的交联，他们可能会储存在-80℃无限期冻结。 </li></ol> 准备磁珠 （步骤都执行在4 ° C） <ol><li>加入50μLDYNAL珠1.5毫升低保留微管。设置1％免疫珠管。添加1毫升座溶液。 </li><li>收集珠使用DYNAL MPC。机架放置在磁场的管。允许珠收集管侧。这应采取约15秒。反转机架两次，以帮助收集珠。用移液器取出上清液。 </li><li>添加删除从机架的机架和反相磁条1毫升座的溶液轻轻悬浮珠，仍实行管 - 无论是10-20倍，或直至珠均匀悬浮。收集以上（步骤2）珠。用移液器取出上清液。 </li><li>在1.5毫升座溶液清洗珠，在第3步，再一次。 </li><li>重悬珠在750μL座解决方案，并添加10微克的抗体。最低为6h，或在4℃孵育过夜，上肩。 </li><li>洗净珠三个吨1毫升座解决方案中的IME，在第3步。 </li></ol>细胞超声<ol><li>从-80 ° C和转移细胞对超声管取出冷冻细胞颗粒。目前，我们宁愿使用超声一个标准polupropylene的底部，15毫升锥形管。我们切管两件在5毫升标记和丢弃的上半部。管，可覆盖封口膜或管帽而设立。 </li><li>使用离心管架，管的位置，所以坐在sonicator探头约0.5-1.0厘米以上的试管底部。小心探头为中心，不接触管双方。 （影响的解决方案是否泡沫或没有在超声探头定位，通常情况下，发泡表明，超声的DNA将会很差剪​​切。） </li><li>超声期间暂停第20号第6次样品应存放在一个冰水浴在超声。为了减少起泡，最初设定的输出功率为0，并增加手动先是一阵，最终的功率。 （如果有显着的泡沫，所有泡沫可以被删除，离心20,000克，温柔的所有材料的再悬浮，没有留下泡沫的气泡。） </li><li> 20,000克的自旋为10分钟，4 ° C至颗粒碎片和收获上清液1.5毫升管。 </li><li>从每个输入的DNA样本保存50μL细胞裂解液。储存在-20 ° C。至少有一个输入的DNA分装应保持每批超声裂解。注意：只能交联逆转和DNA纯化后检查，超声和产生的片段大小分布的影响的影响。 </li></ol>染色质免疫沉淀<ol><li>添加染色质等值金额的5 × 10 7 - 1 × 10 8细胞从细胞超声，第4步到50μL抗体/磁珠组合最终体积为1 mL（可从准备磁珠 ，第6步裂解液，如果有的话）可能调整。上肩孵育过夜，4 ° C。 </li></ol>洗<ol><li>收集珠使用DYNAL MPC。在机架上放置管。允许珠收集管侧。这应该是采取约20秒。反转机架两次帮助收集所有珠。用移液器取出上清液，改变样本之间的秘诀。 </li><li>加入1毫升每管的细胞裂解液，轻轻悬浮珠。这可以通过删除从机架的机架和反相磁条，仍然在地方管 - 10-20次，或直至均匀重悬珠。收集珠子。由移液器取出上清液。重复洗5次以上，改变之间的洗涤提示。 </li><li>洗净珠在1毫升IP1缓冲区，在步骤2中所述，的6倍。 </li><li> 1毫升IP2的缓冲液洗珠的6倍，在步骤2中所述。 </li><li>洗净珠在1毫升TE 8.0缓冲区，在步骤2中所述，的6倍。 </li><li>在960克自旋为3分4 ° C和清除残留的TE缓冲液。 </li></ol>洗脱<ol><li>从珠中添加了65℃，15分钟的水浴中孵育管210μL洗脱缓冲液洗脱材料。涡简要每2分钟。这潜伏期可以延长，只要30分钟，这可以帮助提高回收洗脱液。 </li><li>在室温下1分钟在16000克离心珠。 </li><li>取出200μL上清转移到新管。材料可以被冻结在-20 ° C，存储在一夜之间。 </li></ol>交联逆转<ol><li>反向交联的免疫DNA 洗脱 ，第3步，在65℃孵育至少6小时和15小时（交联逆转更长的时间通常会导致增加的微阵列分析噪声）的最大。这潜伏期可以做到的烘箱中，使管受热均匀，有凝结而成较少。 </li><li>解冻后超声保留输入DNA（13步）50μL，加150μL洗脱缓冲液和混合（3卷）。逆向孵化在65 ° C的交联逆转 ，第1步输入的DNA交联。从这一点来说，每管免疫或输入的DNA被认为是一个单独的管或以后的处理步骤的样品。 </li></ol>纯化的DNA <ol><li>添加10毫克ml - 1的RNaseA（0.2毫克ml - 1的最终浓度），8μL混合颠倒几次管和37 ° C为2小时。 </li><li>加入4μL20毫克ML - 1蛋白酶K（ML - 1终浓度为0.2微克）和混合逆变管数次，在55 ° C孵育2小时。 </li><li>加入400μL酚：氯仿：异戊醇（P：C：IA），涡和2毫升重Phaselock管（Eppendorf公司提供的指示）不同的阶段。 </li><li>如果P：C：IA解决方案是旧的，或在低pH值条件下，将会有degradation的DNA，造成噪音在微阵列分析和检测的有效目标的损失。 </li><li>水层转移到新的离心管中，含有16微升5M氯化钠（200毫米）的最终浓度）和1.5μL20μμL- 1的糖原（30μg总）。 </li><li>加入800μL乙醇。孵育过夜，在-20 ° C或30分钟在-80 ° C。 </li><li>旋转20,000 10分钟克在4 ° C沉淀DNA。通过加入500μL80％乙醇，涡旋重新悬浮颗粒和纺纱再次在20000克在4 ° C 5分钟洗净颗粒</li><li>删除任何剩余的80％乙醇。旋转管简要收取任何剩余的液体，并删除液体pipetteman，避免沉淀。让管空气干燥，直到颗粒干燥颗粒仍然保留着湿润的外观。在70 10毫米的Tris - HCl，pH值8.0μL重悬沉淀。 </li><li> Overdrying这些颗粒可以使他们很难重悬，或片状剥离管方承担责任。 </li><li>可选：15μl免疫沉淀样品的保存供日后使用。这种材料可以用来进行基因特异性PCR基因芯片结果的确认。 </li><li>量化紫外吸收光谱（260纳米）的浓度。 </li></ol>注：库准备和扩增使用自Sigma 10X dNTPs混合放大器在不加修改的GenomePLex WGA的工具包，包括以下步骤： 图书馆准备可在自Sigma 10X放大器组合GenomePLex WGA的试剂盒<ol><li> 1X图书馆制备缓冲液中加入2μL，10μLDNA样本的纯化 DNA，在PCR管中的步骤11。 </li><li>添加1μL图书馆稳定解决方案。 </li><li>涡彻底，离心巩固，并在95个热循环地点° C为2分钟。 </li><li>清凉冰样品，巩固离心，离心，然后返回冰。 </li><li>新增图书馆制备酶1μL，旋涡彻底，然后短暂离心。 </li><li>放置在一个热循环仪和孵化为样本如下： <ul><li> 16℃20分钟</li><li> 24 ° C为20分钟</li><li> 37℃20分钟</li><li> 75℃5分钟</li><li> 4℃举行</li></ul></li><li>从热循环仪和离心样品。样品可立即放大，或储存在20 ° C三天。 </li></ol>放大可在自Sigma 10X放大器组合GenomePLex WGA的试剂盒<ol><li>一个主混音准备从第3步到15μl反应体系中加入以下试剂，如下： <ul><li> 7.5μl的10X放大器组合（无dNTPs） </li><li> WGA的DNA聚合酶5.0μL </li><li> 3.0μL10毫米的dNTP（每件）股票（终浓度为0.4毫米） </li><li>使最终反应体积为75μL无核酸酶水</li></ul></li><li>涡彻底。短暂离心，并开始热循环。 <ul><li>预变性95℃3分钟</li><li>执行14个周期如下： </li><li>变性94℃15秒</li><li>退火/延伸65℃5分钟</li></ul>骑自行车是完成后，保持在4反应° C或储存在-20 ° C，直到准备进行分析或净化。 </li><li>净化从Quiagen ®试剂盒PCR纯化的DNA，根据制造商的指示和量化紫外吸收光谱（260纳米）的浓度。 </li><li>再次执行周期从图书馆准备扩增，通过扩增 ，第2步1步，与下面的修改。 •关于从第3步，上面开始碎裂过程所需的DNA量：如果DNA的浓度大约是200-300微克/毫升，使用2.5μL的样品和水调整最终体积为10μL。 如果DNA浓度约50-60微克/毫升，使用5μL的样品和水调整最终体积为10μL。 •在这第​​二个放大过程中，与主结构dTTPs dNTPs是一个包括10毫米的dATP，dGTP 10毫米，10毫米dCTP和8毫米dTTP的和在相同浓度下，前面的混合2毫米的dUTP混合交换。 </li><li>测量DNA用紫外可见分光光度计（260纳米）。通常情况下，大于9微克扩增的DNA是从每一个反应。 注：标签的DNA目标是与基因芯片®聚双链DNA终端Affymetrix公司的标签根据试剂盒制造商的指示，分述如下： </li></ol>片段扩增目标<ol><li>片段样品，用适当的表格，下面根据数组类型的目标将是混合动力通信指定到。 表1。单一阵列的碎片混合<table border="1"><tbody><tr><td class="jove_content"> 组件数量/金额1 RXN </td></tr><tr><td class="jove_content">双链DNA 7.5微克</td></tr><tr><td class="jove_content"> 10X的基因碎片4.8μL </td></tr><tr><td class="jove_content"> UDG的，10 U /μL1.5μL </td></tr><tr><td class="jove_content"> APE 1，100 U /μL2.25μL </td></tr><tr><td class="jove_content">无核酸酶水至48μL </td></tr></tbody></table>采用的基因芯片®聚双链DNA终端从Affymetrix公司的标签套件 表2。多阵列组的碎片混合<table border="1"><tbody><tr><td> 组件数量/金额1 RXN </td></tr><tr><td>双链DNA 9微克</td></tr><tr><td> 10X的基因碎片4.8μL </td></tr><tr><td> UDG的，10 U /μL1.5μL </td></tr><tr><td> APE 1，100 U /μL2.25μL </td></tr><tr><td>无核酸酶水多达48个</td></tr></tbody></table>采用的基因芯片®聚双链DNA终端从Affymetrix公司的标签套件</li><li>根据以上任一表碎片混合。弗里克组合和自旋向下的管子。 </li><li>孵化的反应： <ul><li> 37℃1小时。 </li><li> 93℃2分钟。 </li><li> 4℃至少2分钟。 </li></ul></li><li>弗里克的组合，自旋向下的管道，并转移到一个新的管45μL的样品。 </li><li>取出凝胶移位分析每个样品2μL。 </li></ol>标签零散的双链DNA <ol><li>准备的双链DNA标签组合如下表所述： 表3。双链DNA标签混合组成<table border="1"><tbody><tr><td> 组件数量/金额1 RXN </td></tr><tr><td> 5X TDT缓冲区12μL </td></tr><tr><td> TDT 2μL </td></tr><tr><td> DNA标签试剂，5毫米1μL </td></tr><tr><td>总体积15μL </td></tr></tbody></table>采用的基因芯片®聚双链DNA终端从Affymetrix公司的标签套件</li><li>加入15μL的双链DNA标记混合的DNA样本，弹簧组合，旋转下来。 </li><li>孵化的反应： <ul><li> 37℃60分钟。 </li><li> 70 ° C时为10分钟。 </li><li> 4℃至少2分钟。 </li></ul></li><li>取出凝胶移位分析每个样品2μL。 </li></ol>凝胶移位分析<ol><li>准备了4％的琼脂糖凝胶。 </li><li>孵育样本片段扩增的目标 ，步骤5 和标签零散的双链DNA，第4步在65 ° C为2分。 </li><li>添加10μL链霉亲和素（1毫克/毫升），并在室温下5分钟的孵育。 </li><li>运行步骤3在4％琼脂糖凝胶（ 凝胶移位分析 ，第1步）的样本，以检查的标志产品的转变迁移。 </li></ol>阵列杂交及洗涤阵列由加州大学河滨基因组的中心。 阵列分析执行的计算分析。 缓冲组合物： 座解决方案：PBS + 0.5％牛血清白蛋白（BSA）。 裂解缓冲液：50毫米的HEPES - KOH，pH值7.5 140毫米氯化钠 1毫米EDTA 1％的Triton X - 100 0.1％SDS 1MM PMSF 最终pH值7.5 IP1：裂解液+ 500毫米氯化钠 IP2：10毫米的Tris - HCl 250 mM的氯化锂 1毫米EDTA 0.5％NP - 40 0.5％的钠Dioxycholat 最终pH值8.0 TE：10毫米的Tris，pH值7.4 1毫米EDTA 最终pH值8.0 洗脱缓冲液：TE缓冲液+ 1％SDS。

<ol>
	<li>Evans, M. J., Kaufman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+in+culture+of+pluripotential+cells+from+mouse+embryos.">Establishment in culture of pluripotential cells from mouse embryos.</a> Nature. 292, 154-156 (1981).</li><li>Boyer, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polycomb+complexes+repress+developmental+regulators+in+murine+embryonic+stem+cells.">Polycomb complexes repress developmental regulators in murine embryonic stem cells.</a> Nature. 441, 349-353 (2006).</li><li>Lee, T. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+developmental+regulators+by+Polycomb+in+human+embryonic+stem+cells.">Control of developmental regulators by Polycomb in human embryonic stem cells.</a> Cell. 125, 301-313 (2006).</li><li>Kornberg, R. D., Lorch, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Twenty-five+years+of+the+nucleosome,+fundamental+particle+of+the+eukaryotic+chromosome.">Twenty-five years of the nucleosome, fundamental particle of the eukaryotic chromosome.</a> Cell. 98, 285-294 (1999).</li><li>Guenther, M. G., Jenner, R. G., Chevalier, B., Nakamura, T., Croce, C. M., Canaani, E., Young, R. A. <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+Hox-specific+roles+for+the+MLL1+methyltransferase.">Global and Hox-specific roles for the MLL1 methyltransferase.</a> Proc. Natl. Acad. Sci. U. S. A.. 102, 8603-8608 (2005).</li><li>Lee, T. I., Johnstone, S. E., Young, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+immunoprecipitation+and+microarray-based+analysis+of+protein+location.">Chromatin immunoprecipitation and microarray-based analysis of protein location.</a> Nat. Protoc. 1, 729-748 (2006).</li><li>Sanchez-Elsner, T., Gou, D., Kremmer, E., Sauer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noncoding+RNAs+of+trithorax+response+elements+recruit+Drosophila+Ash1+to+Ultrabithorax.">Noncoding RNAs of trithorax response elements recruit Drosophila Ash1 to Ultrabithorax.</a> Science. 311, 1118-1123 (2006).</li></ol>

染色质免疫沉淀（ChIP）提供了有价值的技术的染色质为主的过程，在细胞分化中的夹层。这种方法的成功的先决条件是良好的抗体和控制的细胞或组织，缺乏利益抗原染色质的可用性。可结合的DNA微阵列技术，大量的信息芯片。验证芯片结果取决于合适的测试系​​统的可用性，可以链接与动态关联的蛋白质，蛋白质修饰和/或RNA的生物过程在细胞发育的执行。

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Poteinase-K</td>
<td>Reagent</td>
<td>Roche</td>
<td>#EO0491</td>
<td>&nbsp;</td>
<tr>
<td>RNase-A</td>
<td>Reagent</td>
<td>Fermentas</td>
<td>#EN0531</td>
<td>&nbsp;</td>
<tr>
<td>formaldehyde 37%</td>
<td>Reagent</td>
<td>EMD</td>
<td>FX040-13</td>
<td>&nbsp;</td>
<tr>
<td>Chloroform</td>
<td>Reagent</td>
<td>EMD</td>
<td>3150</td>
<td>&nbsp;</td>
<tr>
<td>Ethanol </td>
<td>Reagent</td>
<td>Goldshield</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>phenol:chloroform:isoamyl alcohol</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15593-031</td>
<td>&nbsp;</td>
<tr>
<td>SA, fraction V</td>
<td>Reagent</td>
<td>EMD</td>
<td>2930</td>
<td>&nbsp;</td>
<tr>
<td>Dubecco's Phosphate Buffered Saline</td>
<td>Reagent</td>
<td>Gibco</td>
<td>14190</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>Reagent</td>
<td>EMD</td>
<td>5330</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Chloride</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>S271-10</td>
<td>&nbsp;</td>
<tr>
<td>EDTA</td>
<td>Reagent</td>
<td>EMD</td>
<td>4050</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100 </td>
<td>Reagent</td>
<td>EMD</td>
<td>9410</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Dodecyl Sulfate</td>
<td>Reagent</td>
<td>J.T Baker</td>
<td>4095-02</td>
<td>&nbsp;</td>
<tr>
<td>Lithium chloride</td>
<td>Reagent</td>
<td>EMD</td>
<td>5910</td>
<td>&nbsp;</td>
<tr>
<td>Tris-HCl</td>
<td>Reagent</td>
<td>EMD</td>
<td>9230</td>
<td>&nbsp;</td>
<tr>
<td>NP-40</td>
<td>Reagent</td>
<td>Calbiochem</td>
<td>492015</td>
<td>&nbsp;</td>
<tr>
<td>Deoxycholic acid, sodium salt</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>BP349-100</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium acetate</td>
<td>Reagent</td>
<td>Applichem</td>
<td>631-61-8</td>
<td>&nbsp;</td>
<tr>
<td>Glycine</td>
<td>Reagent</td>
<td>EMD</td>
<td>4840</td>
<td>&nbsp;</td>
<tr>
<td>Glycine</td>
<td>Reagent</td>
<td>EMD</td>
<td>4840</td>
<td>&nbsp;</td>
<tr>
<td>dynalbeads, protein A</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>100.02D</td>
<td>&nbsp;</td>
<tr>
<td>dynalbeads, protein G</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>100.04D</td>
<td>&nbsp;</td>
<tr>
<td>1.5ml Low Retention Microtubes, Xtreme</td>
<td>Other</td>
<td>Phenix Research Products</td>
<td>MAX-815S</td>
<td>&nbsp;</td>
<tr>
<td>Phase Lock Gel Light, 2.0 ml</td>
<td>Reagent</td>
<td>Eppendorf</td>
<td>955154037</td>
<td>&nbsp;</td>		</tbody>
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chromatin immunoprecipitation from human embryonic stem cells

无数后生生物体的细胞功能和结构的复杂性来自少数的干细胞。干细胞是由两个基本属性：自我更新和多能，让一个干细胞分化成几乎任何类型的细胞1的特点。进展干细胞分化的细胞的特点是多能，结构和形态的变化和分层活动的转录因子和信号分子，其活动建立和维持细胞类型特异性基因表达模式的损失。在分子水平，细胞分化涉及到染色质的结构和组成的动态变化，这些动态变化的检测，可以提供有价值的见解干细胞和细胞分化过程中2,3的功能特性。染色质是一个高度压缩的DNA -蛋白质复合物的形式时，细胞包染色体DNA与蛋白质结合，主要是组蛋白4。已与特定阵列调节蛋白，如表观遗传因素，组蛋白变体，和转录因子 2,3,5存在Stemcellness和细胞分化。 染色质免疫沉淀（ChIP）提供了一个宝贵的方法来监视存在的RNA，蛋白质，以及蛋白质修饰在染色质6,7。从不同类型的细胞染色质的比较，可以澄清在细胞分化过程中发生的蛋白染色协会中的动态变化。 染色质免疫沉淀涉及在体内交联的染色质净化。孤立的染色质酶消化或机械力降低到更小的片段。使用特异性抗体沉淀目标蛋白质或蛋白质和DNA修改的染色质片段。净化和沉淀的DNA或RNA作为模板，PCR或DNA芯片检测。一个成功的芯片的先决条件是高品质的抗体所需的抗原和不明确的目标分子控制细胞的染色质可用性。芯片可以关联的蛋白质，蛋白质和RNA的修改，并与特定的目标DNA的RNA的存在，取决于outread工具的选择上，检测目标分子在特定的靶基因在整个基因组的范围内的协会。分化细胞的染色质的蛋白质分布的比较，可以澄清配合沿细胞谱系细胞的进展的染色质成分的动态变化。

Watch this Scientific Journal Video about Chromatin Immunoprecipitation from Human Embryonic Stem Cells at JoVE.com

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

恰逢的ESC分化与细胞类型的具体结构的变化和染色质组成。定义stemcellness和细胞分化的机制，这些变化的检测提供了有价值的见解。染色质免疫沉淀（ChIP）代表一个有价值的方法来剖析底层干细胞分化的分子机制。

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-from-human-embryonic-stem-cells

Research

JoVE Journal

Biology

20.6K 次观看.  University of California - Riverside. 恰逢的ESC分化与细胞类型的具体结构的变化和染色质组成。定义stemcellness和细胞分化的机制，这些变化的检测提供了有价值的见解。染色质免疫沉淀（ChIP）代表一个有价值的方法来剖析底层干细胞分化的分子机制。

视频: Chromatin Immunoprecipitation from Human Embryonic Stem Cells

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Yeah, so I'm Lauren Deon. I'm in charge of the Human Amgen stem cell co facility at MGH. Yeah, so I'm Lauren, I'm in charge of the Human AM stem cell co facility at MGH.So I've been working with human Amgen stem cells for years now since I was a postdoc in George De Lab before, from four, four years. And so right now we have two cell lines, H one and H nine, which are both from Y cell. And so the way we are expanding or maintaining these cells in culture is by, at least the way we are splitting these cells is by using collagenous.So what I usually do is for the first few passage after throwing the cells, I use a technical, the picking colonies. So in this case, I can check under the microscope which colonies are good and differentiate and nice shape, and I will collect only these colonies and I will cut them in few pieces and put them back in, in a fresh flat. And the, the colonies that look kind of differentiate it are just left them alone.The other way to pass the cells is by using trypsin. So I don't use this technique here. I think collagenase is a little bit safer because human being stem cells don't like to be individualized or they don't like to be alone.So if you do trypsin, if you use trypsin for a little bit too long, if you use it for like three to four minutes, which is the way we use strips in for other type of cells, human stem cells will be individualized and most of them will die or just differentiate or will not attach to the myth. So the idea is you have to keep the cells as clumps. You don't want them to be alone in the in, in the flask.So using collagenase, the advantage is to keep cells as plants. Using chipsy, you just select for cells that will survive this very, very harsh process of g opsonization. So these cells are usually very highly proliferative.So you get, you can adapt your cells to this tripsin process. The problem is that you have cells that they behave differently, they go much faster. And one risk is that you can get some commercial abnormality to, to this, to this very high division process.So you say that ization can cause selection of cells in culture for most proliferative cells.Right. More ative and probably also higher survival rates than other cells. So after proliferation, basically culture after proliferation in tripsin, after several passages become a different culture, these different properties.So it's, it's hard to figure out if they are exactly identical. If you do the test, the usual test, let's say you check markers, regular markers of end differentiated cells like OC four, nano one 60 SC four C, C3, all this molecule might still be expressed. If you do TER formation, this cells might still differentiate and be formed like al like normal cells.If you look at the karyotype, you might have some abnormality, but you might still have a normal karyotype. The, the, the selective advantage might be due to just a mutation that you cannot see by doing a karyotype. So the fact that they grow much faster is by itself I think a little bit worser.And if we want to use these cells for transplantation at some point, I would definitely not use this type of cells.Okay. So would you like to tell me what are the main technical problems with using this technique for, for passages of cells? So using S, yes.It's just a little bit longer and also it's, since the cells are not going as fast, it's just much longer to get a high number of cells to do some experiment. So it's just less convenient I would say. Would you like to tell me about few words about the goals of your experiment?How do you use CS cells? Okay, so my main project here is to get endothelial cells from human AB stem cells. And the idea is to be able to make blood.And we are working a lot with tissue bioengineering labs and one of their goal is to make tissue in vitro. But one of the limitation to this is the lack of blood vessels going through this tissues. So if we can bring endothelial cells or put endothelial cells in this tissue, we would have better growth of this tissue.So the idea few sources of cells are known for endothelial cells like cold blood or pressure blood, but it would be interesting to have another source of cells like human years. And the advantage is since human years can be expanded and most infinitely, you could modify these cells, make some inducible system and be able to use it. Then in your final product, your material cells, in this case.

人类胚胎干细胞（ES细胞）的繁殖

科研

生物学

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

从小鼠胚胎干细胞的造血干细胞的推导

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

人类胚胎干细胞的推导Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

文化和维护人类胚胎干细胞

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

为核型分析人类胚胎干细胞的染色体蔓延制备

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

人类胚胎干细胞冷冻和解冻

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

胚胎干细胞分化成少突胶质前体

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation 

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

人类胚胎干细胞畸胎瘤形成命运的映射

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

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 ...

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.

Photobleaching Assays FRAP & FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.

漂白实验（FRAP和FLIP）来衡量生活胚胎干细胞的染色质的蛋白质动力学

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...