The authors would like to acknowledge Dr. Bence Daniel, Matt Peloquin, Dr. Endre Barta, Dr. Balint L Balint and members of the Nagy laboratory for discussions and comments on the manuscript. L.N is supported by grants from the Hungarian Scientific Research Fund (OTKA K100196 and K111941) and co-financed by the European Social Fund and the European Regional Development Fund and Hungarian Brain Research Program - Grant No. KTIA_13_NAP-A-I/9.

1.基于芯片序列分析选择增强<ol><li>从http://ngsdebftp.med.unideb.hu/bioinformatics/下载RXR芯片起原始数据FASTQ文件（mm_ES_RXR_24h_ATRA.fastq.gz） </li><li>下载并提取对准所需的BWA索引文件（在我们的例子: Mus_musculus_UCSC_mm10).(ftp://igenome:G3nom3s4u@ussd-ftp.illumina.com/Mus_musculus/UCSC/mm10/Mus_musculus_UCSC_mm10.tar.gz 注:关于生物信息学分析的步骤参观https://github.com/ahorvath/Bioinformatics_scripts获取更多信息，并下载下面使用的脚本。 </li><li>对准例子FASTQ文件MM10基因组（使用脚本:perform_alignment.sh）。这将创建一个.bam文件和比对统计数据的文件夹中。不结盟RXR芯片起的数据（mm_ES_RXR_24h_ATRA.bam），以及相应的索引文件（.bai）都可以在这里: http://ngsdebftp.med.unideb.hu/bioinformatics/ 注:BWA是一个软件包，对齐RELAtively短序列（ 例如，芯片起的结果），以一序列数据库，如鼠标参照基因组（ 例如，MM10）13。 </li><li>润峰主叫和从头主题分析脚本（callpeaks.sh）。使用.bam文件作为输入。该脚本是基于荷马findPeaks 14。 注意:输出文件给我们关于峰的总数，富集的图案，背景的百分比，并与基序的靶序列等相关信息。 （ 图1）。通常几个主题中列出了它们的重要性顺序。 </li><li>重新映射排名第一的基序（NR半`AGGTCA`）（使用脚本:remap_motif.sh） 注意:作为结果，脚本生成.bed文件，该文件的ChIP-峰覆盖包含感兴趣的转录因子的规范结合位点的基因组区域，将显示。 </li><li>下载并可视化对准RXR芯片起读取结果（mm_ES_RXR_24h_ATRA.bam（也下载.bai））和AGGTCA基序发生（mm_ES_RXR_24h_ATRA_homerpeaks_motif1_mm10s_200_remaped_mbed.bed）通过结合基因组浏览器（IGV）15，以确定推定RAR / RXR结合位点（ 图2和图3）。 注:各种芯片起的数据整合，将有助于更精确地预测好的候选增强16,17。最近的研究表明，活性增强剂通常富集P300和H3K4me1和H3K27ac相关，并位于开放染色质区域，从而显示DNase的我超敏反应还建议18-21。 </li><li>使用在IGV 15" 定义的感兴趣区域 "，以获得200 -所选择的基因组区域的400bp的序列和用于随后的引物设计的序列。 </li></ol> 2.报告基因检测注:萤光素/萤光素酶系统用于作为一个非常敏感的报告基因检测的转录调控。取决于增强子活性萤光素酶是产生将催化萤光素氧化成氧化萤光素导致bioluminescense，它可以被检测到。作为第一步骤，确定推定的增强子序列应当亚克隆到报告载体（ 例如，TK萤光素酶22的pGL3或NanoLuc）。 <ol><li> 引物设计 <ol><li>设计PCR引物200扩增-通过引物3假定增强地区的400基点（可在这里:http://bioinfo.ut.ee/primer3/）23 </li><li>复制 - 粘贴IGV基因组序列，其中包括假定的结合位点为:引物（见步骤1.7）。集产品尺寸范围250 - 在引物3 300基点。 </li><li>验证使用UCSC基因组浏览器的PCR引物在硅片 PCR工具（https://genome.ucsc.edu/cgi-bin/hgPcr）24 注意:此协议在后面的步骤用HindIII和BamHI限制性内切酶进行克隆。检查是否由UCSC 的In-硅片的PCR工具预测将包含AAGCTT或GGATCC限制性位点的PCR扩增序列（ 例如，http://nc2.neb.com/NEBcutter2/）。 </li><li>获得从商业供应商所需的引物。 </li></ol></li><li> 从基因组DNA增强子序列的PCR扩增 <ol><li>从净化主要细胞（ 如初级胚胎成纤维细胞）的模板基因组DNA。使用基因组DNA的分离商业工具并按照制造商的说明。 注:基因组DNA的高品质是成功的PCR扩增至关重要。如果PCR是不从基因组DNA容易适当BAC克隆都可以使用。 </li><li>制备含有100ng的基因组DNA，5μl的10×缓冲液，3微升用MgSO 4（25毫摩尔），5微升的dNTP（2mM的各），1.5微升正向引物（10μM），1.5微升启引物50微升的PCR反应（10μM） ，1微升DNA聚合酶 <li>设置PCR循环（2分钟95℃（20秒95℃，10秒58 - 65℃，18秒70℃）X25重复，2分钟70°C，永远4℃）。设置退火温度为审议底漆的预计值的Tm值。 </li><li>净化与商业PCR纯化试剂盒PCR产物，并按照制造商的说明。 </li><li>通过用引物重复PCR如上使用，但对它们的5添加突出端引入用于克隆的限制性位点&#39;末端（ 例如，正向:5&#39;-ATAT AAGCTT XXXXXXXXXXXXXXXXXXXX-3&#39;（HindⅢ位），版本:5&#39;-TATA GGATCC XXXXXXXXXXXXXXXXXXXX -3&#39;（BamHⅠ位））。 </li><li>运行5微升在琼脂糖凝胶上对PCR产物的检查非特异性PCR产物。 注:如果检测到更多的频段，从而使整个PCR产物和由商业凝胶提取试剂盒净化合适的频段。 </li></o升&gt; </li><li> 限制 注意:要插入克隆TK启动22纯化的PCR产物和载体（ 例如，TK-吕克）的上游侧应用HindIII和BamHI消化。 <ol><li>制备含1微克纯化的PCR产物，1微升的HindIII（20U /微升），1微升的BamHI（20U /微升）和5微升10X缓冲液50微升反应混合物。 </li><li>制备250μl的反应混合物含有5μg的向量（TK吕克，或替代报告载体），5微升的HindIII（20U /微升），5微升的BamHI（20U /微升），5微升的热敏碱性磷酸酶（1U /微升）和25微升10X缓冲。 注:AP治疗载体的大大降低单消化的载体的自身连接，从而所述背景。 </li><li>孵育4小时，反应混合物在37℃，然后纯化消化的PCR产物和用市售的PCR纯化试剂盒的载体。 </li></ol></li><li> Ligatioñ <ol><li>设置用于连接在PCR管包括2微升10×T4 DNA连接酶缓冲液，10毫微克TK-吕克矢量，50纳克刀片（消化的PCR产物），1微升T4 DNA连接酶（400单位/微升）和无核酸酶水，反应多达20微升。 注:准备一个阴性对照，其中在反应中不包含插入片段。 </li><li>轻轻吹打起来并混合反应下来，后短暂离心使用mini-离心机的PCR管，然后在室温孵育10分钟。 </li><li>热灭活在65℃下10分钟，然后冰浴，并使用5微升用于转化的产物进50微升感受态细胞（ 例如，DH5α）。 </li><li>使用标准的热休克过程转化细菌，拿起殖民地和隔离质粒DNA与商业套件。通过之前的下一步测序验证所有的结构。 </li></ol></li><li> ES细胞的转染 <ol><li>使用48孔板。加入200微升0.1％明胶SOLUT离子/孔30分钟前，细胞电镀。 </li><li>板胚胎干细胞（ES细胞）转染的前一天。使用无饲养ES细胞和板以每孔3×10 4个细胞在250μl的ES介质/孔的密度。 </li><li>制备质粒混合物（每个组合计算了8个孔，4未处理和4视黄酸处理的） 混合1:1250纳克TK-吕克空（阴性对照），950纳克β半乳糖苷酶编码向量（的βGal）， 混合2 :1.250纳克TK-吕克的Hoxa1增强（阳性对照），950毫微克的βGal， 混合3:1.250纳克TK-吕克PRMT8增强剂（感兴趣区域），950毫微克的βGal。 注:ES细胞表达所需的转录因子（RAR / RXR）。离开井未转染的过程中的荧光素酶和β半乳糖苷酶的测量后，确定基值。 </li><li>转染的品质降低血清培养基添加到每个质粒混合高达106微升总体积（足够用于8孔）。 </li><li>加入4.5微升ES质量染离子试剂通过移液15倍上下仔细混合。在室温下孵育10分钟的转染混合物。 </li><li>加入13微升转染混合物，以每口井的细胞，吹打调匀。将细胞放回孵化器（37°C）O /配体治疗N前。 </li><li>从细胞中吸小心取出媒体，并添加250微升新鲜培养基/孔含有0.01 - 1μM全反式维甲酸（RA）作为最终的浓度或DMSO作为溶媒。孵育细胞24 - 48小时。 注:维甲酸对光敏感。 </li></ol></li><li> 细胞裂解液的制备 <ol><li>稀释5倍裂解缓冲液（1.25毫升中含0.5摩尔Tris pH值= 7.8，加入1ml的1M二硫苏糖醇（DTT）（以H 2 O），将10毫升的0.1M的EDTA的pH = 8.0，将50ml甘油，加入5ml的Triton X-100，无菌加水至100毫升）至1x使用无菌水，加入20微升的1M DTT / 10毫升并在使用前平衡至室温。制备出10毫升在测定当天的48孔板中。 </li&gt; <li>从细胞中吸小心取出介质。用1X PBS冲洗细胞一次。 200μl的1×裂解缓冲液/孔直接添加到细胞中。 </li><li>在RT（化学裂解）摇动2小时。冻结在平板上的细胞裂解物在-80℃（机械裂解）。裂解物可以储存在-80℃几天。 </li><li>解冻的裂解。完成解冻后，使用电子多道移液器转移细胞裂解物至96孔板中。转移80微升细胞溶胞产物的96孔透明板β半乳糖苷酶测定法和40微升细胞裂解物，以白色板为萤光素酶测定。避免形成任何气泡。 </li></ol></li><li> β半乳糖苷酶测定法 注:β-半乳糖苷酶或替代性的第二记者应该被用作内部对照，以占非特异性的实验变异。 <ol><li>准备β半乳糖苷酶底物缓冲液80.0克Na 2 HPO 4•7H 2 O，3.8克和 NaH 2 PO 4•H 2 0，30.0克氯化钾，将1.0g硫酸镁4•7H 2 O，使多达1000毫升无菌水。调节pH至7.4。过滤消毒（0.2微米），并存储在4℃ </li><li>混合1毫升β半乳糖苷酶底物缓冲液与4毫克ONPG（邻 - 硝基苯基β-D-半乳糖苷酶）。使用前添加3.5微升每1毫升缓冲液2-巯基乙醇（βME）的。 100μl的β半乳糖苷酶底物缓冲液添加到80μl的细胞裂解物。 </li><li>在37℃孵育反应，直到昏黄色开发。阅读在OD 420处的吸光度的酶标仪和出口数据。 注:时间取决于转染效率，通常是超过30分钟不再。 </li></ol></li><li> 萤光素酶检测 <ol><li>准备50毫升萤光素底物试剂:15.1毫克D-荧光素盐，82.7毫克ATP盐，185毫克硫酸镁4•7H 2 O，加至50ml聚丙烯管，然后加入1.5毫升1M的HEPES缓冲液，48​​.5毫升ATP免费的水，旋涡，确保所有的化合物完全溶解。保持5 - - 10毫升分装，在-80℃。 </li><li>温馨5毫升萤光素底物试剂，以RT在开始之前。吸移管将100μl底物试剂到含有40微升细胞溶胞产物的白色板的每个孔中，并立即用发光计数器机器测量信号。 </li><li>从一式三份转染至少三个独立的实验获得的活动。 </li><li>首先计算每个井的基部标准化荧光素酶活性和碱规范化β半乳糖苷酶值减去用于从测得的各值的非转染的细胞测得的平均值。然后计算萤光素酶/β半乳糖苷酶的活性的比值。 </li></ol></li></ol> 3.增强RNA的表征注:增强活动的一个更直接的指标已经从最近的基因组-W涌现该确定的许多短的非编码RNA，从50至2000个核苷酸，这是从增强剂转录大小不等，和被称为增强剂的RNA（eRNAs）IDE研究16,25,26（ 图4）。尔娜感应与相邻的外显子编码基因的诱导高度相关。因此，与信号相关 ​​的增强子活性可以在体内通过比较用RT-qPCR的各种条件之间尔娜生产定量。 <ol><li> 指引引物设计用于检测尔娜用RT-qPCR的 <ol><li>选择尔娜测量基因组区域是至少1.5 - 2 kb的距离注释转录起始位点。 注:重要的是，高含量的整个基因内增强剂的mRNA转录防止尔娜的准确定量的有义方向，在基因内增强剂反义eRNAs是可检测的，并且在基因外增强剂水平eRNAs相似。 </li><li>作为一般规则，使用地区200 - 1,000 bp的往复m为转录因子结合的任一对有义或反义链（ 图4和图6），用于引物设计的兴趣点的中心。 注意:如果GRO-以次，DNase的以次，H3K4me1，H3K4me3的或H3K27ac芯片起数据可从所需细胞类型和条件下，它可以用于更准确的引物设计。 eRNAs从特征在于高水平的H3K4me1和ME3的推定增强子区转录。 eRNAs的表达与活化蛋白增强标记H3K27ac的富集正相关。 </li><li>设计PCR引物eRNAs的基于染料的荧光RT-qPCR的测量由引物3（http://bioinfo.ut.ee/primer3/）23生成用于引物设计为每反义序列（反向有义链的互补）: http://www.bioinformatics.org/sms/rev_comp.html。插入200 - 引物设计300bp的有义或反义序列的。 </li></ol></li><li> 尔娜转录的测量 </s仲&gt; <ol><li>根据制造商的建议从处理的细胞与商业酸性苯酚/基于氯仿提取试剂分离总RNA。 </li><li>使用DNA酶I，以逆转录反应使用它们之前，对待分离的RNA。根据逆转录现有的制造商的建议，灭活脱氧核糖核酸酶。 </li><li>测量RNA浓度的DNase I处理后，用每RT反应1000纳克。使用高品质的逆转录酶。 注:由于大量eRNAs可能无法聚腺苷酸化，反转录需要使用随机引物。 </li><li>检测用标准程序逆转录尔娜用RT-qPCR的。测量用于归一非治疗依赖性的mRNA（如 36B4，PPIA，GAPDH，Actb的）。 </li></ol></li></ol>

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T., 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=Integrative+genomics+viewer.">Integrative genomics viewer.</a> Nat Biotechnol. 29, 24-26 (2011).</li><li>Daniel, B., 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=The+active+enhancer+network+operated+by+liganded+RXR+supports+angiogenic+activity+in+macrophages.">The active enhancer network operated by liganded RXR supports angiogenic activity in macrophages.</a> Genes Dev. 28, 1562-1577 (2013).</li><li>Zhu, Y., 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=Predicting+enhancer+transcription+and+activity+from+chromatin+modifications.">Predicting enhancer transcription and activity from chromatin modifications.</a> Nucleic Acids Res. 41, 10032-10043 (2013).</li><li>Visel, 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=ChIP-seq+accurately+predicts+tissue-specific+activity+of+enhancers.">ChIP-seq accurately predicts tissue-specific activity of enhancers.</a> Nature. 457, 854-858 (2009).</li><li>Heintzman, N. D., 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=Distinct+and+predictive+chromatin+signatures+of+transcriptional+promoters+and+enhancers+in+the+human+genome.">Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome.</a> Nat Genet. 39, 311-318 (2007).</li><li>Rada-Iglesias, 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=A+unique+chromatin+signature+uncovers+early+developmental+enhancers+in+humans.">A unique chromatin signature uncovers early developmental enhancers in humans.</a> Nature. 470, 279-283 (2010).</li><li>Bonn, S., 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=Tissue-specific+analysis+of+chromatin+state+identifies+temporal+signatures+of+enhancer+activity+during+embryonic+development.">Tissue-specific analysis of chromatin state identifies temporal signatures of enhancer activity during embryonic development.</a> Nat Genet. 44, 148-156 (2012).</li><li>Hollenberg, S. 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K., 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=Widespread+transcription+at+neuronal+activity-regulated+enhancers.">Widespread transcription at neuronal activity-regulated enhancers.</a> Nature. 465, 182-187 (2010).</li><li>Wang, D., 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=Reprogramming+transcription+by+distinct+classes+of+enhancers+functionally+defined+by+eRNA.">Reprogramming transcription by distinct classes of enhancers functionally defined by eRNA.</a> Nature. 474, 390-394 (2011).</li><li>Simandi, Z., 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=PRMT1+and+PRMT8+regulate+retinoic+acid-dependent+neuronal+differentiation+with+implications+to+neuropathology.">PRMT1 and PRMT8 regulate retinoic acid-dependent neuronal differentiation with implications to neuropathology.</a> Stem Cells. 33, 726-741 (2014).</li><li>Zhou, H. Y., 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=A+Sox2+distal+enhancer+cluster+regulates+embryonic+stem+cell+differentiation+potential.">A Sox2 distal enhancer cluster regulates embryonic stem cell differentiation potential.</a> Genes Dev. 28, 2699-2711 (2014).</li></ol>

The authors have nothing to disclose.

In recent years, advances in sequencing technology have allowed large-scale predictions of enhancers in many cell types and tissues 7-9. The workflow described above allows one to perform primary characterization of candidate enhancers chosen based on ChIP-seq data. The detailed steps and notes will help anyone to set up a routine enhancer validation in the lab.
The most critical step in the luciferase reporter assay is the transfection efficiency. It is recommended to include a GFP...

Development of a multicellular organism requires precisely regulated expression of thousands of genes across developing tissues. Regulation of gene expression is accomplished in large part by enhancers. Enhancers are short non-coding DNA elements that can be bound with transcription factors (TFs) and act from a distance to activate transcription of a target gene1. Enhancers are generally cis-acting and most frequently found just upstream of the transcription start site (TSS), but recent studies also described examples where enhancers were found much further upstream, on the 3' of the gene or even within the introns and exons2. 
There are hundreds of thousands of potential enhancers in the vertebrate genomes1. Recent methods based on chromatin immunoprecipitation (ChIP) provide high-throughput data of the whole genome that can be used for enhancer analysis3-9. Though data obtained by ChIP-seq experiments greatly increases the likelihood to identify cell and tissue-specific enhancers, it is important to keep in mind that detected binding sites do not necessarily identify direct DNA binding and/or functional enhancers. Thus, further functional analysis of newly identified enhancers is indispensable. In this work, we present a basic three-step process of putative active enhancer identification and validation. This includes: 1) selection of putative transcription factor binding sites by bioinformatics analysis of ChIP-seq data, 2) cloning and validation of these regulatory sequences in reporter constructs, and 3) measurement of enhancer RNA (eRNA). 
Exposure of embryonic stem (ES) cells to retinoic acid (RA) is frequently used to promote neural differentiation of the pluripotent cells 10. RA exerts its effects by binding to RA receptors (RAR&#945;, &#946;, &#947;) and retinoid X receptors (RXR&#945;, &#946;, &#947;). RARs and RXRs in a form of heterodimer bind to DNA motifs called RA-response elements, that is typically arranged as direct repeats of AGGTCA sequence (called as half site) and regulate transcription. Ligand-treatment experiments allowed the identification of several retinoic acid regulated genes in ES cells 11,12. However, enhancer elements for many of these genes has not been described yet. To demonstrate how the here-described workflow can be used for enhancer identification and validation we show step-by-step the selection and characterization of two retinoic acid-dependent enhancers in embryonic stem cells.

<table border="0" cellpadding="0" cellspacing="0" width="862">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">KOD DNA polymerase</td>
			<td style="width:121px;">Merck Millipore</td>
			<td style="width:119px;">71085-3</td>
			<td style="width:323px;">for PCR amplification of enhancer from gDNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNeasy Blood &#38; Tissue kit&#160;</td>
			<td>Qiagen</td>
			<td>69504</td>
			<td>for genomic DNA isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAquick PCR Purification kit</td>
			<td>Qiagen</td>
			<td>28106</td>
			<td>for PCR product purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gel extraction kit&#160;</td>
			<td>Qiagen</td>
			<td>28706</td>
			<td>for gel extraction if there are more PCR product</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HindIII</td>
			<td style="width:121px;">NEB</td>
			<td>R3104L</td>
			<td>restriction enzyme</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BamHI</td>
			<td style="width:121px;">NEB</td>
			<td>R3136L</td>
			<td>restriction enzyme</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FastAP</td>
			<td>Thermo Scientific</td>
			<td>EF0651</td>
			<td>release of 5&#39;- and 3&#39;-phosphate groups from DNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">T4 DNA ligase</td>
			<td style="width:121px;">NEB</td>
			<td>M0202</td>
			<td>for ligation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAprep Spin Miniprep kit</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td>for plasmid isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">DMEM</td>
			<td style="width:121px;">Gibco</td>
			<td style="width:119px;">31966-021</td>
			<td>ES media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">FBS</td>
			<td style="width:121px;">Hyclone</td>
			<td style="width:119px;">SH30070.03</td>
			<td>ES media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">MEM Non-Essential Amino Acid</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">M7145</td>
			<td>ES media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Penicillin-Streptomycin</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">P4333</td>
			<td>ES media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Beta Mercaptoethanol</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">M6250</td>
			<td>ES media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FuGENE HD&#160;</td>
			<td style="width:121px;">Promega</td>
			<td style="width:119px;">E2311</td>
			<td>transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Opti-MEM&#174; I Reduced Serum Medium</td>
			<td>Life Technologies</td>
			<td style="width:119px;">31985-062</td>
			<td>for transfection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">All-trans retinoic acid</td>
			<td style="width:121px;">Sigma</td>
			<td>R2625</td>
			<td>ligand, for activation of RAR/RXR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">96-well clear plate</td>
			<td style="width:121px;">Greiner</td>
			<td>655101</td>
			<td>for Beta galactosidase assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">96-well white plate</td>
			<td style="width:121px;">Greiner</td>
			<td>655075</td>
			<td>for Luciferase assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-luciferin, potassium salt</td>
			<td style="width:121px;">Goldbio.com</td>
			<td style="width:119px;">115144-35-9</td>
			<td>for Luciferase assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ATP salt</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">A7699-1G</td>
			<td>for Luciferase assay</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MgSO4x 7H2O</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">230391-25G</td>
			<td>for Luciferase assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">HEPES</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">H3375-25G</td>
			<td>for Luciferase assay</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Na2HPO4 x 7H2O</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">431478-50G</td>
			<td>for Beta galactosidase assay</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">NaH2PO4 x H2O</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">S9638-25G</td>
			<td>for Beta galactosidase assay</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MgSO4 x 7H2O</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">230391-25G</td>
			<td>for Beta galactosidase assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">KCl</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">P9541-500G</td>
			<td>for Beta galactosidase assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ONPG (o-nitrophenyl-&#946;-D-galactosidase)</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">N1127-1G</td>
			<td>for Beta galactosidase assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TRIzol&#174;</td>
			<td>Life Technologies</td>
			<td>15596-026</td>
			<td>RNA isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">High-Capacity cDNA Reverse Transcription Kit</td>
			<td>Life Technologies</td>
			<td>4368814</td>
			<td>reverse transcription of eRNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Rnase-free Dnase</td>
			<td style="width:121px;">Promega</td>
			<td>M6101</td>
			<td>Dnase treatment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">SsoFast Eva Green</td>
			<td style="width:121px;">BioRad</td>
			<td style="width:119px;">750000105</td>
			<td>RT-qPCR mastermix</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:300px;">CFX384 Touch&#8482; Real-Time PCR Detection System</td>
			<td style="width:121px;">BioRad</td>
			<td style="width:119px;"></td>
			<td>qPCR machine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">BioTek Synergy 4 microplate reader</td>
			<td style="width:121px;">BioTek</td>
			<td style="width:119px;"></td>
			<td>luminescent counter</td>
		</tr>
	</tbody>
</table>

prediction and validation of gene regulatory elements activated during retinoic acid induced embryonic stem cell differentiation

胚胎发育是涉及到许多基因的激活和镇压一个多步骤的过程。在基因组中的增强子元件是公知的细胞分化过程中以促进基因表达的组织和细胞类型特异性调节。因此，他们的鉴定和进一步调查，以了解细胞的命运是如何决定的重要。基因表达数据（ 例如，微阵列或RNA-SEQ）和染色质免疫沉淀（ChIP）为基础的全基因组研究（芯片起）结果的整合使得这些调控区域大规模鉴定。然而，细胞类型特异性增强剂的功能验证进一步要求在体外和体内实验程序。在这里，我们描述了活性增强剂如何可以识别和实验验证。通过芯片起数据分析，2）克隆和EXPER 1）鉴定调节区:此协议提供了一步一步的工作流程，其包括在报道实验所识别的基因组序列的推定的调节潜力imental验证，和3）测定增强子活性的体内通过测量增强RNA转录水平。所提出的协议是不够详细，帮助任何人设立这个工作流程中的实验室。重要的是，该协议可以很容易地适应，并在任何细胞模型系统中使用。

我们使用了泛特异性RXR抗体以鉴定的全基因组其类风湿性关节炎调节基因在它们的靠近受体富集。从用视黄酸处理的ES细胞获得RXR芯片起数据的生物信息学分析揭示了核受体半位点（AGGTCA）下的RXR占用位点（ 图1）的富集。使用生物信息学算法我们映射回的基序，检索结果为半位点的RXR芯片起数据（ 图2）。这种分析有助于我们准确地识别，用规范...

Watch this Scientific Journal Video about Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation at JoVE.com

Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation

In this work we provide an experimental workflow of how active enhancers can be identified and experimentally validated.

预测与基因调控元件的验证激活过程中维甲酸诱导胚胎干细胞分化

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to ...

prediction-validation-gene-regulatory-elements-activated-during

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

8.1K 次观看.  Sanford-Burnham-Prebys Medical Discovery Institute at Lake Nona. In this work we provide an experimental workflow of how active enhancers can be identified and experimentally validated.

视频: Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation

Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes available for basic research and translational applications. To differentiate hiPSCs into cardiomyocytes, various protocols including embryoid body (EB)-based differentiation and growth factor induction have been developed. However, these protocols are inefficient and highly variable in their ability to generate purified cardiomyocytes. Recently, a small molecule-based protocol utilizing modulation of Wnt/&#946;-Catenin signaling was shown to promote cardiac differentiation with high efficiency. With this protocol, greater than 50%-60% of differentiated cells were cardiac troponin-positive cardiomyocytes were consistently observed. To further increase cardiomyocyte purity, the differentiated cells were subjected to glucose starvation to specifically eliminate non-cardiomyocytes based on the metabolic differences between cardiomyocytes and non-cardiomyocytes. Using this selection strategy, we consistently obtained a greater than 30% increase in the ratio of cardiomyocytes to non-cardiomyocytes in a population of differentiated cells. These highly purified cardiomyocytes should enhance the reliability of results from human iPSC-based in vitro disease modeling studies and drug screening assays.

Here, we describe a robust protocol for human cardiomyocyte derivation that combines small molecule-modulated cardiac differentiation and glucose deprivation-mediated cardiomyocyte purification, enabling production of purified cardiomyocytes for the purposes of cardiovascular disease modeling and drug screening.

从人类诱导多能干细胞使用小分子调制分化和随后的葡萄糖饥饿高度纯化心肌推导

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes ...

科研

发育生物学

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

小鼠胚胎干细胞的无血清单层培养神经细胞分化

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

心脏分化的表观遗传调控胚胎干细胞和组织

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

二维和三维胚体小鼠胚胎干细胞的维甲酸诱导神经分化的分析

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early embryonic development, kinase activity is highly dynamic and widespread across the embryo. Pharmacological and genetic approaches are commonly used to probe kinase activities. Unfortunately, it is challenging to achieve superior spatial and temporal resolution using these strategies. Furthermore, it is not feasible to control the kinase activity in a reversible fashion in live cells and multicellular organisms. Such a limitation remains a bottleneck for achieving a quantitative understanding of kinase activity during development and differentiation. This work presents an optogenetic strategy that takes advantage of a bicistronic system containing photoactivatable proteins Arabidopsis thaliana cryptochrome 2 (CRY2) and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). Reversible activation of the mitogen-activated protein kinase (MAPK) signaling pathway is achieved through light-mediated protein translocation in live cells. This approach can be applied to mammalian cell cultures and live vertebrate embryos. This bicistronic system can be generalized to control the activity of other kinases with similar activation mechanisms and can be applied to other model systems.

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

This protocol describes an optogenetic strategy to modulate mitogen-activated protein kinase (MAPK) activity during cell differentiation and Xenopus embryonic development. This method allows for the reversible activation of the MAPK signaling pathway in mammalian cell culture and in multicellular live organisms, like Xenopus embryos, with high spatial and temporal resolution.

在细胞分化过程中光介导的可逆调节丝裂原活化蛋白激酶途径<em&gt;爪蟾</em&gt;胚胎发育

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

人胚胎或诱导多能干细胞对视网膜色素上皮细胞的快速定向分化

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

有丝分裂过程中染色体分离的活体细胞成像

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

人类多能干细胞有效分化为肝细胞

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during heart development using ventricular specific fluorescent reporter knock-in mice (MLC-2v-tdTomato mice). Heart development involves a linear heart tube formation, the heart tube looping, and four chamber septation. These complex processes are highly conserved in all vertebrates. The mouse embryonic heart has been widely used for heart developmental studies. However, due to their extremely small size, dissecting mouse embryonic hearts is technically challenging. In addition, visualization of cardiac chamber formation often needs in situ hybridization, beta-galactosidase staining using LacZ reporter mice, or immunostaining of sectioned embryonic hearts. Here, we describe how to dissect mouse embryonic hearts and directly visualize ventricular chamber formation of MLC-2v-tdTomato mice using whole mount epifluorescent microscopy. With this method, it is possible to directly examine heart tube formation and looping, and four chamber formation without further experimental manipulation of mouse embryos. Although the MLC-2v-tdTomato reporter knock-in mouse line is used in this protocol as an example, this protocol can be applied to other heart-specific fluorescent reporter transgenic mouse lines.

We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods.

全山荧光在小鼠胚胎发生过程中心脏室发育的分析

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral mesencephalon (VM). Cell replacement therapy holds great promise for treatment of PD. Recently, induced neural stem cells (iNSCs) have emerged as a potential candidate for cell replacement therapy due to the reduced risk of tumor formation and the plasticity to give rise to region-specific neurons and glia. iNSCs can be reprogrammed from autologous somatic cellular sources, such as fibroblasts, peripheral blood mononuclear cells (PBMNCs) and various other types of cells. Compared with other types of somatic cells, PBMNCs are an appealing starter cell type because of the ease to access and expand in culture. Sendai virus (SeV), an RNA non-integrative virus, encoding reprogramming factors including human OCT3/4, SOX2, KLF4 and c-MYC, has a negative-sense, single-stranded, non-segmented genome that does not integrate into host genome, but only replicates in the cytoplasm of infected cells, offering an efficient and safe vehicle for reprogramming. In this study, we describe a protocol in which iNSCs are obtained by reprogramming PBMNCs, and differentiated into specialized VM DA neurons by a two-stage method. Then DA precursors are transplanted into unilaterally 6-hyroxydopamine (6-OHDA)-lesioned PD mouse models to evaluate the safety and efficacy for treatment of PD. This method provides a platform to investigate the functions and therapeutic effects of patient-specific DA neural cells in vitro and in vivo.

The protocol presents the reprogramming of peripheral blood mononuclear cells to induce neural stem cells by Sendai virus infection, differentiation of iNSCs into dopaminergic neurons, transplantation of DA precursors into the unilaterally-lesioned Parkinson&#39;s disease mouse models, and evaluation of the safety and efficacy of iNSC-derived DA precursors for PD treatment.

预测与基因调控元件的验证激活过程中维甲酸诱导胚胎干细胞分化

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