Name: prediction and validation of gene regulatory elements activated during retinoic acid induced embryonic stem cell differentiation
Uploaded: 2016-06-21T13:00:00
Description: 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

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.

チップ-seqの分析に基づく1エンハンサーの選択<ol><li> http://ngsdebftp.med.unideb.hu/bioinformatics/からRXRのChIP-seqの生データFASTQファイル（mm_ES_RXR_24h_ATRA.fastq.gz）をダウンロード</li><li>我々の場合で（位置合わせのために必要なBWAインデックスファイルをダウンロードし、解凍します。 Mus_musculus_UCSC_mm10).(ftp://igenome:G3nom3s4u@ussd-ftp.illumina.com/Mus_musculus/UCSC/mm10/Mus_musculus_UCSC_mm10.tar.gz 注：バイオインフ?...

<ol>
	<li>Wamstad, J. A., Wang, X., Demuren, O. O., Boyer, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distal+enhancers:+new+insights+into+heart+development+and+disease.">Distal enhancers: new insights into heart development and disease.</a> Trends in cell biology. 24, 294-302 (2014).</li><li>Pennacchio, L. A., Bickmore, W., Dean, A., Nobrega, M. A., Bejerano, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancers:+five+essential+questions.">Enhancers: five essential questions.</a> Nat Rev Genet. 14, 288-295 (2013).</li><li>Mardis, E. R. <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:+welcome+to+the+new+frontier.">ChIP-seq: welcome to the new frontier.</a> Nature methods. 4, 613-614 (2007).</li><li>Muller, F., Tora, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+and+DNA+sequences+in+defining+promoters+for+transcription+initiation.">Chromatin and DNA sequences in defining promoters for transcription initiation.</a> Biochim Biophys Acta. 1839, 118-128 (2013).</li><li>Ounzain, S., Pedrazzini, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+promise+of+enhancer-associated+long+noncoding+RNAs+in+cardiac+regeneration.">The promise of enhancer-associated long noncoding RNAs in cardiac regeneration.</a> Trends Cardiovasc Med. , (2015).</li><li>Lam, M. T., Li, W., Rosenfeld, M. G., Glass, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancer+RNAs+and+regulated+transcriptional+programs.">Enhancer RNAs and regulated transcriptional programs.</a> Trends Biochem Sci. 39, 170-182 (2014).</li><li>Dickel, D. E., 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=Function-based+identification+of+mammalian+enhancers+using+site-specific+integration.">Function-based identification of mammalian enhancers using site-specific integration.</a> Nature methods. 11, 566-571 (2014).</li><li>Blum, R., Vethantham, V., Bowman, C., Rudnicki, M., 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=Genome-wide+identification+of+enhancers+in+skeletal+muscle:+the+role+of+MyoD1.">Genome-wide identification of enhancers in skeletal muscle: the role of MyoD1.</a> Genes Dev. 26, 2763-2779 (2012).</li><li>Vermunt, M. W., 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=Large-scale+identification+of+coregulated+enhancer+networks+in+the+adult+human+brain.">Large-scale identification of coregulated enhancer networks in the adult human brain.</a> Cell reports 9. , 767-779 (2014).</li><li>Bain, G., Kitchens, D., Yao, M., Huettner, J. E., Gottlieb, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cells+express+neuronal+properties+in+vitro.">Embryonic stem cells express neuronal properties in vitro.</a> Dev Biol. 168, 342-357 (1995).</li><li>Mahony, 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=Ligand-dependent+dynamics+of+retinoic+acid+receptor+binding+during+early+neurogenesis.">Ligand-dependent dynamics of retinoic acid receptor binding during early neurogenesis.</a> Genome Biol. 12 (R2), (2011).</li><li>Simandi, Z., Balint, B. L., Poliska, S., Ruhl, R., Nagy, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+retinoic+acid+receptor+signaling+coordinates+lineage+commitment+of+spontaneously+differentiating+mouse+embryonic+stem+cells+in+embryoid+bodies.">Activation of retinoic acid receptor signaling coordinates lineage commitment of spontaneously differentiating mouse embryonic stem cells in embryoid bodies.</a> FEBS Lett. 584, 3123-3130 (2010).</li><li>Li, H., Durbin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+and+accurate+short+read+alignment+with+Burrows-Wheeler+transform.">Fast and accurate short read alignment with Burrows-Wheeler transform.</a> Bioinformatics. 25, 1754-1760 (2009).</li><li>Heinz, 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=Simple+combinations+of+lineage-determining+transcription+factors+prime+cis-regulatory+elements+required+for+macrophage+and+B+cell+identities.">Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities.</a> Mol Cell. 38, 576-589 (2010).</li><li>Robinson, J. 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. M., Giguere, V., Segui, P., Evans, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colocalization+of+DNA-binding+and+transcriptional+activation+functions+in+the+human+glucocorticoid+receptor.">Colocalization of DNA-binding and transcriptional activation functions in the human glucocorticoid receptor.</a> Cell. 49, 39-46 (1987).</li><li>Untergasser, 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=Primer3--new+capabilities+and+interfaces.">Primer3--new capabilities and interfaces.</a> Nucleic Acids Res. 40, e115 (2012).</li><li>Rhead, 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+UCSC+Genome+Browser+database:+update+2010.">The UCSC Genome Browser database: update 2010.</a> Nucleic Acids Res. 38, D613-D619 (2010).</li><li>Kim, T. 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>

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

胚発生は、多くの遺伝子の活性化と抑制を含む多段階プロセスです。ゲノム中のエンハンサーエレメントは、組織及び細胞分化の間の遺伝子発現の細胞型特異的調節に寄与することが知られています。したがって、それらの同定およびさらなる調査は、細胞の運命を決定する方法を理解するために重要です。遺伝子発現データ（ 例えば、マイクロアレイまたはRNA-seqの）およびクロマチン免疫沈降（ChIPの）ベースのゲノムワイドな研究（チップ配列）の結果の統合は、これらの調節領域の大規模な同定を可能にします。しかしながら、細胞型特異的エンハンサーの機能的検証は、in vitroおよびin vivo実験手順でさらに必要とします。ここでは、アクティブなエンハンサーを同定し、実験的に検証できる方法について説明します。このプロトコルは、ステップバイステップのワークフローを提供します：1）のChIP-seqのデータ解析による調節領域の同定、2）クローニングおよびEXPERimentalレポーターアッセイで同定されたゲノム配列の推定調節の可能性の検証、およびエンハンサーRNA転写物のレベルを測定することにより、in vivoでのエンハンサー活性の3）決意。提示されたプロトコルは、ラボでは、このワークフローを設定するために誰を助けるために十分に詳述されています。重要なことに、このプロトコルは容易に適応し、任意の細胞モデル系において使用することができます。

我々はゲノムワイドRA調節遺伝子は、その近接して受容体濃縮を持っている識別するために、汎特異的RXR抗体を使用します。レチノイン酸で処理されたES細胞から得られたRXRチップ、配列データのバイオインフォマティクス解析は、RXR部位を占領下で核内受容体半部位（AGGTCA）（ 図1）の濃縮が明らかになりました。バイオインフォマティクスのアルゴリ?...

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.

レチノイン酸誘導された胚性幹細胞分化の間に活性化された予測と遺伝子調節要素の検証

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

Research

JoVE Journal

Developmental Biology

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

小分子変調分化とその後のグルコース飢餓を用いたヒト人工多能性幹細胞からの高度に精製された心筋細胞の導出

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

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

無血清単層培養中のマウス胚性幹細胞の神経分化

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

胚性幹細胞や組織の心臓分化のエピジェネティック制御

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

2および3次元の胚様体中のマウス胚性幹細胞のレチノイン酸誘発神経分化の分析

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

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

細胞分化中のマイトジェン活性化プロテインキナーゼ経路の光媒介可逆的調節<em&gt;アフリカツメガエル</em&gt;胚発生

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

人間の胚または誘導多能性幹細胞からの網膜色素上皮細胞の急速な監督の分化

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

細胞分裂時の染色体分配の生きているセルイメージ投射

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

ヒト多能性幹細胞の肝細胞への効率的な分化

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

Isolation, Propagation, and Prion Protein Expression During Neuronal Differentiation of Human Dental Pulp Stem Cells

分離、伝達、およびプリオン蛋白質発現ひと歯髄幹細胞の神経分化

Bioethical issues related to the manipulation of embryonic stem cells have hindered advances in the field of medical research. For this reason, it is very ...

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

落射蛍光マウント全体を用いたマウス胚発生中に心臓商工会議所発展の分析

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