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.

Chip-seq Analiz dayanarak 1. Artırıcı Seçimi <ol><li> http://ngsdebftp.med.unideb.hu/bioinformatics/ gelen RXR ChIP seq ham veri fastq dosyası (mm_ES_RXR_24h_ATRA.fastq.gz) now </li><li> Indirin ve bizim durumumuzda (uyum için gerekli BWA dizin dosyası ayıklamak: Mus_musculus_UCSC_mm10).(ftp://igenome:G3nom3s4u@ussd-ftp.illumina.com/Mus_musculus/UCSC/mm10/Mus_musculus_UCSC_mm10.tar.gz NOT: biyoinformatik analiz aşamaları hakkında daha fazla bilgi için https://github.com/ahorvath/Bioinformatics_scrip...

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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

Embriyonik gelişim, birçok genlerin aktivasyonunu ve baskıyı içeren çok aşamalı bir süreçtir. genomu içinde güçlendirici elemanlar hücre farklılaşması esnasında gen sentezlenmesinin doku ve hücre tipi özel düzenlemeye katkıda bulunduğu bilinmektedir. Böylece, kendi kimlik ve daha fazla araştırma hücre kaderi nasıl belirlendiğini anlamak için önemlidir. Gen ifadesi verileri (örneğin, mikrodizi veya RNA-seq) ve kromatin immunoprecipitation (ChIP) tabanlı genom çalışmaları (ChIP-seq) sonuçlarının entegrasyonu bu düzenleyici bölgelerin büyük ölçekli belirlenmesini sağlar. Ancak, hücre türüne özgü arttırıcılar işlevsel doğrulama in vitro ve in vivo deney prosedürü de gerektirir. Burada aktif artırıcılar tespit ve deneysel olarak doğrulanmış nasıl açıklar. ChIP-seq veri analizi, 2) klonlama ve exper tarafından düzenleyici bölgelerin 1) tanımlanması: Bu protokol içeren bir adım-adım iş akışı sağlarimental bir raportör analizinde tanımlanmış olan genom dizilerinin varsayımsal düzenleme potansiyeli doğrulama ve güçlendirici RNA transkript seviyesinin ölçülmesiyle in vivo güçlendirici aktivitesinin 3) belirlenmesi. sunulan protokol laboratuarında bu iş akışını kurmak için kimseye yardımcı olmak için yeterli ayrıntılı. Önemli olarak, iletişim kuralı, kolayca adapte ve herhangi bir hücresel model sistemde kullanılabilir.

Biz genom hangi RA-düzenlenen genler kendi yakın reseptör zenginleştirme var tanımlamak amacıyla bir pan-spesifik RXR antikor kullanıldı. Retinoik asit ile tedavi ES hücrelerinden elde RXR ChIP seq veri Biyoinformatik analiz RXR sitesi işgal altında nükleer reseptör yarım ekibi (AGGTCA) (Şekil 1) zenginleştirme saptandı. Bir biyoinformatik algoritması kullanarak biz RXR ChIP-seq verilere yarım sitesinden (Şekil 2) için motif arama so...

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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.

Tahmin ve Gen Düzenleme Elementlerin Doğrulama Retinoik Asit Kaynaklı Embriyonik sırasında Aktif Hücre Farklılaşma Kök

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

Küçük Molekül-modüle Farklılaşma ve sonraki Glikoz şiddetli açlık kullanma İnsan uyarılmış pluripotent kök hücrelerin yüksek oranda saf kardiyomiyositlerde türetilmesi

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

Serum serbest Tek tabakalı Kültüründe Fare Embriyonik Kök Hücre Sinir Farklılaşma

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

Embriyonik Kök Hücre ve Doku Kardiyak Farklılaşma epigenetik Yönetmelik

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

İki ve Üç boyutlu Embriyoid Oluşumu Fare Embriyonik Kök Hücreler Retinoik Asit kaynaklı sinir Farklılaşma analizi

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

Hücre Farklılaştırması Sırasında Mitojenle Aktive Edilen Protein Kinaz Yolunun Hafif-Uyumlu Geri Dönüşümlü Modülasyonu ve<em&gt; Xenopus</em&gt; Embriyonik Gelişim

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

Hızlı, yönlendirilmiş farklılaşma insan embriyonik veya İndüklenmiş Pluripotent kök hücrelerinden retina Pigment epitel hücrelerinin

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

Kromozom segregasyon mitoz sırasında canlı hücre görüntüleme

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

Insan pluripotent kök hücrelerinin karaciğer hücrelerine verimli farklılaşma

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

Yalıtım, yayma ve insan diş Pulp kök hücrelerin Nöronal Farklılaşma sırasında Prion Protein ifade

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

Bütün Mount Epifluorescence kullanarak fare embriyogenez sırasında kalp odası gelişimin analizi

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