Name: chromatin immunoprecipitation (chip) to assay dynamic histone modification in activated gene expression in human cells
Uploaded: 2010-07-29T17:00:00
Description: Watch this Scientific Journal Video about Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells at JoVE.com

この作品は、バージニア大学、バージニア大学がんセンターとトーマスF.とケイトミラーJeffressメモリアルトラストによってサポートされています。我々は、細胞株についてはジェームズE.ダーネルラボ（ロックフェラー大学）ロバートロスラボ（フォーダム大学）に感謝します。

プランニング ChIP実験が予定されている日前に、細胞は十分な数が利用できるように、培養すべきである。各ChIPアッセイでは〜1から1.5 × 10 7個の細胞が必要になることを前提としています。常に負（IgG抗体）と正（パンヒストン抗体）のコントロールと重複実験の両方を行う予定です。 ヒント - 2FTGHセルの場合、各ChIPアッセイは、80％コンフルエ...

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	<li>Levy, D., Darnell, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signalling:+Stats:+transcriptional+control+and+biological+impact.">Signalling: Stats: transcriptional control and biological impact.</a> Nature Reviews Molecular Cell Biology. 3, 651-662 (2002).</li><li>Bromberg, J., Darnell, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+STATs+in+transcriptional+control+and+their+impact+on+cellular+function.">The role of STATs in transcriptional control and their impact on cellular function.</a> Oncogene. 19, 2468-2473 (2000).</li><li>Hu, X., Ivashkiv, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-regulation+of+signaling+pathways+by+interferon-gamma:+implications+for+immune+responses+and+autoimmune+diseases.">Cross-regulation of signaling pathways by interferon-gamma: implications for immune responses and autoimmune diseases.</a> Immunity. 31, 539-550 (2009).</li><li>Campos, E. I., Reinberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histones:+annotating+chromatin.">Histones: annotating chromatin.</a> Annu Rev Genet. 43, 559-599 (2009).</li><li>Kouzarides, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+Modifications+and+Their+Function.">Chromatin Modifications and Their Function.</a> Cell. 128, 693-705 (2007).</li><li>Jenuwein, T., Allis, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translating+the+histone+code.">Translating the histone code.</a> Science. 293, 1074-1080 (2001).</li><li>Wittwer, C. T., Herrmann, M. G., Moss, A. A., Rasmussen, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+fluorescence+monitoring+of+rapid+cycle+DNA+amplification.">Continuous fluorescence monitoring of rapid cycle DNA amplification.</a> Biotechniques. 22 (130-131), 134-138 (1997).</li><li>Higuchi, R., Dollinger, G., Walsh, P. S., Griffith, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+amplification+and+detection+of+specific+DNA+sequences.">Simultaneous amplification and detection of specific DNA sequences.</a> Biotechnology (N Y). 10, 413-417 (1992).</li><li>Kroger, A., Koster, M., Schroeder, K., Hauser, H., Mueller, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activities+of+IRF-1.">Activities of IRF-1.</a> J Interferon Cytokine Res. 22, 5-14 (2002).</li><li>Ross, R. A., Spengler, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+neuroblastoma+stem+cells.">Human neuroblastoma stem cells.</a> Seminars in cancer biology. 17, 241-247 (2007).</li></ol>

概説ChIPのプロトコールは、20を超える異なるヒストン修飾アッセイするために、ラボで正常に使用されています。さらに、我々は転写因子の占有率の変化、いくつかのIFN -γ誘導される遺伝子におけるヒストン修飾酵素、ヒストン修飾やRNAポリメラーゼIIの転写機構を認識するタンパク質を、特徴付けている。我々はまた、癌幹細胞10の分化中にヒストン修飾の変化を特徴づけるための...

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<td>Fisher</td>
<td>BP118-500</td>
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<td>Pharmco</td>
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<td>Roche</td>
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<td>HEPES</td>
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<td>H3784</td>
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<td>R&amp;D Systems</td>
<td>285-IF-100</td>
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<td>Jackson Immunoresearch</td>
<td> 109-005-003</td>
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<td>N3500</td>
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<td>Anti-Histone H3, CT, pan</td>
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<td>Millipore</td>
<td>07-690</td>
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<td>Proteinase K</td>
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<td>5-Prime</td>
<td>2500140</td>
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<td>RNAse A</td>
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<td>5-Prime</td>
<td>2500130</td>
<td>&nbsp;</td>
<tr>
<td>Salmon Sperm DNA/Protein A Agarose</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>16-157</td>
<td>&nbsp;</td>
<tr>
<td>Salmon Sperm DNA/Protein G Agaorse</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>16-201</td>
<td>&nbsp;</td>
<tr>
<td>SDS</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP166-500</td>
<td>&nbsp;</td>
<tr>
<td>Tris-Cl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T5941</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Acros</td>
<td>327372500</td>
<td>&nbsp;</td>
<tr>
<td>Anti-K36me3</td>
<td>&nbsp;</td>
<td>Abcam</td>
<td>ab9050</td>
<td>&nbsp;</td>
<tr>
<td>Anti-STAT1</td>
<td>&nbsp;</td>
<td>Santa Cruz</td>
<td>sc-345X</td>
<td>&nbsp;</td>
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<td>Anti-RNA Polymerase II</td>
<td>&nbsp;</td>
<td>Santa Cruz</td>
<td>sc-899</td>
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chromatin immunoprecipitation (chip) to assay dynamic histone modification in activated gene expression in human cells

細胞外リガンドの種類に応じで、STAT（転写のシグナルトランスデューサーおよびアクチベーター）の転写因子は、急速にそれらが単一のチロシン残基1のリン酸化によって活性化される細胞表面の受容体の細胞質に彼らの潜在的な状態から採用されています。そして、彼らは二量体化と成長、分化、恒常性と免疫応答に影響を及ぼす、標的遺伝子の転写を駆動するために核内に移行。当然のことながら、正常な細胞プロセスにおけるそれらの広範な関与を考えると、STATの機能の調節不全は、特に癌2および自己免疫疾患3に、人間の病気に貢献しています。 それはよく転写が4,5クロマチン鋳型への変化によって調節されることを確立されている。これらの変化はATP依存性複合体だけでなく、共有結合性のヒストン修飾とDNAメチル化6の活動が含まれています。遺伝子発現のSTATの活性化が急速かつ一過性でもあるので、STAT依存性遺伝子座における変調クロマチンテンプレートの特定のメカニズムが必要です。これらのメカニズムを定義するには、我々は、ヒストン修飾とSTATシグナルに応答する遺​​伝子座でそれらを生成する酵素活性を特徴づける。このプロトコルは、クロマチン免疫沈降、STATが活性化する遺伝子の発現にクロマチンにシグナル伝達の研究のための貴重な方法を説明します。

Watch this Scientific Journal Video about Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells at JoVE.com

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

このプロトコルは、クロマチン免疫沈降（ChIP）はシグナル伝達経路によって誘導される転写を調節するクロマチンテンプレートに動的な変化を研究するために使用される方法について説明します。

ヒト細胞において活性化遺伝子発現のアッセイのダイナミックヒストン修飾のクロマチン免疫沈降（ChIP）

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

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Biology

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

ヒト胚性幹細胞からクロマチン免疫沈降

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

高効率で初代培養細胞をトランスフェクトするためにジーンパルサーMXcellのエレクトロポレーションシステムの使用

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

ロボット工学と植物組織における遺伝子発現の研究​​のための動的画像解析

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

ナマルバ細胞におけるIL - 4依存性遺伝子発現のsiRNAのノックダウン：遺伝子発現研究を簡素化する自動化セルカウンターの使い方

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

遺伝的関連の機能的基準をテストするための対立遺伝子特異的遺伝子発現アッセイ

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

Clonogenic Assay: Adherent Cells

クローン原性アッセイ：付着細胞

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

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

初期段階のマウス胚を用いて組織特異的遺伝子のためのクロマチン免疫沈降アッセイ

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

In vitro Reconstitution of the Active T. castaneum Telomerase

In vitro Reconstitution of the Active T. castaneum Telomerase

アクティブT.の試験管内再構成でcastaneumのテロメラーゼ

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Chromatin Immunoprecipitation ChIP using Drosophila tissue

を使用してクロマチン免疫沈降（ChIP）ショウジョウバエ組織

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

リボソーム結合したポリペプチドを単離するためのツールと​​してSECM逮捕シーケンスを使用して、

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...