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％コンフルエントで15約一cmの組織培養皿が必要です。 PART 1：クロマチンを調製し、チップのPERFORM <ol><li>培地を取り出し、5 ng / mLのでIFN -γを含有する10％宇宙の子牛血清（CCS）/ DMEM 20mLで必要な時間のために細胞を誘導する。 CCS / DMEMに加え10％の20mLで非誘導プレート上でメディアを交換してください。 </li><li>ドラフトでは、15cmの皿の中でメディアを20mL（1％最終濃度）に540μLの37％ホルムアルデヒドを追加。 ヒント -チルトプレートとメディアプールにホルムアルデヒドを追加-均等にそれを広めるためにしてロックプレート。 </li><li>室温で10分間進行さ架橋を許可する。 </li><li>架橋を停止するには、0.125 Mの最終濃度にグリシンを追加。 </li><li>真空は、培地を吸引除去し、緩衝生理食塩水（PBS）を10mLの氷冷リン酸で細胞を1回洗浄する。 </li><li> 〜7 mLのPBSでセルスクレーパーで細胞を回収し、氷上で50 mLコニカルチューブに同じ処理した細胞を（6プレートから）組み合わせる。 </li><li> 4℃で1800rpmで遠心分離（ベックマンCoutlerアレグラ12 - R遠心）℃で5分間とPBSを吸引除去する。 ヒント -スナップ凍結細胞ペレットであればここに停止し、-80℃で保存します。 </li><li> 4 mLの膨潤バッファー（25mMのHEPES pHは7.8、1.5mMのMgCl 2、10mMの塩化カリウム、0.1％NP40。1mMのDTT、完全なプロテアーゼ阻害剤と使用前に氷上で寒さを追加）で細胞ペレットを再懸濁し、15に転送するmLコニカルチューブ。 ヒント -この膨潤バッファーの量は、6プレートプール（〜6月9日× 10 7細胞）に適しています。多かれ少なかれ、バッファがdouncing効率が変わることがあります。 </li><li> 10分間氷上で細胞懸濁液をインキュベートする。 </li><li>あらかじめ冷却した15 mLのdouncer（ウィートン）に懸濁液を移すと乳棒Aで20ストロークをダウンス</li><li>転送には氷の上に15 mLコニカルチューブに戻し、細胞をdounced。 </li><li> 4℃で5分間2500rpmで遠心℃、上清を捨てる。 </li><li>超音波の緩衝液3mlで核ペレットを（6枚から〜200μL）（15ペレットの量）（50mMのHEPES pHは7.9、140mMのNaCl、1mMのEDTA、1％トリトンX - 100、0.1％デオキシコール酸ナトリウム、再懸濁します。 0.1％SDS。）使用直前に氷の上で完全なプロテアーゼ阻害剤と寒さを追加。 ヒント -この超音波処理バッファーの量は、6プレートプール（〜6月9日× 10 7細胞）に適しています。多かれ少なかれバッファは、超音波処理の効率が変わることがあります。 </li><li>マイクロチップのついたMisonixのソニケーター3000を使用して10サイクル（20秒8の設定on/40秒オフ/振幅）を氷上で核懸濁液を超音波処理してください。これは、通常サイズで200〜1000 bpsを平均化クロマチンの断片をもたらす。 ヒント - sonicatorsが異なるため、超音波処理の効率が経験的に決定する必要があります。そのためには、超音波処理の前に、各超音波処理のサイクル後に20μLのアリコートを取る。 4時間65℃で、これらのサンプルを加熱する。 DNAのローディングバッファーを追加し、DNA断片を可視化するために標準の1％アガロース/ TBEゲルを実行してください。） </li><li>転送は、4℃で20分間SS34遠心管と遠心する材料を超音波処理° SS34ローター（ソーバルRC6プラス遠心）で13000rpmでCペレットに、通常細胞の残骸の小さい量が何であるか。 </li><li>慎重に氷の上に15 mLコニカルチューブに上清を移す。 </li><li> 360μLサケ精子DNA /プロテインAまたはGアガロースビーズのスラリーを追加し、少なくとも30分間、4℃で揺れによる事前クリア。 ヒント -この手順では、非特異的な結合事象から発生するアッセイでのノイズを減少させる。 </li><li>ペレットアガロースビーズへの5分間4℃で1000rpmで遠心する。 </li><li>氷の上に別の15 mLコニカルチューブに上清をあらかじめクリアに転送します。 </li><li>事前にクリアクロマチンのA260ユニットを測定し、4（4）A260ユニット（0.2 mg / mLの1 A260ユニット= 0.050 mg / mlのを前提）に超音波処理バッファーを使用して、音量を調節する ヒント - A260の単位を測定するには、190μlのDDWで事前にクリアクロマチンの10μLの希釈し、190μlのDDWで10μL超音波処理のバッファを持つ空白の分光光度計を。 </li><li> 20℃入力のサンプルとストアとしてクロマチンの75μLを（4 A260単位で）保存° C架橋を逆にする準備が整うまで。 ヒント -この手順では、データ解析のための非常に重要です。入力のアリコートを取ることを無視しないでください。 </li><li>私上を1.7 mLマイクロチューブに分注して調製したクロマチンを1mLのアリコートとしてCE。 ヒント -ここに停止する場合は、-80℃ドライアイスとストアの準備クロマチンを凍結スナップ℃にNBは：可能であれば保存されているクロマチンが少なく、最適な結果よりも生産する、低下することができるので、避けてください。 </li><li>重複の誘導および非誘導サンプルセットへのChIPグレード抗体を追加。誘導および非誘導サンプルセットへの陰性および陽性対照としてIgG抗体（2μg）を、パンヒストンH3抗体（15μL）を追加します。 ヒント -使用する抗体の量は、経験的に決定するかあるいは提案された量のために供給業者の製品のシートを参照してくださいする必要があります。のChIPグレード抗体の一般的に1〜2μgのが適しています。適切なボリュームは、血清として供給される抗体のために経験的に決定する必要があります。 </li><li>一晩4のチューブを揺する℃に</li></ol> PART 2：免疫複合体のCOLLECT <ol><li>すべてのチューブにサケ精子DNA /タンパク質またはプロテインGアガロースビーズのスラリー60μLを加える。 マウスモノクローナル抗体のウサギポリクローナル抗体とプロテインGのためのヒントを使用するタンパク質。 </li><li>少なくとも60分間、4 ° Cでロック。 </li><li> 4℃で2000rpmで静かにmicrofugingによるペレットは免疫複合体（アガロースビーズ）℃で3分間。 </li><li>上清を吸引除去する。 </li><li> 4℃を揺動し、10分間、次洗浄バッファーの各1mLで免疫複合体を洗う4℃、2000rpmで3分間、マイクロ·各洗浄し、上清を吸引間C。氷上で冷却し、サンプルをしてください。使用直前にPMSFを追加。 <ul><li>低塩ウォッシュ - 0.1％SDS、1％トリトンX - 100、2mMのEDTA、20mMのトリス- Cl、pHは8.0、150mMのNaCl、1mMのPMSF </li><li>高ソルトウォッシュ - 0.1％SDS、1％トリトンX - 100、2mMのEDTA、20mMのトリス- Cl、pHは8.0、500mMのNaCl、1mMのPMSF </li><li>塩化リチウムウォッシュ - 0.25 M塩化リチウム、1％NP - 40、1％デオキシコール酸ナトリウム、1mMのEDTA、10mMのトリス- Cl、pH8.0の、1mMのPMSF </li><li> TEウォッシュ - TEバッファー-10 mMのトリス- Cl、pH8.0の、1mMのEDTA、1mMのPMSFで2回洗浄</li></ul></li><li>最後のTEバッファー洗浄を削除し、ビーズに、500μlの溶出バッファー（50mMのトリス- Cl、pH8.0の、10mMのEDTA、1％SDS）を追加します。 ヒント -溶出バッファーを新しく調製する必要がある。 </li><li> 65℃30分間溶出緩衝液と熱にアガロースビーズを混在させる渦℃に ヒント -この30分間、各チューブは、少なくとも10分おきにボルテックス。 </li><li> 2000rpmで30秒間、マイクロと新しいマイクロ遠心チューブに溶離液を移す。 ヒント -溶出時には、ご入力のサンプルを解凍し、それぞれに500μlの溶出バッファーを追加します。今後すべてのステップでこれらのサンプルが含まれています。 </li><li>すべてのサンプルと渦〜200 mMの最終濃度を5 M NaClを追加します。 </li><li> 65℃で一晩（または少なくとも4時間）。 </li></ol> PART 3：PurifyをChIP&#39;dと入力のDNA <ol><li>蓋に結露を収集するために30秒間のすべてのサンプルをマイクロフュージ。 </li><li> 15分間室温で各チューブとインキュベートしてRNase Aを1μlの（10 U /μL）を追加します。 </li><li> 10μLの0.5 M EDTA、40μL、0.5 Mトリス- Cl pH6.5で、2μLプロテイナーゼK（10 mg / mL）に追加し、45℃で60分間、℃でインキュベートする。ヒントは、これらの試薬のマスターミックスを調製し、各チューブに52μLを加える。 </li><li>フェノール/クロロホルム/イソアミルアルコール抽出し、クロロホルム/イソアミルアルコールは、サンプルを抽出。 </li><li> 2μLグリコーゲン（25μg/μLの）と3M酢酸Na液（pH5.2）と渦0.1ボリュームを追加します。 </li><li> 100％エタノールと渦の2.5倍量を追加。 </li><li> 20℃で一晩。 </li></ol>パート4：PurifyをChIP&#39;dと入力のDNAつづき<ol><li> 4℃13000rpmで20分以上のための微量サンプル° C DNA /グリコーゲンペレットを沈殿させる。 </li><li> 70％エタノール1mLでDNA /グリコーゲンペレットを洗浄する。 </li><li>空気は、DNA Savant社の高速真空遠心分離でペレットまたは迅速に乾燥して乾燥させる。 ヒント -上のDNAを乾燥させないでください。 </li><li> 200μLのTE緩衝液にDNA /グリコーゲンペレットを再懸濁します。 </li><li> DNA（1μL）は、リアルタイムPCRによる定量のための準備ができています。 20店舗、残りのDNA℃に ヒント - DNA以上の1μLを使用して、リアルタイムPCR反応を阻害することができるので、注意してスケールアップ。 </li></ol>パート5：代表的な結果： チップと入力DNAサンプルは、目的の遺伝子座に特異的なプライマーを用いたリアルタイムPCR 7,8（アプライドバイオシステムズ7500 FastリアルタイムPCRシステムを応用）を用いて定量されています。に濃縮またはアッセイされるヒストン修飾に欠けていることが知られているターゲットのゲノム配列が同様に陽性および陰性コントロールとして使用することができるプライマー。データは入力のパーセントとして表されます。 ChIPの手順は完全にしてくださいR生物はヒストン修飾で報告された変化は統計的に有意であることを保証するために複製うち3 epeated。 プライマー設計、PCRの効率、および入力と正規化手順のパーセントを計算するために適切な方法を含むDNAを、定量化するためにリアルタイムPCRを使用する際に考慮すべきいくつかの重要な問題があります。遺伝子を越えヒストン修飾の占有率をプロファイル化する場合、PCRの効率は、全てのプライマーペア間で一貫している必要があります。リアルタイムPCRシステムのメーカーは、必要な情報とトレーニングを提供することができます。 図1に、クロマチンの代表的な結果は、IFN -γによって引き起こされる、IRF1遺伝子9のSTAT1活性化中に免疫沈降を示しています。 <img src="/files/ftp_upload/2053/2053fig1.jpg" alt="図1" /> 図1。 STAT1、RNAポリメラーゼIIとH3K36me3の占有はIRF1遺伝子のSTAT1活性化中に動的です。 （A、B、C）α- H3K36me3、α- STAT1と2FTGH細胞から採取したクロマチンのα-ポルIIを持ったチップはIFN -γのための30分（赤い四角）、1.5時間（青丸）、5時間（で誘導緑色の三角形）または非誘導（ブラックダイヤモンド）。 IgGは、ネガティブコントロール（グレー×印）です。（D）パンH3は、ポジティブコントロールであると誘導（青丸）での遺伝子座でヒストン占有および非誘導条件（ブラックダイヤモンド）を示しています。 -5 BP周りディップでは、転写開始部位で発見されたヌクレオソーム枯渇によるものです。

<ol>
	<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>

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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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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>37% Formaldehyde</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP531-500</td>
<td>&nbsp;</td>
<tr>
<td>Chloroform/Isoamyl alcohol</td>
<td>&nbsp;</td>
<td>Acros</td>
<td>AC32715-5000</td>
<td>&nbsp;</td>
<tr>
<td>Complete Mini Protease Inhibitors</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>1836153</td>
<td>&nbsp;</td>
<tr>
<td>Cosmic Calf Serum</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30087.04N</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30243</td>
<td>&nbsp;</td>
<tr>
<td>DTT</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D0632</td>
<td>&nbsp;</td>
<tr>
<td>EDTA</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP118-500</td>
<td>&nbsp;</td>
<tr>
<td>Ethanol</td>
<td>&nbsp;</td>
<td>Pharmco</td>
<td>zp1110EP</td>
<td>&nbsp;</td>
<tr>
<td>Glycine</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>100149</td>
<td>&nbsp;</td>
<tr>
<td>Glycogen</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>901393</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3784</td>
<td>&nbsp;</td>
<tr>
<td>IFN-gamma</td>
<td>&nbsp;</td>
<td>R&amp;D Systems</td>
<td>285-IF-100</td>
<td>&nbsp;</td>
<tr>
<td>IgG</td>
<td>&nbsp;</td>
<td>Jackson Immunoresearch</td>
<td> 109-005-003</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>&nbsp;</td>
<td>MP Biologicals</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>LiCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L4408</td>
<td>&nbsp;</td>
<tr>
<td>MgCl2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M8266</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Acetate</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP333-500</td>
<td>&nbsp;</td>
<tr>
<td>Deoxycholic Acid Sodium Salt</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP349-100</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>7647-14-5</td>
<td>&nbsp;</td>
<tr>
<td>NP40</td>
<td>&nbsp;</td>
<td>US Biological</td>
<td>N3500</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Histone H3, CT, pan</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>07-690</td>
<td>&nbsp;</td>
<tr>
<td>Phenol/chloroform/isoamyl</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>108-95-2</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate Buffered Saline</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>7647-14-5</td>
<td>&nbsp;</td>
<tr>
<td>PMSF</td>
<td>&nbsp;</td>
<td>EMD</td>
<td>50-230-0316</td>
<td>&nbsp;</td>
<tr>
<td>Proteinase K</td>
<td>&nbsp;</td>
<td>5-Prime</td>
<td>2500140</td>
<td>&nbsp;</td>
<tr>
<td>RNAse A</td>
<td>&nbsp;</td>
<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>
<tr>
<td>Anti-RNA Polymerase II</td>
<td>&nbsp;</td>
<td>Santa Cruz</td>
<td>sc-899</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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 ...

chromatin-immunoprecipitation-chip-to-assay-dynamic-histone

Research

JoVE Journal

Biology

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

28.5K ビュー.  University of Virginia. 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。

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

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

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 ...

生物学

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

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

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

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

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

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 to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

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

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits1.
This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants)2,3. Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells.
DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers4. The primer extension assay is carried out using the MassARRAY (Sequenom)5 platform according to the manufacturer's specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results.

Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.

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

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with 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

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

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

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

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

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at different developmental stages. Epigenetic regulation contributes to a broad spectrum of biological processes, including cellular differentiation during embryonic development and homeostasis in adulthood. A critical strategy in epigenetic studies is to examine how various histone modifications and chromatin factors regulate gene expression. To address this, Chromatin Immunoprecipitation (ChIP) is used widely to obtain a snapshot of the association of particular factors with DNA in the cells of interest.
ChIP technique commonly uses cultured cells as starting material, which can be obtained in abundance and homogeneity to generate reproducible data. However, there are several caveats: First, the environment to grow cells in Petri dish is different from that in vivo, thus may not reflect the endogenous chromatin state of cells in a living organism. Second, not all types of cells can be cultured ex vivo. There are only a limited number of cell lines, from which people can obtain enough material for ChIP assay.
Here we describe a method to do ChIP experiment using Drosophila tissues. The starting material is dissected tissue from a living animal, thus can accurately reflect the endogenous chromatin state. The adaptability of this method with many different types of tissue will allow researchers to address a lot more biologically relevant questions regarding epigenetic regulation in vivo1, 2. Combining this method with high-throughput sequencing (ChIP-seq) will further allow researchers to obtain an epigenomic landscape.

Chromatin Immunoprecipitation ChIP using Drosophila tissue

Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system.

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

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

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 ...