この作品は、国立精神衛生研究所（5R01MH071476）からの助成金によって支えられて。

手順： 第1回日 1。バッファをDouncingと冷凍事後灰白質組織の50から500 mgをホモジナイズする。 ！注意 - ！ヒト組織は、厳格な安全条件の下で慎重に取り扱う必要があります。それは、BSL - 2以上の安全基準で処理する必要があります。 <ol><li>以前に解剖、検死-80〜脳° C、バッファをDouncingの5倍脳容積でそれらをダウンス、および2.0 mLのマイクロ遠心チューブに行われます。マッチしたサンプルと対照のペアが同時に処理されます。 </li></ol> 2。ミクロコッカスヌクレアーゼ（MN）消化<ol><li>氷の上に配置する前にピペッティングして、ソリューション内のサンプルと混合するミクロコッカスヌクレアーゼの5U/mLを追加。 *重要なステップ- MNが偶数の4で行動する能力を持っているので、すぐにこの手順を実行することが重要℃である</li><li> 37℃で7分間サンプルをインキュベート℃に</li><li> 7分のインキュベーションの後、MNaseの活動を停止するには10mMの濃度に0.5M EDTAを加える。 </li></ol> 3。 Hypotonisation <ol><li> 15 mLのファルコンチューブにサンプルを置きます。 0.2mmのEDTA、0.2Mベンズアミジン及び0.1Mのphenylmethanesulphonylfluorideの1 / 1000のサンプルの量（PMSF）の1 / 2000サンプルの体積の10倍のサンプルボリュームを追加します。後者の二つの化合物は、プロテアーゼ阻害剤として使用されています。 * 重要なステップ-すべての手順の間に氷上でサンプルを維持することが重要です。 </li><li>それは10分ごとにボルテックスしながら、1時間サンプルをインキュベートする。 </li><li>時間長いインキュベーションの終わりに、1 / 2000 3M DTTのサンプルの量、さらに別のプロテアーゼ阻害剤を追加。 </li><li>渦のサンプルは、もう一度、4℃で10分間3175 RCFで遠心 *オプションのステップ-プロテインGアガロースでPrecleansing。 <ol><li>上清を取り出し、新しい15 mLのファルコンチューブに入れ。 </li><li>プロテインGアガロースの500μLを追加します。 </li><li> 30分間室温で回転させます。 </li><li>で10分間4℃で4000rpmで遠心する</li></ol></li><li> 500μLを入力コントロール（のみゲノムDNAを含む）として使用されるように上清を配布し、残りはサンプルごとの1600μLを含む2つのチューブに分割されます - クロマチン免疫沈降（ChIP）サンプル用です。 </li><li>入力コントロールは、さらに使用するまで-80℃CO / Nに配置されます。 </li><li> ChIPサンプルに - して抗体の10XFSBと4μgの1:10体積、渦のサンプルを追加し、4℃で一晩、それらを回転させる。 ！注意 - ！抗体の量と希釈は、最適化が必要な場合があります。 </li></ol>第2日目 ！注意！ -ヌクレオソームDNAを単離するために使用されるプロテインGアガロースを洗浄することによって第2日が始まります 。アガロースビーズは非常に敏感であるので、アガロースビーズを含むソリューションをピペッティングするたびにヒントの頭を切断する必要があります。 1。 DNAにプロテインGアガロースビーズをプロービング<ol><li> 2 mlのマイクロチューブに245μLのプロテインG -アガロース（イナフ4チューブ用）（ループ）に1.6mlの1XFSBを追加。 </li><li> 2つの2 mLチューブと補充1X FSBでそれぞれ最大1.6 mLまでに解決策を区切ります。 </li><li> 30秒間室温で回転させると30秒間に0.1 RCFで遠心。 </li><li>真空を用いて上清を取り除く。 </li><li>もう一度1.6mlの1X FSBを追加。 30秒間のサンプルを回転させ、その後30秒間に0.1 RCFで遠心。 </li><li>再び上清を除き、および1.5 mLの1XFSBで両方のチューブを組み合わせる。 </li><li> 15μLサケ精子DNA（10mg/mL）を追加します。 ！注意 - ！このステップは、原理的には、ビーズの免疫沈降物の非特異的結合を減らす必要があります。しかし、これはまた、（人間の）DNA配列のいくつかの誤検知につながる可能性があります。 </li><li> 30秒間に0.1 RCFで遠心分離し、室温で30分間回転させます。 </li><li>上清を取り除く。 1XFSB 200μLを加える。 </li><li>各ChIPのサンプルにアガロースビーズを90μLを加える。 1時間4℃で回転させる。 </li><li>ネガティブコントロールとして機能し、4で回転℃で1時間に残っているアガロースビーズに1XFSB液1mLを追加。 </li><li>時間の長いインキュベーション後、30秒間に0.1 RCFでサンプルとネガティブコントロールを遠心してください。上清を捨てる。 </li></ol> 2。ビーズを洗浄 ！注意！ -洗浄バッファーは使用まで4℃で保管し、古いか月未満の場合にのみ使用することがありますする必要があります。 <ol><li>ビーズにそれぞれの洗浄溶液1 mLを加え、室温で3分間回転させます。 </li><li> 30秒で0.1 RCFで遠心。 </li><li>上清を使用して真空を捨てる。 </li></ol> *各洗浄液 - 低塩の洗浄バッファー - 高塩分洗浄バッファ</p&gt; -塩化リチウム溶液- わずか1分、室温で回転させる！ - TE緩衝液（10mMトリス、1mMのEDTA液pH = 8） 3。溶出 *重要なステップは -それを使用する必要がある日にすべての実験のために新鮮な溶出バッファーを加えます。 <ol><li>各サンプルに、新たに調製した溶出緩衝液250μLを加える。 </li><li> 1分間に0.4 RCFで遠心分離し、RTで15分間回転させます。 </li><li> 2.0 mlのマイクロ遠心チューブ（ループ）で上清を保存します。 </li><li>数秒間手でそれぞれのサンプルと渦に溶出バッファー250μLを加える。その後、mulitvortexer上で15分間ボルテックスサンプル。 </li><li> 4分間に16 RCF rpmで遠心し、同じ2.0mLのチューブ（ループ）で上清を保存する。 </li></ol> 4。ダイジェストプロテイン<ol><li>各チップのサンプルに10μlの0.5M EDTA、25μlの0.8Mのトリス- HCl、pH6.5で、10 mg / mlのプロテ​​イナーゼK（1 / 200サンプル）を追加します。 </li><li>各入力の制御に、プロテイナーゼK消化（サンプルバッファーの1 / 10）及び10mg/mlプロテイナーゼK（サンプルバッファーの1 / 200）のために溶解バッファーを追加します。 </li><li>少なくとも3時間、52℃での入力とChIPサンプルをインキュベートする。 </li></ol> 5。フェノール/クロロホルム抽出 ！注意 - ！実験はフェノール/クロロホルムを必要とは、フードの下で実行する必要があります。フェノール/クロロホルム処理する際にニトリルの手袋を使用してください。 <ol><li> 3時間のインキュベーションの後、我々は、各サンプルに500μlのフェノールクロロホルムを追加してください。 </li><li>数秒間ボルテックスすべてのサンプルは、その後5分、13 RCFのrpmで遠心。 </li><li>この時点で、2つのフェーズが存在するであろうと我々はトップフェーズの内容に興味を持っています。上部相を取り出し、2 mlのマイクロチューブに入れて。 </li><li> 100％エタノール1.375 mLのと一緒に各サンプルに3M酢酸ナトリウムのグリコーゲンおよび50μLの2μlの混合物を追加。 </li><li>激しくボルテックスし、すべてのサンプル。 ℃で一晩-80に配置します。 </li></ol> 3日目<ol><li>削除するサンプルフォーム-80 ° Cとそれらを解凍するために氷の上に置きます。 </li><li> 4時10分15 RCFで遠心分離℃に</li><li>慎重にチューブの底にペレットが剥がれないように上清を取り除く。 </li><li>各サンプルに冷75％エタノール1 mlを追加し、次にそれらを4-6回転倒混和します。 </li><li> 4℃で5分間18 RCFで遠心℃、 </li><li>もう一度上清を除去し、ペレットを空気乾燥することができます。 </li><li> -80 50 4mmのトリス- HCl、PH8のμLとストアに乾燥ペレットを溶解℃でさらに使用するまで。 </li></ol>

<ol>
	<li>Huang, H. S., Matevossian, A., Jiang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Akbarian+S.+Chromatin+immunoprecipitation+in+postmortem+brain.">Akbarian S. Chromatin immunoprecipitation in postmortem brain.</a> J Neurosci Methods. 156, 284-292 .</li><li>Huang, H. S., Matevossian, A., Whittle, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prefrontal+dysfunction+in+schizophrenia+involves+mixed-lineage+leukemia+1-regulated+histone+methylation+at+GABAergic+gene+promoters.">Prefrontal dysfunction in schizophrenia involves mixed-lineage leukemia 1-regulated histone methylation at GABAergic gene promoters.</a> J Neurosci. 27, 11254-11262 .</li><li>O'Neill, L. P., Turner, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoprecipitation+of+native+chromatin:+NChIP.">Immunoprecipitation of native chromatin: NChIP.</a> Methods. 31, 76-82 .</li><li>Spiteri, E., Konopka, G., Coppola, G., 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=Identification+of+the+transcriptional+targets+of+FOXP2,+a+gene+linked+to+speech+and+language,+in+developing+human+brain.">Identification of the transcriptional targets of FOXP2, a gene linked to speech and language, in developing human brain.</a> Am J Hum Genet. 81, 1144-1157 .</li><li>Huang, H. u. a. n. g., Akbarian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GAD1+mRNA+expression+and+DNA+methylation+in+prefrontal+cortex+of+subjects+with+schizophrenia.">GAD1 mRNA expression and DNA methylation in prefrontal cortex of subjects with schizophrenia.</a> PLoS ONE. 2, .</li><li>Stadler, F., Kolb, G., Rubusch, L., Baker, S. P., Jones, E. G., Akbarian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+methylation+at+gene+promoters+is+associated+with+developmental+regulation+and+region-specific+expression+of+ionotropic+and+metabotropic+glutamate+receptors+in+human+brain.">Histone methylation at gene promoters is associated with developmental regulation and region-specific expression of ionotropic and metabotropic glutamate receptors in human brain.</a> J Neurochem. 94, 324-336 .</li></ol>

プロトコルは、これらのクロマチンのマーキングは、（ヒストン）アセチル化とリン酸化1を含む変更の他のタイプと比較して、死後のアーチファクトが発生しにくくなる可能性があるため、人間の脳のヒストンおよび/ ​​またはDNAメチル化の署名に興味がある研究者のために特に有用であるここで概要を説明2。死後脳では、モノヌクレオソームの調製の研究に適している、DNAは、...

ソリューション： <ul><li> nChIP Douncingバッファー：10mMトリス、4 mMのMgCl 2、1mMのCaCl 2は、pHを7.5に調整する</li><li> 10倍FSB：50mMのEDTA、200mMのトリス、500mMのNaClは、pHを7.5に調整する</li><li>低塩洗浄緩衝液：0.1％SDS、1％トリトンX - 100、2mMのEDTA、20mMトリス（pH 8）を、150mMのNaCl </li><li>高い塩洗浄緩衝液：0.1％SDS、1％トリトンX - 100、2mMのEDTA、20mMトリス（pHを8）、500mMのNaCl </li><li>塩化リチウム緩衝液：1％IGEPAL - CA 630、1％デオキシコール酸、1mMのEDTA（pH8）に、10mMのトリス（pH8）に、0.25 M塩化リチウム</li><li> TEバッファー：10mMトリス（pHを8）、1mMのEDTA </li><li>溶出バッファー：0.1M NaHCO 3を 、1％SDS </li><li>プロテイナーゼK消化バッファー（10X）：1Mトリス、50mMのEDTA、2％SDS、2M NaClを8.0にpHを調整する</li><li> 3 M酢酸ナトリウム：オートクレーブ滅菌蒸留水100mLで24.61グラム無水酢酸ナトリウム（MW = 82.03）。濃縮された酢酸を用いて5.2にpHを調整する。 </li></ul>

a chromatin assay for human brain tissue

このような統合失調症、双極性疾患や自閉症などの慢性神経精神疾患は、遺伝子発現と他の分子病理学のエピジェネティックな変化を引き起こす可能性がある遺伝的要因と環境要因の組み合わせに起因すると考えられています。伝統的に、しかし、死後脳での発現の研究​​は、mRNAやタンパク質の定量化に限られていた。そのような自己消化時間や組織の健全性のばらつきなどの死後脳研究で遭遇する制限はまたより高次のクロマチン構造のいずれかの研究に影響を与える可能性があります。しかし、DNAを含むゲノムDNAのヌクレオソームの組織：結合コアヒストンは - 主に様々な脳の銀行が提供する代表的なサンプルで保存されるように見えます。そのため、死後脳で定義されたゲノムの遺伝子座でのメチル化パターンとコアヒストンの他の共有結合修飾を研究することが可能です。ここで、我々は凍結（固定なし）人間の脳の標本用に簡略化したネイティブクロマチン免疫沈降（NChIP）プロトコルを提示する。脳ホモジネートのミクロコッカスヌクレアーゼの消化から始まる、NChIPは、3日以内に完了することができる定量PCRが続きます。ここで紹介する方法は、通常と病気のヒトの脳における遺伝子発現のエピジェネティックな機構を解明するために有用なはずです。

Watch this Scientific Journal Video about A Chromatin Assay for Human Brain Tissue at JoVE.com

A Chromatin Assay for Human Brain Tissue

最近まで、人間の脳での発現の研究​​は、RNAやタンパク質の定量化に限られていた。このホワイトペーパーで説明されているクロマチン免疫沈降の技術と、それは死後脳における遺伝子発現のヒストンメチル化と他のエピジェネティックな調節因子をマッピングすることが可能となります。

人間の脳組織のためのクロマチンアッセイ

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental ...

Research

JoVE Journal

Biology

12.8K ビュー.  University of Massachusetts Medical School. 最近まで、人間の脳での発現の研究​​は、RNAやタンパク質の定量化に限られていた。このホワイトペーパーで説明されているクロマチン免疫沈降の技術と、それは死後脳における遺伝子発現のヒストンメチル化と他のエピジェネティックな調節因子をマッピングすることが可能となります。

ビデオ: A Chromatin Assay for Human Brain Tissue

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

アクチン共沈降アッセイ、F -アクチンへの結合タンパク質の解析のための

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

生物学

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 ...

Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, little is known about changes in neuronal chromatin and nuclear organization during the course of development and aging, or in chronic neuropsychiatric disease. However, to date most chromatin and DNA based assays (other than FISH) lack single cell resolution. To this end, the considerable cellular heterogeneity of brain tissue poses a significant limitation, because typically various subpopulations of neurons are intermingled with different types of glia and other non-neuronal cells. One possible solution would be to grow cell-type specific cultures, but most CNS cells, including neurons, are ex vivo sustainable, at best, for only a few weeks and thus would provide an incomplete model for epigenetic mechanisms potentially operating across the full lifespan.  Here, we provide a protocol to extract and purify nuclei from frozen (never fixed) human postmortem brain. The method involves extraction of nuclei in hypotonic lysis buffer, followed by ultracentrifugation and immunotagging with anti-NeuN antibody. Labeled neuronal nuclei are then collected separately using fluorescence-activated sorting. This method should be applicable to any brain region in a wide range of species and suitable for chromatin immunoprecipitation studies with site- and modification-specific anti-histone antibodies, and for DNA methylation and other assays. 

The cellular heterogeneity of brain tissue poses a significant limitation for the study of epigenetic markings in chromatin because most assays lack single cell resolution. Neurons typically are intermingled with glia and other non-neuronal cells. We provide a protocol to extract and collect neuronal nuclei from human brain.

人間の死後の脳組織から神経細胞核の単離

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, ...

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

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

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

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

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 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 ...

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 ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

ハイスループットシーケンシングのための高品質クロマチン免疫沈降DNAテンプレート（チップ-SEQ）の生成

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 complexes consist of 14 protein subunits including Ino80, a SNF2-like ATPase, which serves both as the catalytic subunit and the scaffold for assembly of the complexes. Functions of the other subunits and the mechanisms by which they contribute to the INO80 complex&#39;s chromatin remodeling activity remain poorly understood, in part due to the challenge of generating INO80 subassemblies in human cells or heterologous expression systems. This JOVE protocol describes a procedure that allows purification of human INO80 chromatin remodeling subcomplexes that are lacking a subunit or a subset of subunits. N-terminally FLAG epitope tagged Ino80 cDNA are stably introduced into human embryonic kidney (HEK) 293 cell lines using Flp-mediated recombination. In the event that a subset of subunits of the INO80 complex is to be deleted, one expresses instead mutant Ino80 proteins that lack the platform needed for assembly of those subunits. In the event an individual subunit is to be depleted, one transfects siRNAs targeting this subunit into an HEK 293 cell line stably expressing FLAG tagged Ino80 ATPase. Nuclear extracts are prepared, and FLAG immunoprecipitation is performed to enrich protein fractions containing Ino80 derivatives. The compositions of purified INO80 subcomplexes can then be analyzed using methods such as immunoblotting, silver staining, and mass spectrometry. The INO80 and INO80 subcomplexes generated according to this protocol can be further analyzed using various biochemical assays, which are described in the accompanying JOVE protocol. The methods described here can be adapted for studies of the structural and functional properties of any mammalian multi-subunit chromatin remodeling and modifying complexes.

This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.

発生と人間INO80クロマチンリモデリング複合体とSubcomplexesの精製

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

比色金ナノ粒子アッセイに適用される構造スイッチングアプタマーを選択するための方法

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod shaped chromosomes which are moved individually by the mitotic spindle apparatus. Mitotic chromatin condensation yields chromosomes compacted fifty-fold denser as in interphase. During exit from mitosis, chromosomes have to re-establish their functional interphase state, which is enclosed by a nuclear envelope and is competent for replication and transcription. The decondensation process is morphologically well described, but in molecular terms poorly understood: We lack knowledge about the underlying molecular events and to a large extent the factors involved as well as their regulation. We describe here a cell-free system that faithfully recapitulates chromatin decondensation in vitro, based on mitotic chromatin clusters purified from synchronized HeLa cells and X. laevis egg extract. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its co-ordination with processes simultaneously occurring during mitotic exit such as nuclear envelope and pore complex re-assembly.

The molecular mechanisms of the decondensation of highly compacted mitotic chromatin are ill-defined. We present a cell-free assay based on mitotic chromatin clusters isolated from HeLa cells and Xenopus laevis egg extract that faithfully reconstitutes the decondensation process in vitro.

無細胞アッセイは、有糸分裂の終了時クロマチン脱凝縮を勉強します

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod ...