AKSは1＆2が発達セル31（3）、Magalskaから転載されている図にこの作品は、ドイツの研究財団とERC（WAにAN377 / 3-2と309528はCHROMDECON）とベーリンガーインゲルハイムフォンの博士フェローシップによってサポートされていましたら 、有糸分裂、305から318まで、2014年の終わりにクロマチン脱凝縮で、RuvBのようなATPアーゼ機能、エルゼビアからの親切な許可を得て。

<ol>
	<li>Lohka, M. J., Masui, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+in+vitro+of+sperm+pronuclei+and+mitotic+chromosomes+induced+by+amphibian+ooplasmic+components.">Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components.</a> Science. 220, 719-721 (1983).</li><li>de la Barre, A. E., Robert-Nicoud, M., Dimitrov, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+mitotic+chromosomes+in+Xenopus+egg+extract.">Assembly of mitotic chromosomes in Xenopus egg extract.</a> Methods Mol Biol. 119, 219-229 (1999).</li><li>Maresca, T. J., Heald, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+studying+spindle+assembly+and+chromosome+condensation+in+Xenopus+egg+extracts.">Methods for studying spindle assembly and chromosome condensation in Xenopus egg extracts.</a> Methods Mol Biol. 322, 459-474 (2006).</li><li>Galy, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+gp210+in+mitotic+nuclear-envelope+breakdown.">A role for gp210 in mitotic nuclear-envelope breakdown.</a> J Cell Sci. 121, 317-328 (2008).</li><li>Chan, R. C., Forbes, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+study+of+nuclear+assembly+and+nuclear+import+using+Xenopus+egg+extracts.">In vitro study of nuclear assembly and nuclear import using Xenopus egg extracts.</a> Methods Mol Biol. 322, 289-300 (2006).</li><li>Gillespie, P. J., Gambus, A., Blow, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+use+of+Xenopus+egg+extracts+to+study+DNA+replication+and+chromatin+associated+proteins.">Preparation and use of Xenopus egg extracts to study DNA replication and chromatin associated proteins.</a> Methods. 57, 203-213 (2012).</li><li>Gant, T. M., Wilson, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+assembly.">Nuclear assembly.</a> Annu Rev Cell Dev Biol. 13, 669-695 (1997).</li><li>Magalska, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RuvB-like+ATPases+function+in+chromatin+decondensation+at+the+end+of+mitosis.">RuvB-like ATPases function in chromatin decondensation at the end of mitosis.</a> Developmental Cell. 31, 305-318 (2014).</li><li>Belmont, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitotic+chromosome+structure+and+condensation.">Mitotic chromosome structure and condensation.</a> Curr Opin Cell Biol. 18, 632-638 (2006).</li><li>Lavoie, B. D., Tuffo, K. M., Oh, S., Koshland, D., Holm, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitotic+chromosome+condensation+requires+Brn1p,+the+yeast+homologue+of+Barren.">Mitotic chromosome condensation requires Brn1p, the yeast homologue of Barren.</a> Mol Biol Cell. 11, 1293-1304 (2000).</li><li>Philpott, A., Leno, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleoplasmin+remodels+sperm+chromatin+in+Xenopus+egg+extracts.">Nucleoplasmin remodels sperm chromatin in Xenopus egg extracts.</a> Cell. 69, 759-767 (1992).</li><li>Philpott, A., Leno, G. H., Laskey, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm+decondensation+in+Xenopus+egg+cytoplasm+is+mediated+by+nucleoplasmin.">Sperm decondensation in Xenopus egg cytoplasm is mediated by nucleoplasmin.</a> Cell. 65, 569-578 (1991).</li><li>Burglin, T. R., Mattaj, I. W., Newmeyer, D. D., Zeller, R., De Robertis, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+of+nucleoplasmin+from+Xenopus+laevis+oocytes+and+analysis+of+its+developmental+expression.">Cloning of nucleoplasmin from Xenopus laevis oocytes and analysis of its developmental expression.</a> Genes Dev. 1, 97-107 (1987).</li><li>Whitaker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+at+fertilization+and+in+early+development.">Calcium at fertilization and in early development.</a> Physiological reviews. 86, 25-88 (2006).</li><li>Gasser, S. M., Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+methods+for+the+isolation+of+individual+and+clustered+mitotic+chromosomes.">Improved methods for the isolation of individual and clustered mitotic chromosomes.</a> Exp Cell Res. 173, 85-98 (1987).</li><li>Eisenhardt, N., Schooley, A., Antonin, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+in+vitro+assays+to+analyze+the+function+of+transmembrane+nucleoporins+and+targeting+of+inner+nuclear+membrane+proteins.">Xenopus in vitro assays to analyze the function of transmembrane nucleoporins and targeting of inner nuclear membrane proteins.</a> Methods Cell Biol. 122, 193-218 (2014).</li><li>Wignall, S. M., Deehan, R., Maresca, T. J., Heald, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+condensin+complex+is+required+for+proper+spindle+assembly+and+chromosome+segregation+in+Xenopus+egg+extracts.">The condensin complex is required for proper spindle assembly and chromosome segregation in Xenopus egg extracts.</a> J Cell Biol. 161, 1041-1051 (2003).</li><li>Forbes, D. J., Kirschner, M. W., Newport, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+formation+of+nucleus-like+structures+around+bacteriophage+DNA+microinjected+into+Xenopus+eggs.">Spontaneous formation of nucleus-like structures around bacteriophage DNA microinjected into Xenopus eggs.</a> Cell. 34, 13-23 (1983).</li><li>Hartl, P., Olson, E., Dang, T., Forbes, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+assembly+with+lambda+DNA+in+fractionated+Xenopus+egg+extracts:+an+unexpected+role+for+glycogen+in+formation+of+a+higher+order+chromatin+intermediate.">Nuclear assembly with lambda DNA in fractionated Xenopus egg extracts: an unexpected role for glycogen in formation of a higher order chromatin intermediate.</a> J Cell Biol. 124, 235-248 (1994).</li><li>Sandaltzopoulos, R., Blank, T., Becker, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+repression+by+nucleosomes+but+not+H1+in+reconstituted+preblastoderm+Drosophila+chromatin.">Transcriptional repression by nucleosomes but not H1 in reconstituted preblastoderm Drosophila chromatin.</a> EMBO J. 13, 373-379 (1994).</li><li>Ulbert, S., Platani, M., Boue, S., Mattaj, I. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+membrane+protein-DNA+interactions+required+early+in+nuclear+envelope+assembly.">Direct membrane protein-DNA interactions required early in nuclear envelope assembly.</a> J Cell Biol. 173, 469-476 (2006).</li><li>Zhang, C., Clarke, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin-independent+nuclear+envelope+assembly+induced+by+Ran+GTPase+in+Xenopus+egg+extracts.">Chromatin-independent nuclear envelope assembly induced by Ran GTPase in Xenopus egg extracts.</a> Science. 288, 1429-1432 (2000).</li><li>Paulson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+chromosome+clusters+from+metaphase-arrested+HeLa+cells.">Isolation of chromosome clusters from metaphase-arrested HeLa cells.</a> Chromosoma. 85, 571-581 (1982).</li><li>Ramadan, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cdc48/p97+promotes+reformation+of+the+nucleus+by+extracting+the+kinase+Aurora+B+from+chromatin.">Cdc48/p97 promotes reformation of the nucleus by extracting the kinase Aurora B from chromatin.</a> Nature. 450, 1258-1262 (2007).</li></ol>

The authors have nothing to disclose.

アフリカツメガエル卵抽出物を忠実にインビトロで細胞プロセスを再現するための非常に有用なツールであり、このシステムは正常細胞周期および細胞分裂事象2、3,5,6,17の特徴付けに使用したアフリカツメガエル 。原因卵形成中の卵に隔離核成分の大型店に、卵抽出物は、細胞成分の優れた供給源です。哺乳動物組織細胞株または遺伝子操作上のRNAiのような他のアプローチと比較すると、それはいくつかの利点があります。無細胞系は、細胞の生存率が他の方法で制限されるであろうで細胞プロセスを研究することができます。さらに複雑なプロセスの一つのステップは、単純なアッセイで分析することができます。ここで紹介する脱凝縮アッセイはそれぞれ、他の有糸分裂のイベントからの干渉なしで有糸分裂後の脱凝縮の分子メカニズムを研究することができます。 アフリカツメガエル卵抽出物は、特定のタンパク質の枯渇により操作が容易ですおよび阻害剤または変異タンパク質8を追加。例えば、 図2は、脱凝縮アッセイに非加水分解性ATPまたはGTPアナログ、ATPγSとGTPγSを加えた結果を示しています。 アフリカツメガエル卵の希釈および分画遠心によって膜および細胞質ゾルのような構成要素は、16を分離することができる。 図3は、膜の存在下または非存在下で行われる脱凝縮アッセイを示します。最後に、無細胞アッセイはまた、分画法により、例えば新規の因子を同定するために使用することができます。我々が発見したような戦略を使用して重要な脱凝縮などのAAA + -ATPアーゼRuvBL1 / RUVBL2は8要因。 in vitro系 X.に基づきますフォーブスら未受精Xに λファージのDNAの注入を示した：卵は異なるDNAテンプレートで使用されてきたアフリカツメガエル ツメガエルの卵は、NAKのクロマチンのアセンブリを誘導しましたEDファージλDNA。ウイルスDNAの注入卵を活性化するように、クロマチンのアセンブリは、核様構造体18の形成を行ったと同様にλファージDNA は 、インビトロ19 における構造体のような核を生成するために、卵抽出物と組み合わせて使用することができます。 DNAでコーティングされた磁気ビーズは、DNA 20のchromatinizationと核膜21の動員を勉強だけでなく、ために使用されていることが、これは正真正銘の核再組み立てプロセスに似ていたどの程度に開いたままであるが、核エンベロープのアセンブリおよび細孔は、22錯体。ここで紹介するプロトコルは、活性化したアフリカツメガエルの卵から生成された抽出物を用いたHeLa細胞から分離された有糸分裂クロマチンクラスターの脱凝縮することができます。それは徹底的にinterphasic核8の改革につながるイベントを再構築します。核膜の形成を研究するために使用広く適用核集合反応に比べると代わりに精子DNAの脱凝縮アッセイヒーラ有糸分裂クロマチンクラスタ内の有糸分裂の終わりに核膜孔複合体は、使用されています。精子のDNAは、有糸分裂クロマチンまたは未受精および非活性卵3から調製された抽出物とのインキュベーションの際にも、個々の染色体に組み込むことができます。私たちは、手続きを簡素化し、クロマチン凝縮からの干渉を避けるために、クロマチン源として有糸分裂のクラスタを使用することにしました。また、卵抽出物の調製は、わずかに修正されています。固定角ローター内の2つの高速遠心分離工程によってクリアクロマチン脱凝縮低速エキスが使用されます。低速抽出物は、その活性を失うことなく、液体窒素中に最大6ヶ月間保存することができます。これとは対照的に、核アセンブリ反応において、細胞質ゾルと浮い膜は、凍結の可能な前希釈および差動高速遠心分離により低速抽出物から生成されている（Eisenhardt ら 16 detai用）プロトコルを導きました。我々のアッセイ系では、膜の添加は、核膜孔複合体を含む閉じた核膜の形成を可能にします。結果の核は核のインポートおよびエクスポートの8のための能力があります。したがって、このシステムは、クロマチン脱凝縮及び核膜の改革の両方をサポートしています。興味深いことに、クロマチン脱凝縮は、膜が存在しない（ 図3）も可能です。しかし、膜の添加は大きめの核になります。ほとんどの場合、核膜の改革は、核輸入に依存し、まだ未定義のメカニズムによる二次脱凝縮ステップを誘導します。 HeLa細胞の有糸分裂クロマチンクラスターの単離のために、ガッサーとレムリ15によって確立されたプロトコルの修正版を使用しました。同期された有糸分裂細胞は、非イオン性界面活性剤のジギトニンを含有する緩衝液および機械的力によって溶解されます。クロマチンを含むクラスタとして単離します1核からの全ての染色体。単一の染色体の分離プロトコルに比べて決定的な違いは、細胞が低張腫れはありませんが、溶解の前に4℃に冷却しているという事実です。これは、個々の染色体15、23の断線を防ぐことができます。全クロマチンクラスターの分離の利点を認識しJRポールソン23によって公開されたプロトコルに比べ、ガッサー・レムリはキナーゼ、ヌクレアーゼ、プロテアーゼおよびホスファターゼの活性を低減するためにEDTAを含む代わりにMgをポリアミンバッファ2+ベースのバッファを使用するとこのことにより、分離工程15の間に存在するタンパク質とDNAの変更の量を減少させます。また、微分遠心分離中のコロイダルシリカ粒子の濃度勾配を使用して、非常に質の汚染を減少させます。プロトコルは、チャイニーズハムスター卵巣細胞、マウス15からの有糸分裂クロマチンクラスターを単離することができます。 <pそれは、有糸分裂の終了時に起こるようにクラス= &quot;jove_contentは&quot;&gt;要するに、我々のプロトコルを忠実クロマチン脱凝縮を再構成します。 in vitroでのATP依存性は、クロマチン脱凝縮は、少なくとも部分的にRuvBL1 / 2の関与だけでなく、有糸分裂出口24の間にクロマチンから有糸分裂キナーゼオーロラBを削除し、別のAAA + -ATPase、P97、によって説明することができます。プロセスは、GTP加水分解を必要とするなぜ私たちは、このセットアップを使用して答えるつもり未解決の問題の一つです。

アフリカツメガエルの卵抽出物は、無細胞アッセイのシンプルで複雑 ​​な細胞事象を研究するための強力かつ広く適用するツールです。 Lohka＆増井1によるそれらの最初の記述以来、彼らは広く、このようなクロマチン凝縮2、スピンドルアセンブリ3、核膜崩壊4だけでなく、核-細胞質間輸送5またはDNA複製6などの有糸分裂のプロセスを研究するために使用されています。例えば、核エンベロープ改革と核膜孔複合体の再構築などinterphasic核の改革のために必要な有糸分裂の終わりで行われているイベントは、はるかに少ない初期の有糸分裂のイベントに比べて理解されているが、同様にアフリカツメガエルの卵抽出物7を使用して研究することができます。我々は最近、有糸分裂8の最後にクロマチン脱凝縮を研究するためにアフリカツメガエルの卵抽出物に基づくアッセイを確立した、アンダー調べプロセスは、私が待っています詳細な特徴づけをtsで。 後生動物では、クロマチンは高度に遺伝物質の忠実な分離を行うために、有糸分裂のエントリに集光されます。クロマチンは、間期中の遺伝子発現およびDNA複製のためにアクセス可能であることを保証するために、それは、有糸分裂の終了時に脱圧縮される必要があります。脊椎動物では、クロマチンは 、Sにおける有糸分裂圧縮は通常はるかに低い酵母、 例えば、2つだけ倍とは対照的に、相間9に比べて有糸分裂の間に複数の圧縮された50倍までですセレビシエ 10。脊椎動物クロマチン脱凝縮は、主に卵の受精後の精子DNAの再編との関連で検討されています。分子機構は、どのヌ、豊富な卵母細胞のタンパク質で、卵に保存されたヒストンH2AとH2Bに精子固有のプロタミンを交換します。このプロセスはまた、 アフリカツメガエル卵抽出物11、12を用いて解明されました。しかし、表現ヌの卵母細胞13と、有糸分裂クロマチンに限定されているこれらの精子固有のプロタミンが含まれていません。したがって、有糸分裂の最後に脱凝縮クロマチンは、8つの独立したヌクレオです。 インビトロ脱凝縮反応のために我々は同期のHeLa細胞から単離された活性化したアフリカツメガエル の卵とクロマチンクラスターから生成されたエキスを使用しています。カルシウムイオノフォアと卵の治療は、受精時の精子のエントリによって生成された卵母細胞へのカルシウム放出を模倣。カルシウム波は最初相間14に進行し、減数分裂の第二中期で逮捕され、細胞周期の再開と卵をトリガします。そのため、フォームアクティブに卵準備卵抽出物は、有糸分裂終了/相間状態を表し、クロマチン脱凝縮、核膜などの有糸分裂終了のための特定のイベントを誘導する能力があると複雑な改革を細孔。有糸分裂CHの単離のためにクラスタromatin我々は、染色体のクラスターが有糸分裂に同期したHeLa細胞からの溶解により放出され、勾配遠心分離によってバッファを含むポリアミンで単離されるガッサー＆レムリ15、によって公開されたプロトコルを少し変更したバージョンを使用していました。

<table><tbody><tr><td>spermine tetrahydrochloride</td><td>Fluka analytical</td><td>85610-25G</td><td></td></tr><tr><td>spermidine trihydrochloride</td><td>Sigma&amp;nbsp;</td><td>S2501-5G</td><td></td></tr><tr><td>high-purity digitonin</td><td>Millipore</td><td>300410-1GM</td><td>toxic</td></tr><tr><td>PMSF</td><td>Applichem</td><td>A0999,0100</td><td>toxic</td></tr><tr><td>thymidine</td><td>Calbiochem</td><td>6060</td><td></td></tr><tr><td>nocodazole</td><td>Calbiochem</td><td>487928</td><td>toxic</td></tr><tr><td>37% formaldehyde solution</td><td>Roth</td><td>7398-1</td><td>toxic</td></tr><tr><td>trypan blue solution (0.4%)</td><td>Sigma</td><td>T8154</td><td>toxic</td></tr><tr><td>1,4-dithiothreitol (DTT)</td><td>Roth</td><td>6908.2</td><td></td></tr><tr><td>AEBSF hydrochloride</td><td>Applichem</td><td>A1421,0001</td><td></td></tr><tr><td>pepstatin</td><td>Roth</td><td>2936.1/2/3</td><td></td></tr><tr><td>leupeptin</td><td>Roth</td><td>CN334</td><td></td></tr><tr><td>aprotinin</td><td>Roth</td><td>A162.3</td><td></td></tr><tr><td>Percoll (colloidal silica particles solution)</td><td>GE Healthcare</td><td>17-0891-01</td><td></td></tr><tr><td>glutamine</td><td>Gibco</td><td>25030-024</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>75 cm&amp;sup2; tissue culture flasks</td><td>Greiner Bio-one</td><td>658175</td><td></td></tr><tr><td>heat-inactivated fetal bovine serum (FBS)</td><td>Gibco</td><td>10500-064</td><td></td></tr><tr><td>Homogenizer (40 ml tissue grinder)</td><td>Wheaton</td><td>357546</td><td></td></tr><tr><td>Neubauer chamber</td><td>Assistent</td><td>441/1</td><td></td></tr><tr><td>&amp;nbsp;Oak Ridge Centrifuge Tubes, polycarbonate (50 ml)</td><td>Nalgene</td><td>3118-0050</td><td></td></tr><tr><td>100 &amp;micro;m cell strainer, nylon</td><td>BD Falcon</td><td>352360</td><td></td></tr><tr><td>cytochalasin B</td><td>Applichem</td><td>A7657,0010</td><td>toxic</td></tr><tr><td>cycloheximide</td><td>Roth</td><td>8682.3</td><td>toxic</td></tr><tr><td>L-cystein</td><td>Merck</td><td>1,028,381,000</td><td></td></tr><tr><td>hCG available as Ovogest</td><td>MSD</td><td>1431593</td><td></td></tr><tr><td>PMSG available as Intergonan</td><td>MSD</td><td>1431015</td><td></td></tr><tr><td>A23187 (mixed calcium-magnesium-salt)</td><td>Enzo</td><td>ALX-450-002-M010</td><td>toxic</td></tr><tr><td>syringe needles (1.20 x 40 mm, 18 G x 1 1/2&quot;)</td><td>Braun</td><td>4665120</td><td></td></tr><tr><td>ATP</td><td>Serva</td><td>10920.03</td><td></td></tr><tr><td>GTP, 2 Na x 3 H&lt;sub&gt;2&lt;/sub&gt;0</td><td>Roth</td><td>K056.1/2/3/4</td><td></td></tr><tr><td>creatine phosphat disodium salt</td><td>Calbiochem</td><td>2380</td><td></td></tr><tr><td>creatine phosphokinase</td><td>Sigma</td><td>C3755-35KU</td><td></td></tr><tr><td>DMAP</td><td>Sigma</td><td>D2629-1G</td><td></td></tr><tr><td>DAPI&amp;nbsp;</td><td>Roche</td><td>10236276001</td><td></td></tr><tr><td>PFA</td><td>Sigma</td><td>P-6148</td><td>toxic</td></tr><tr><td>centrifugation tubes for TLA 100 (7 x 10 mm, 5/16 x 13/16 in.)</td><td>Beckman Coulter</td><td>343775</td><td></td></tr><tr><td>&quot;Cell-Saver&quot; (tips with wide opening, 1,000 &amp;micro;l)</td><td>Biozym</td><td>729065</td><td></td></tr><tr><td>50% glutaraldehyde solution, grade I</td><td>Sigma alderich</td><td>G7651-10 ml</td><td>toxic</td></tr><tr><td>0.1% (w/v) poly-L-lysine solution</td><td>Sigma</td><td>P8920-100 ml</td><td></td></tr><tr><td>flat-bottom tubes (6 ml, 16.0/55 mm)</td><td>Greiner Bio-one</td><td>145211</td><td></td></tr><tr><td>Vectashield mounting medium</td><td>Vector laboratories</td><td>H1000</td><td></td></tr><tr><td>tubes for TLA120 (11 x 34 mm, 7/16 x 1 3/8 in.)</td><td>Beckman Coulter</td><td>343778</td><td></td></tr><tr><td>&quot;Cell-Saver&quot; (tips with wide opening, 200 &amp;micro;l)</td><td>Biozym</td><td>729055</td><td></td></tr><tr><td>12 mm coverslips</td><td>Thermo Scientific</td><td>0784 #1</td><td></td></tr></tbody></table>

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.

脱凝縮反応の時間依存性 図1は、脱凝縮アッセイの典型的な時間経過を示します。反応の開始時に目に見える染色体のクラスターがdecondenses、シングル、ラウンドと滑らかな核に合流。卵抽出物は、スクロースで置き換えられたときに染色体クラスタバッファ脱凝縮活性は卵抽出物中に存在することを示唆している、縮合したままです。 クロマチン脱凝縮は、エネルギー依存性のプロセスであります インビトロ脱凝縮反応は、好都合には阻害剤を添加することにより、例えば操作することができます。 図2に示した実験では、非加水分解性ATPまたはGTP類似体、ATPγSまたはGTPγSを反応に添加しました。両方は、ATPとGTP依存、アクティブなプロセス（ 図2）であると、脱凝縮を示すを阻害します。 脱凝縮アッセイは、膜（ 図3）の存在下または非存在下で行いました。両方の条件にクロマチンしかし、大きな核内膜結果の追加を脱凝縮を受けることに注意してください。おそらく、核膜の改革は、核輸送に依存してさらに別の機構による二次脱凝縮ステップを誘導します。 <img alt="図1" src="/files/ftp_upload/53407/53407fig1.jpg" /> 体外 脱凝縮反応における 番目 の電子 の図1時間経過 。HeLa細胞由来の有糸分裂クロマチンクラスタがinterphasic アフリカツメガエルの卵抽出物とインキュベートしました。サンプルは、DAPIで染色し、共により分析し、4％PFAおよび0.5％グルタルアルデヒドで示された時点で固定されました<html> nfocal顕微鏡。再印刷Magalska ら 8から。スケールバーは5μmである。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53407/53407fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図2" src="/files/ftp_upload/53407/53407fig2.jpg" /> 図2クロマチン脱凝縮は、ATPとGTP加水分解を必要とする。クロマチン脱凝縮を10mMATPγS、10mMのGTPγSまたは対照緩衝液の存在下で行いました。サンプルは、示された時点で、4％PFAおよび0.5％グルタルアルデヒドで固定し、共焦点顕微鏡法により分析しました。再印刷Magalska ら 8から。スケールバーは5μmである。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53407/53407fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> 図3「SRC = &quot;/ファイル/ ftp_upload / 53407 / 53407fig3.jpg&quot; /&gt; 膜の存在下および非存在下で、図3クロマチン脱凝縮。クロマチン脱凝縮は、存在しない（A）または 120分間浮遊精製膜の存在（B）で行いました。サンプルを4％PFAおよび0.5％グルタルアルデヒドで固定し、共焦点顕微鏡法により分析しました。クロマチンは、DAPI、DiIC 18（1,1&#39;-ジオクタデシル-3,3,3 &#39;、3&#39;-テトラメチル過塩素酸塩）を有する膜を用いて染色します。スケールバーは5μmである。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53407/53407fig3large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

Watch this Scientific Journal Video about A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis at JoVE.com

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

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 ...

a-cell-free-assay-to-study-chromatin-decondensation-at-end

Nanyang Technological University

Research

JoVE Journal

Biology

10.2K ビュー.  Max Planck Society. 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.

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

Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for spermatogenesis. These techniques allow for visualization of protein and DNA localization patterns during meiotic events such as homologous chromosome pairing, synapsis and DNA repair. While a few protocols have been described in the literature, general chromatin spread techniques using mammalian prophase oocytes are limited and difficult due to the timing of meiosis initiation in fetal ovaries. In comparison, prophase spermatocytes can be collected from juvenile male mice with higher yields without the need for microdissection. However, it is difficult to obtain a pure synchronized population of cells at specific stages due to the heterogeneity of meiotic and post-meiotic germ cell populations in the juvenile and adult testis. For later stages of meiosis, it is advantageous to assess oocytes undergoing meiosis I (MI) or meiosis II (MII), because groups of mature oocytes can be collected from adult female mice and stimulated to resume meiosis in culture. Here, methods for meiotic chromatin spread preparations using oocytes dissected from fetal, neonatal and adult ovaries are described with accompanying video demonstrations. Chromosome missegregation events in mammalian oocytes are frequent, particularly during MI. These techniques can be used to assess and characterize the effects of different mutations or environmental exposures during various stages of oogenesis. As there are distinct differences between oogenesis and spermatogenesis, the techniques described within are invaluable to increase our understanding of mammalian oogenesis and the sexually dimorphic features of chromosome and protein dynamics during meiosis.

Oogenesis in mammals is known to be error-prone, particularly due to chromosome missegregation. This manuscript describes chromatin spread preparation methods for mouse prophase, metaphase I and II-staged oocytes. These fundamental techniques allow for the study of chromatin-bound proteins and chromosome morphology throughout mammalian oogenesis.

クロマチン広がるマウス卵母細胞の進行を前期から中期 II の分析のための準備

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for ...

遺伝学

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

転写因子の結合、細胞活性中に事象を表示する転写、翻訳、回転率のリアルタイム解析

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over the past decade, new and innovative technologies have greatly enhanced the practicality of live-cell imaging. Cells can now be kept in focus and continuously imaged over several days while maintained under 37 &#176;C and 5% CO2 cell culture conditions. Moreover, multiple fields of view representing different experimental conditions can be acquired simultaneously, thus providing high-throughput experimental data. Live-cell imaging provides a significant advantage over fixed-cell imaging by allowing for the direct visualization and temporal quantitation of dynamic cellular events. Live-cell imaging can also identify variation in the behavior of single cells that would otherwise have been missed using population-based assays. Here, we describe live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel. We demonstrate methods to visualize whether mitotically arrested cells die directly from mitosis or slip back into interphase. We also describe how the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system can be used to assess the fraction of interphase cells born from mitotic slippage that are capable of re-entering the cell cycle. Finally, we describe a live-cell imaging method to identify nuclear envelope rupture events.

Live-cell imaging provides a wealth of information on single cells or whole populations that is unattainable by fixed cell imaging alone. Here, live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel are described.

長期的な住セルイメージ投射細胞の運命をパクリタ キセルに応答を評価するために

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over ...

がん研究

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene expression. The cellular transcriptional machinery and its chromatin modification proteins coordinate to regulate transcription. To analyze transcriptional regulation at the molecular level, several experimental methods such as electrophoretic mobility shift, transient reporter and chromatin immunoprecipitation (ChIP) assays are available. We describe a modified ChIP assay in detail in this article because of its advantages in directly showing histone modifications and the interactions between proteins and DNA in cells. One of the key steps in a successful ChIP assay is chromatin shearing. Although sonication is commonly used for shearing chromatin, it is difficult to identify reproducible conditions. Instead of shearing chromatin by sonication, we utilized enzymatic digestion with micrococcal nuclease (MNase) to obtain more reproducible results. In this article, we provide a straightforward ChIP assay protocol using MNase.

Chromatin immunoprecipitation (ChIP) is a powerful tool for understanding the molecular mechanisms of gene regulation. However, the method involves difficulties in obtaining reproducible chromatin fragmentation by mechanical shearing. Here, we provide an improved protocol for a ChIP assay using enzymatic digestion.

哺乳類細胞における微小コッカルヌクレアーゼを用いてクロマチン免疫沈殿アッセイ

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

細胞分裂時の染色体分配の生きているセルイメージ投射

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

発生生物学

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 ...

生物学

Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such architectures are not random and involve interactions between gene promoters and regulatory elements 13. The binding of transcription factors to specific regulatory sequences brings about a network of transcription regulation and coordination 1, 14. 
Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) was developed to identify these higher-order chromatin structures 5,6. Cells are fixed and interacting loci are captured by covalent DNA-protein cross-links. To minimize non-specific noise and reduce complexity, as well as to increase the specificity of the chromatin interaction analysis, chromatin immunoprecipitation (ChIP) is used against specific protein factors to enrich chromatin fragments of interest before proximity ligation. Ligation involving half-linkers subsequently forms covalent links between pairs of DNA fragments tethered together within individual chromatin complexes. The flanking MmeI restriction enzyme sites in the half-linkers allow extraction of paired end tag-linker-tag constructs (PETs) upon MmeI digestion. As the half-linkers are biotinylated, these PET constructs are purified using streptavidin-magnetic beads. The purified PETs are ligated with next-generation sequencing adaptors and a catalog of interacting fragments is generated via next-generation sequencers such as the Illumina Genome Analyzer. Mapping and bioinformatics analysis is then performed to identify ChIP-enriched binding sites and ChIP-enriched chromatin interactions 8. 
We have produced a video to demonstrate critical aspects of the ChIA-PET protocol, especially the preparation of ChIP as the quality of ChIP plays a major role in the outcome of a ChIA-PET library. As the protocols are very long, only the critical steps are shown in the video.

Chromatin Interaction Analysis with Paired-End Tag Sequencing ChIA-PET for Mapping Chromatin Interactions and Understanding Transcription Regulation

Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a method for de novo detection of chromatin interactions, for better understanding of transcriptional control.

クロマチンの相互作用のマッピングと転写調節を理解するためのペアエンドタグシーケンシング（CHIA-PET）によるクロマチンの相互作用の解析

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such ...

Chromosome Preparation From Cultured Cells

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as fluorescence in situ hybridization (FISH), this assay has the powerful ability to analyze individual cells for aberrations that involve gains or losses of portions of the genome&#160;and rearrangements involving one or more chromosomes. In humans, chromosome abnormalities occur in approximately 1 per 160 live births1,2, 60-80% of all miscarriages3,4, 10% of stillbirths2,5, 13% of individuals with congenital heart disease6, 3-6% of infertility cases2, and in many patients with developmental delay&#160;and birth defects7. Cytogenetic analysis of malignancy is routinely used by researchers and clinicians, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance8,9.&#160; Chromosome isolation is invaluable for gene therapy and stem cell research of organisms including nonhuman primates and rodents10-13.
Chromosomes can be isolated from cells of live tissues, including blood lymphocytes, skin fibroblasts, amniocytes, placenta, bone marrow, and tumor specimens. Chromosomes are analyzed at the metaphase stage of mitosis, when they are most condensed and therefore more clearly visible. The first step of the chromosome isolation technique involves the disruption of the spindle fibers by incubation with Colcemid, to prevent the cells from proceeding to the subsequent anaphase stage. The cells are then treated with a hypotonic solution and preserved in their swollen state with Carnoy&#39;s fixative. The cells are then dropped on to slides and can then be utilized for a variety of procedures. G-banding involves trypsin treatment followed by staining with Giemsa to create characteristic light and dark bands. The same procedure to isolate chromosomes can be used for the preparation of cells for procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)14,15.

Chromosomes can be isolated from live cells such as lymphocytes or skin fibroblasts, and from organisms including humans or mice. These chromosome preparations can be further utilized for routine G-banding and molecular cytogenetic procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY).

培養細胞からの染色体の準備

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

enChIPによって特定のゲノム領域と関連する分子の同定の単離

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Use of Time-Lapse Microscopy and Stage-Specific Nuclear Depletion of Proteins to Study Meiosis in S. cerevisiae

Time-lapse fluorescence microscopy has revolutionized the understanding of meiotic cell-cycle events by providing temporal and spatial data that is often not seen by imaging fixed cells. Budding yeast has proved to be an important model organism to study meiotic chromosome segregation because many meiotic genes are highly conserved. Time-lapse microscopy of meiosis in budding yeast allows the monitoring of different meiotic mutants to show how the mutation disrupts meiotic processes. However, many proteins function at multiple points in meiosis. The use of loss-of-function or meiotic null mutants can therefore disrupt an early process, blocking or disturbing the later process and making it difficult to determine the phenotypes associated with each individual role. To circumvent this challenge, this protocol describes how the proteins can be conditionally depleted from the nucleus at specific stages of meiosis while monitoring meiotic events using time-lapse microscopy. Specifically, this protocol describes how the cells are synchronized in prophase I, how the anchor away technique is used to deplete proteins from the nucleus at specific meiotic stages, and how time-lapse imaging is used to monitor meiotic chromosome segregation. As an example of the usefulness of the technique, the kinetochore protein Ctf19 was depleted from the nucleus at different time points during meiosis, and the number of chromatin masses was analyzed at the end of meiosis II. Overall, this protocol can be adapted to deplete different nuclear proteins from the nucleus while monitoring the meiotic divisions.

Use of Time-Lapse Microscopy and Stage-Specific Nuclear Depletion of Proteins to Study Meiosis in S. cerevisiae

Time-lapse microscopy is a valuable tool for studying meiosis in budding yeast. This protocol describes a method that combines cell-cycle synchronization, time-lapse microscopy, and conditional depletion of a target protein to demonstrate how to study the function of a specific protein during meiotic chromosome segregation.

S. cerevisiaeの減数分裂を研究するためのタイムラプス顕微鏡とタンパク質のステージ特異的核枯渇の使用

Time-lapse fluorescence microscopy has revolutionized the understanding of meiotic cell-cycle events by providing temporal and spatial data that is often ...

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

要約

さらに動画を探す

この動画の章

クロマチン広がるマウス卵母細胞の進行を前期から中期 II の分析のための準備

転写因子の結合、細胞活性中に事象を表示する転写、翻訳、回転率のリアルタイム解析

長期的な住セルイメージ投射細胞の運命をパクリタキセルに応答を評価するために

哺乳類細胞における微小コッカルヌクレアーゼを用いてクロマチン免疫沈殿アッセイ

細胞分裂時の染色体分配の生きているセルイメージ投射

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）

クロマチンの相互作用のマッピングと転写調節を理解するためのペアエンドタグシーケンシング（CHIA-PET）によるクロマチンの相互作用の解析

培養細胞からの染色体の準備

enChIPによって特定のゲノム領域と関連する分子の同定の単離

S. cerevisiaeの減数分裂を研究するためのタイムラプス顕微鏡とタンパク質のステージ特異的核枯渇の使用

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

要約

さらに動画を探す

この動画の章

クロマチン広がるマウス卵母細胞の進行を前期から中期 II の分析のための準備

転写因子の結合、細胞活性中に事象を表示する転写、翻訳、回転率のリアルタイム解析

長期的な住セルイメージ投射細胞の運命をパクリタ キセルに応答を評価するために

哺乳類細胞における微小コッカルヌクレアーゼを用いてクロマチン免疫沈殿アッセイ

細胞分裂時の染色体分配の生きているセルイメージ投射

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）

クロマチンの相互作用のマッピングと転写調節を理解するためのペアエンドタグシーケンシング（CHIA-PET）によるクロマチンの相互作用の解析

培養細胞からの染色体の準備

enChIPによって特定のゲノム領域と関連する分子の同定の単離

S. cerevisiaeの減数分裂を研究するためのタイムラプス顕微鏡とタンパク質のステージ特異的核枯渇の使用

長期的な住セルイメージ投射細胞の運命をパクリタキセルに応答を評価するために