Name: isolation and cultivation of neural progenitors followed by chromatin-immunoprecipitation of histone 3 lysine 79 dimethylation mark
Uploaded: 2018-01-26T13:00:00
Description: Watch this Scientific Journal Video about Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark at JoVE.com

アンリエット ・ Bertemes は、ラボ内 CGN 栽培プロトコルを確立する支援を感謝いたします。このメソッドの紙は、DFG 資金 CRC992 医療エピジェネティクスによるテレビへの資金によって支えられました。著者はフライブルクの銀河チームのサポートを認める: 教授 Rolf Backofen、バイオインフォマティクス、アルベルト ・ ルートヴィヒ大学フライブルク、ドイツ共同研究センター 992 医療エピジェネティクス (DFG グラント SFB 992/1 2012) によって資金を供給、Björn Grüning Pavankumar Videmドイツ連邦教育省および研究 (BMBF グラント 031 A538A RBC (de。NBI))。

フライブルク大学と地方自治体の動物福祉委員会は、次のプロトコルに記載されているすべての動物実験 (G12/13、G16/11) を承認しました。
1. 準備
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クリック単価の分離のための準備
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<li>交尾 (E11.5 と E14.5) 野開発のさまざまな段階で胚を取得するタイミングを設定します。少なくとも 8 週齢であるひずみ技研 (海軍医学研究所) のマウスを使用します?...

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	<li>Molyneaux, B. J., Arlotta, P., Menezes, J. R. L., Macklis, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+subtype+specification+in+the+cerebral+cortex.">Neuronal subtype specification in the cerebral cortex.</a> Nat. Rev. Neurosci. 8, 427-437 (2007).</li><li>Kandel, E. R., Squire, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroscience:+breaking+down+scientific+barriers+to+the+study+of+brain+and+mind.">Neuroscience: breaking down scientific barriers to the study of brain and mind.</a> Science. 290, 1113-1120 (2000).</li><li>G&#246;tz, M., Sommer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+development:+the+art+of+generating+cell+diversity.">Cortical development: the art of generating cell diversity.</a> Dev. Camb. Engl. 132, 3327 (2005).</li><li>Hirabayashi, Y., Gotoh, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+control+of+neural+precursor+cell+fate+during+development.">Epigenetic control of neural precursor cell fate during development.</a> Nat. Rev. Neurosci. 11, 377-388 (2010).</li><li>Davis, A. A., Temple, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+self-renewing+multipotential+stem+cell+in+embryonic+rat+cerebral+cortex.">A self-renewing multipotential stem cell in embryonic rat cerebral cortex.</a> Nature. 372, 263-266 (1994).</li><li>Sauvageot, C. M., Stiles, 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=Molecular+mechanisms+controlling+cortical+gliogenesis.">Molecular mechanisms controlling cortical gliogenesis.</a> Curr. Opin. Neurobiol. 12, 244-249 (2002).</li><li>McConnell, S. K., Kaznowski, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+dependence+of+laminar+determination+in+developing+neocortex.">Cell cycle dependence of laminar determination in developing neocortex.</a> Science. 254, 282-285 (1991).</li><li>Desai, A. R., McConnell, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+restriction+in+fate+potential+by+neural+progenitors+during+cerebral+cortical+development.">Progressive restriction in fate potential by neural progenitors during cerebral cortical development.</a> Dev. Camb. Engl. 127, 2863-2872 (2000).</li><li>Glickstein, M., Strata, P., Voogd, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebellum:+history.">Cerebellum: history.</a> Neuroscience. 162, 549-559 (2009).</li><li>Marzban, H., 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=Cellular+commitment+in+the+developing+cerebellum.">Cellular commitment in the developing cerebellum.</a> Front. Cell. Neurosci. 8, (2015).</li><li>Azevedo, F. A. C., 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=Equal+numbers+of+neuronal+and+nonneuronal+cells+make+the+human+brain+an+isometrically+scaled-up+primate+brain.">Equal numbers of neuronal and nonneuronal cells make the human brain an isometrically scaled-up primate brain.</a> J. Comp. Neurol. 513, 532-541 (2009).</li><li>Machold, R., Fishell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Math1+is+expressed+in+temporally+discrete+pools+of+cerebellar+rhombic-lip+neural+progenitors.">Math1 is expressed in temporally discrete pools of cerebellar rhombic-lip neural progenitors.</a> Neuron. 48, 17-24 (2005).</li><li>Luger, K., M&#228;der, A. W., Richmond, R. K., Sargent, D. F., Richmond, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+nucleosome+core+particle+at+2.8+A+resolution.">Crystal structure of the nucleosome core particle at 2.8 A resolution.</a> Nature. 389, 251-260 (1997).</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>Zhang, Q., 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=Histone+modification+mapping+in+human+brain+reveals+aberrant+expression+of+histone+H3+lysine+79+dimethylation+in+neural+tube+defects.">Histone modification mapping in human brain reveals aberrant expression of histone H3 lysine 79 dimethylation in neural tube defects.</a> Neurobiol. Dis. 54, 404-413 (2013).</li><li>Zhang, Y., 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=Transcription+regulation+by+histone+methylation:+interplay+between+different+covalent+modifications+of+the+core+histone+tails.">Transcription regulation by histone methylation: interplay between different covalent modifications of the core histone tails.</a> Genes Dev. 15, 2343-2360 (2001).</li><li>Sanders, S. L., 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=Methylation+of+histone+H4+lysine+20+controls+recruitment+of+Crb2+to+sites+of+DNA+damage.">Methylation of histone H4 lysine 20 controls recruitment of Crb2 to sites of DNA damage.</a> Cell. 119, 603-614 (2004).</li><li>Martin, C., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+diverse+functions+of+histone+lysine+methylation.">The diverse functions of histone lysine methylation.</a> Nat. Rev. Mol. Cell Biol. 6, 838-849 (2005).</li><li>Greer, E. L., Shi, Y. <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:+a+dynamic+mark+in+health,+disease+and+inheritance.">Histone methylation: a dynamic mark in health, disease and inheritance.</a> Nat. Rev. Genet. 13, 343-357 (2012).</li><li>Ng, H. H., 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=Lysine+methylation+within+the+globular+domain+of+histone+H3+by+Dot1+is+important+for+telomeric+silencing+and+Sir+protein+association.">Lysine methylation within the globular domain of histone H3 by Dot1 is important for telomeric silencing and Sir protein association.</a> Genes Dev. 16, 1518-1527 (2002).</li><li>Kebede, A. F., Schneider, R., Daujat, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+types+and+sites+of+histone+modifications+emerge+as+players+in+the+transcriptional+regulation+contest.">Novel types and sites of histone modifications emerge as players in the transcriptional regulation contest.</a> FEBS J. 282, 1658-1674 (2015).</li><li>Roidl, D., Hacker, C. <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+during+neural+development.">Histone methylation during neural development.</a> Cell Tissue Res. 356, 539-552 (2014).</li><li>Mersfelder, E. L., Parthun, M. 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+tale+beyond+the+tail:+histone+core+domain+modifications+and+the+regulation+of+chromatin+structure.">The tale beyond the tail: histone core domain modifications and the regulation of chromatin structure.</a> Nucleic Acids Res. 34, 2653-2662 (2006).</li><li>van Leeuwen, F., Gafken, P. R., Gottschling, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dot1p+Modulates+Silencing+in+Yeast+by+Methylation+of+the+Nucleosome+Core.">Dot1p Modulates Silencing in Yeast by Methylation of the Nucleosome Core.</a> Cell. 109, 745-756 (2002).</li><li>Jones, B., 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=The+histone+H3K79+methyltransferase+Dot1L+is+essential+for+mammalian+development+and+heterochromatin+structure.">The histone H3K79 methyltransferase Dot1L is essential for mammalian development and heterochromatin structure.</a> PLoS Genet. 4, e1000190 (2008).</li><li>Woo Park, J., 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=RE-IIBP+Methylates+H3K79+and+Induces+MEIS1-mediated+Apoptosis+via+H2BK120+Ubiquitination+by+RNF20.">RE-IIBP Methylates H3K79 and Induces MEIS1-mediated Apoptosis via H2BK120 Ubiquitination by RNF20.</a> Sci. Rep. 5, 12485 (2015).</li><li>Vlaming, H., van Leeuwen, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+upstreams+and+downstreams+of+H3K79+methylation+by+DOT1L.">The upstreams and downstreams of H3K79 methylation by DOT1L.</a> Chromosoma. , (2016).</li><li>Jones, B., 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=The+histone+H3K79+methyltransferase+Dot1L+is+essential+for+mammalian+development+and+heterochromatin+structure.">The histone H3K79 methyltransferase Dot1L is essential for mammalian development and heterochromatin structure.</a> PLoS Genet. 4, e1000190 (2008).</li><li>Feng, Y., 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=Early+mammalian+erythropoiesis+requires+the+Dot1L+methyltransferase.">Early mammalian erythropoiesis requires the Dot1L methyltransferase.</a> Blood. 116, 4483-4491 (2010).</li><li>Cattaneo, P., 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=DOT1L-mediated+H3K79me2+modification+critically+regulates+gene+expression+during+cardiomyocyte+differentiation.">DOT1L-mediated H3K79me2 modification critically regulates gene expression during cardiomyocyte differentiation.</a> Cell Death Differ. 23, 555-564 (2016).</li><li>Zhang, Q., 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=Histone+modification+mapping+in+human+brain+reveals+aberrant+expression+of+histone+H3+lysine+79+dimethylation+in+neural+tube+defects.">Histone modification mapping in human brain reveals aberrant expression of histone H3 lysine 79 dimethylation in neural tube defects.</a> Neurobiol. Dis. 54, 404-413 (2013).</li><li>B&#252;ttner, N., Johnsen, S. A., K&#252;gler, S., Vogel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Af9/Mllt3+interferes+with+Tbr1+expression+through+epigenetic+modification+of+histone+H3K79+during+development+of+the+cerebral+cortex.">Af9/Mllt3 interferes with Tbr1 expression through epigenetic modification of histone H3K79 during development of the cerebral cortex.</a> Proc. Natl. Acad. Sci. U. S. A. 107, 7042-7047 (2010).</li><li>Roidl, D., 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=DOT1L+Activity+Promotes+Proliferation+and+Protects+Cortical+Neural+Stem+Cells+from+Activation+of+ATF4-DDIT3-Mediated+ER+Stress+In+Vitro.">DOT1L Activity Promotes Proliferation and Protects Cortical Neural Stem Cells from Activation of ATF4-DDIT3-Mediated ER Stress In Vitro.</a> Stem Cells Dayt. Ohio. 34, 233-245 (2016).</li><li>Robinson, J. T., 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=Integrative+genomics+viewer.">Integrative genomics viewer.</a> Nat. Biotechnol. 29, 24-26 (2011).</li><li>Langmead, B., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+gapped-read+alignment+with+Bowtie+2.">Fast gapped-read alignment with Bowtie 2.</a> Nat. Methods. 9, 357-359 (2012).</li><li>Zhang, Y., 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=Model-based+Analysis+of+ChIP-Seq+(MACS).">Model-based Analysis of ChIP-Seq (MACS).</a> Genome Biol. 9, R137 (2008).</li><li>Ram&#237;rez, F., 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=deepTools2:+a+next+generation+web+server+for+deep-sequencing+data+analysis.">deepTools2: a next generation web server for deep-sequencing data analysis.</a> Nucleic Acids Res. 44, W160-W165 (2016).</li><li>Roidl, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Histone modifications during cerebral cortex development. , (2015).</li><li>Turner, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChIP+with+Native+Chromatin:+Advantages+and+Problems+Relative+to+Methods+Using+Cross-Linked+Material.">ChIP with Native Chromatin: Advantages and Problems Relative to Methods Using Cross-Linked Material.</a> Mapping Protein/DNA Interactions by Cross-Linking. , (2001).</li><li>Dincman, T. A., Beare, J. E., Ohri, S. S., Whittemore, S. 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+cortical+mouse+oligodendrocyte+precursor+cells.">Isolation of cortical mouse oligodendrocyte precursor cells.</a> J. Neurosci. Methods. 209, 219-226 (2012).</li></ol>

ヒストン修飾、転写因子、ヒストン コード リーダー、作家、または消しゴムのゲノムの占有を検出するクロマチン免疫沈降を実行する 2 つの主要な方法があります。ヌクレアーゼ消化、ネイティブのクロマチン免疫沈降を用いたネイティブ チップであると、ヌクレオソームと他の DNA に接続されたタンパク質が DNA にしたバインドされて PFA 固定、せん断されたクロマチンを使用して提案手?...

脳の感覚, モータ, と認知機能が非常に複雑で物理的な環境の変化に影響を受けやすいです。脳は、深くつながっています 3 つの一般的な部分、後肢-、半ばと前脳, で構成されています。前脳, 内終脳は背終 (DT) と腹側終 (VT) に分けることができます。マウスの DT は、「インサイド アウト」方法1で E11.5 と E18.5 間形作られる六つの皮質層で構成されています。VT には、開発後大脳基底核2,3を形成する神経節隆起が含まれています。いくつかの細胞の種類で分類できます哺乳類の中枢神経系神経細胞やアストロ サイト、オリゴデンドロ サイト4など5時間空間的方法を開発します。まず、神経前駆細胞 (Npc) はニューロン、介在ニューロン VT と投射ニューロンの種類に、DT ではグリア細胞 (例えば、アストロ サイト6) に上昇を与えます。皮質過程を最も表面的な層 (層)、最初に形成、カハール ・ レチウス細胞を含みます。Npc を生成 E12.5 と E14.5 の間より深い神経層 (VI, V) 中を生じさせる上層 (第四) ニューロン7,814.5 16.5、前駆細胞の間。軸索の身元は、異なるモルフォゲン誘起の時間空間的転写プログラムおよびまたエピジェネティック プログラム2に指定します。
モーターの調整に関与しているが、小脳は菱脳にありマウス9で E10 と約 P20 の間成長します。小脳皮質と小脳核10が含まれています。成人の小脳皮質は 3 層、最も外側の分子層、プルキンエ細胞層、顆粒ニューロンの10を含む最も内側の顆粒層から成っています。小脳顆粒細胞は最小のニューロンで、脊椎動物の脳11すべての神経細胞の約 80% を表しています。彼らは外部の胚のゾーンに位置する前駆体から開発し、彼らの先12プルキンエ細胞層を移行します。終脳、小脳の開発はいくつかの重要なモルフォゲンによって調整される、ように時間と空間に依存した特定の機能を持っているし、開始は転写プログラム10に定義されています。
大脳皮質と小脳の層の開発は、特定のモルフォゲンの転写式で、したがって、DNA のクロマチンの状態によって制御されます。簡易ビューでクロマチンの状態を転写活性状態としてユークロマチンと転写活性サイレント領域として異質染色質に分割できます。クロマチンの基本単位としてヌクレオソームには、各コアのヒストン H2A、H2B、H3、H4、147 の塩基対の DNA13に囲まれた 2 つのコピーが含まれています。ヒストンは、メチル化、アセチル化、リン酸化、ユビキチン化、sumo 化、ADP リボシル化、脱アミノ化、およびプロリン異性化14,15によって高いファーストクリーニング変更します。ヒストンのリジンのメチル化は転写、複製、組み換え16、DNA 損傷応答17、およびゲノムインプリンティング18制御する最も安定したヒストン修飾と見なされます。リシンはモノラル、ディ、またはトリ メチル19アクセス可能なヒストンの尾にだけでなく、ヒストン20の球状ドメイン内で表示できます。H3K4 H3K36 で特定のメチル化は、主ユークロマチン、H3K9、H3K27、または H4K20 で特定のメチル化は主にヘテロクロマチン領域、すべての残基がヒストンの尾14,内にある19,21。H3K79 のメチル化はヒストンの球状ドメイン内にあるし、転写活性、発現不活性ゲノム領域22とも関連付けられています。それは、酵母や子牛の胸腺、鶏、人間23で観察されているので変更は保存されました。H3K79 モノ, ジおよびトリメチル (H3K79me1、me2、me3) は、ヒストンのメチル化酵素 DOT1L24,25と26核設定ドメインを含むタンパク質 2 (Nsd2) によって触媒されます。DOT1L は、増殖、DNA 修復、27の reprograming 携帯電話で関与しています。マウスでDot1lの損失は、発達段階 E10.528,29周り出生前の死に します。心臓の発達過程および myocardiocyte の分化は、DOT1L は遺伝子発現の規則30に不可欠です。中枢神経系における DOT1L 関数は、神経管開発31で関与する可能性があります、それはTbr1の抑制に関与する-脳開発32中、式小胞体ストレスによる機能可能性があります応答遺伝子33。コンテキスト依存のアクティブ化または中枢神経系の開発のような体内状況に特に H3K79me の抑制作用は、日付には部分的に32を理解専用です。H3K79 のメチル化はヒストン 3 の球状ドメインにあるので、柔軟なヒストンの尾23での変更と比較してアクセスしにくい立体です。H3K79 メチル化の機能を理解し、その場所とゲノム環境を決定する信頼性と再現性の高い分析法が必要です。QPCR や時点の異なるシーケンスを介して H3K79 のメチル化を分析する、異なる神経前駆細胞 (皮質のクリック単価) と小脳の CGNPs、DOT1L 阻害剤治療、チップ メソッドの分離手法を提示するこの方法で大脳皮質と小脳の開発中にポイント。プロトコルとその可能性の概要については、図 1を参照してください。

<table>
	<tbody>
		<tr>
			<td>Anti-GAPDH</td>
			<td>Abcam</td>
			<td>ab8245</td>
			<td>Category: Antibody 
			Abbreviation/Comment: Immunoblot dilution 1:5000</td>
		</tr>
		<tr>
			<td>Anti-H3</td>
			<td>Abcam</td>
			<td>ab1791</td>
			<td>Category: Antibody 
			Abbreviation/Comment: Immunoblot dilution 1:3000</td>
		</tr>
		<tr>
			<td>Anti-H3K79me2</td>
			<td>Diagenode</td>
			<td>pAb-051-050</td>
			<td>Category: Antibody 
			Abbreviation/Comment: ChIP antibody</td>
		</tr>
		<tr>
			<td>Anti-H3K79me2</td>
			<td>Abcam</td>
			<td>ab-051-050</td>
			<td>Category: Antibody 
			Abbreviation/Comment: Immunoblot dilution 1:1000</td>
		</tr>
		<tr>
			<td>Anti-rabbit-IgG</td>
			<td>Diagenode</td>
			<td>C15410206</td>
			<td>Category: Antibody 
			Abbreviation/Comment: ChIP Ctrl antibody</td>
		</tr>
		<tr>
			<td>Anti-Tubulin alpha</td>
			<td>Abcam</td>
			<td>ab108629</td>
			<td>Category: Antibody 
			Abbreviation/Comment: Immunoblot dilution 1:3000</td>
		</tr>
		<tr>
			<td>Apo-Transferrin (1 mg/ml)</td>
			<td>Sigma-Aldrich</td>
			<td>T1147</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CCM</td>
		</tr>
		<tr>
			<td>B27 Supplement (50x)</td>
			<td>Life Technologies</td>
			<td>17504044</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CCM</td>
		</tr>
		<tr>
			<td>Bioanalyzer</td>
			<td>Agilent technologies</td>
			<td>G2940CA</td>
			<td>Category: ChIP 
			Abbreviation/Comment: For analysis of sheared chromatin</td>
		</tr>
		<tr>
			<td>Bioruptor NextGen</td>
			<td>Diagenode</td>
			<td>B01020001</td>
			<td>Category: ChIP 
			Abbreviation/Comment: Ultrasonicator</td>
		</tr>
		<tr>
			<td>Boric acid pH 8.4</td>
			<td>Sigma Aldrich</td>
			<td>B6768</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CPC culturing</td>
		</tr>
		<tr>
			<td>CFX Connect RT PCR Detection System</td>
			<td>Bio-Rad</td>
			<td>1855201</td>
			<td>Category: ChIP Analysis 
			Abbreviation/Comment: Detection system for qPCR</td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>Life Technologies</td>
			<td>11320-033</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CGM</td>
		</tr>
		<tr>
			<td>Dynabeads Protein A</td>
			<td>Invitrogen</td>
			<td>10001D</td>
			<td>Category: ChIP 
			Abbreviation/Comment: Magnetic beads, for ChIP</td>
		</tr>
		<tr>
			<td>EPZ-5676</td>
			<td>Selleckchem</td>
			<td>S7062</td>
			<td>Category: DOT1L inhibition 
			Abbreviation/Comment: For DOT1L inhibition in cell culture</td>
		</tr>
		<tr>
			<td>Ethylenediamine tetraacetic acid</td>
			<td>SERVA</td>
			<td>39760.01</td>
			<td>Category: ChIP 
			Abbreviation/Comment: EDTA</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum 10% (v/v)</td>
			<td>Gibco</td>
			<td>10082147</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CPC isolation and CGM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G5767</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CGNP isolation</td>
		</tr>
		<tr>
			<td>Glutathione (1.25 mg/ml)</td>
			<td>Sigma-Aldrich</td>
			<td>G4251</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CCM</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Carl Roth</td>
			<td>3187</td>
			<td>Category: ChIP 
			Abbreviation/Comment: For cell fixation</td>
		</tr>
		<tr>
			<td>GoTaq mastermix</td>
			<td>Promega</td>
			<td>A6002</td>
			<td>Category: ChIP Analysis 
			Abbreviation/Comment: DNA polymerase master mix for qPCR</td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balanced Salt Solution</td>
			<td>Life Technologies</td>
			<td>14025-100</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: HBSS</td>
		</tr>
		<tr>
			<td>L-glutamine (200 mM)</td>
			<td>Life Technologies</td>
			<td>25030081</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CCM</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma-Aldrich</td>
			<td>L2020</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CPC culturing</td>
		</tr>
		<tr>
			<td>Lithium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>L4408</td>
			<td>Category: ChIP 
			Abbreviation/Comment: LiCl</td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Life Technologies</td>
			<td>17502048</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CGM</td>
		</tr>
		<tr>
			<td>NanoDrop 3300</td>
			<td>Thermo Fisher</td>
			<td>3300</td>
			<td>Category: ChIP 
			Abbreviation/Comment: Fluorospectrometer for DNA quantification</td>
		</tr>
		<tr>
			<td>NEB Next Ultra DNA Library Prep Kit for Illumina</td>
			<td>NEB</td>
			<td>E7645S</td>
			<td>Category: ChIP Analysis 
			Abbreviation/Comment: Kit for Library preparation</td>
		</tr>
		<tr>
			<td>NEBNext Multiplex Oligos for Illumina</td>
			<td>NEB</td>
			<td>E7335</td>
			<td>Category: ChIP Analysis 
			Abbreviation/Comment: Oligos for Library preparation</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CCM</td>
		</tr>
		<tr>
			<td>NP-40 Alternative</td>
			<td>Calbiochem</td>
			<td>492016</td>
			<td>Category: ChIP 
			Abbreviation/Comment: For ChIP buffer</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Carl Roth</td>
			<td>335</td>
			<td>Category: ChIP 
			Abbreviation/Comment: PFA, for cell fixation</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin-Neomycin 1% (v/v)</td>
			<td>Life Technologies</td>
			<td>15640055</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: PSN, for CCM and CGM</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Life Technologies</td>
			<td>10010023</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: PBS, for CPC isolation</td>
		</tr>
		<tr>
			<td>PicoGreen Kit</td>
			<td>Thermo Fisher</td>
			<td>P11496</td>
			<td>Category: ChIP Analysis 
			Abbreviation/Comment: Visualizing dye for DNA quantification</td>
		</tr>
		<tr>
			<td>Poly-D-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P6407</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CGNP isolation</td>
		</tr>
		<tr>
			<td>Poly-L-ornithine hydrobromide</td>
			<td>Sigma Aldrich</td>
			<td>P3655</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CPC culturing</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Thermo Fisher</td>
			<td>AM9640G</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: KCl, for CGM</td>
		</tr>
		<tr>
			<td>Protease inhibitor</td>
			<td>Roche</td>
			<td>4693159001</td>
			<td>Category: ChIP 
			Abbreviation/Comment: For ChIP</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Sigma-Aldrich</td>
			<td>3115879001</td>
			<td>Category: ChIP 
			Abbreviation/Comment: For ChIP</td>
		</tr>
		<tr>
			<td>Qiagen MinElute</td>
			<td>Qiagen</td>
			<td>28004</td>
			<td>Category: ChIP 
			Abbreviation/Comment: Kit for DNA purification</td>
		</tr>
		<tr>
			<td>RNAse</td>
			<td>Sigma-Aldrich</td>
			<td>R6513</td>
			<td>Category: ChIP 
			Abbreviation/Comment: For ChIP</td>
		</tr>
		<tr>
			<td>SGC0946</td>
			<td>Selleckchem</td>
			<td>S7079</td>
			<td>Category: DOT1L inhibition 
			Abbreviation/Comment: For DOT1L inhibition in cell culture</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Carl Roth</td>
			<td>8551.1</td>
			<td>Category: ChIP 
			Abbreviation/Comment: for Elution buffer</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Carl Roth</td>
			<td>9265</td>
			<td>Category: ChIP 
			Abbreviation/Comment: NaCl, for ChIP buffer</td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma-Aldrich</td>
			<td>30970</td>
			<td>Category: ChIP 
			Abbreviation/Comment: For ChIP</td>
		</tr>
		<tr>
			<td>Sodium dodecylsulfate</td>
			<td>Carl Roth</td>
			<td>183</td>
			<td>Category: ChIP 
			Abbreviation/Comment: SDS, for ChIP</td>
		</tr>
		<tr>
			<td>Sonic hedgehock (SHH)</td>
			<td>Sigma-Aldrich</td>
			<td>SRP6004</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CGNP isolation</td>
		</tr>
		<tr>
			<td>Superoxide dismutase (1mg/ml)</td>
			<td>Sigma-Aldrich</td>
			<td>S7571</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CCM</td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)aminomethane</td>
			<td>Carl Roth</td>
			<td>9090</td>
			<td>Category: ChIP 
			Abbreviation/Comment: TRIS, for ChIP buffer</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Carl Roth</td>
			<td>X100</td>
			<td>Category: ChIP 
			Abbreviation/Comment: For ChIP buffer</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0,05% (w/v)</td>
			<td>Sigma Aldrich</td>
			<td>59417C</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CPC isolation</td>
		</tr>
		<tr>
			<td>Tween20</td>
			<td>Carl Roth</td>
			<td>28320</td>
			<td>Category: ChIP 
			Abbreviation/Comment: For bead preparation</td>
		</tr>
		<tr>
			<td colspan="4">Other Lab devices: Neubauer counting chamber, Incubator, Rotator, Shaker, Disection set, Water bath</td>
		</tr>
		<tr>
			<td>CCM: Cortical cell medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CGM: CGNP cell culture medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and cultivation of neural progenitors followed by chromatin-immunoprecipitation of histone 3 lysine 79 dimethylation mark

脳の発達は、時間空間的なモルフォゲンや転写の異なるプログラムの勾配によって制御される複雑なプロセスです。また、ヒストンのメチル化などのエピジェネティックなクロマチン修飾の確立およびこのプロセス内で特定のセル運命を維持する重要な役割があります。大多数のヒストンのメチル化はヒストン、修飾子、消しゴム、およびヒストン リーダー蛋白質にアクセス可能な柔軟なヒストンの尾で発生します。対照的に、H3K79 のメチル化はヒストン 3 の球状ドメインに位置し、さまざまな発達機能に関与しています。H3K79 メチル化は進化的に保存されているし、酵母にホモ ・ サピエンスから種の広い範囲で見つけることができます。神経前駆細胞を含む生物内の異なる細胞数の変更が発生します。H3K79 メチル化ヒストン 3 の球状ドメインの場所すると、評価が難しくなります。ここでは、分離する手法を提示して文化大脳皮質前駆細胞 (CPCs) 胎生期大脳皮質の脳組織 (E11.5-E14.5) または小脳顆粒細胞前駆細胞 (CGNPs) 生後組織 (P5-P7)、効率的に immunoprecipitate量的な PCR (qPCR) とゲノム シーケンスの H3K79me2。

神経前駆細胞の分離、培養、H3K79me2 チップ、チップの解析手法の一般的な方式:産後の段階で胚の脳の発達中の異なる時点で大脳皮質前駆細胞や小脳顆粒細胞前駆細胞の H3K79me2 チップを実行するフローチャートを図 1に示します。最初のステップとして、脳が分離して (E11.5 と E14.5) の間終脳や小脳 (P5 P7) が取得します。DT とバーモン...

Watch this Scientific Journal Video about Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark at JoVE.com

Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark

3 ヒストンのリジン 79 ジメチル化マークのクロマチン免疫沈降に続いて神経前駆細胞の分離と培養

ヒストン 3 リジン 79 ジメチル化 (H3K79me2) - 内にあるヒストン マークのクロマチン免疫沈降 (チップ) の胎生期および生後の脳組織から神経前駆細胞の培養を特定し効果的かつ再現可能な手法を提案しますヒストン 3 の球状ドメインで。

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. ...

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マウス神経堤幹細胞の培養のためのニワトリ胚抽出物の製造

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent ...

Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

運動によって変調された神経細胞の生理的、形態学的および神経化学的キャラクタリゼーション

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor ...

Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

軸索損傷後の脊髄後根神経節の組織からのクロマチン免疫沈降

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

内在性NADH蛍光の二光子イメージングによるマウス大脳皮質におけるMicroregional低酸素の検出

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

ホールマウントマッスル分析による再現性のあるマウスの坐骨神経クラッシュと再生のその後の評価

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

ラット胚神経細胞の分離と文化：クイックプロトコル

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

胎児マウス神経管から神経堤細胞の単離および培養

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

An Optimized Procedure for Fluorescence-activated Cell Sorting (FACS) Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice

An Optimized Procedure for Fluorescence-activated Cell Sorting FACS Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice

蛍光活性化細胞選別（FACS）胎児マウスの内臓からの自律神経前駆細胞の分離用に最適化された手順

During development neural crest (NC)-derived neuronal progenitors migrate away from the neural tube to form autonomic ganglia in visceral organs like the ...

Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

重度の脊髄損傷後のフィブリンおよび成長因子カクテル神経幹細胞の生存と分化の促進

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord ...

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

マウス腸の神経前駆細胞の定量細胞遊走アッセイ

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...