Henriette Bertemes laboratuar CGN ekimi protokolde kurmak için yardım ettiğin için teşekkür ederim. Bu yöntem kağıt DFG tarafından finanse edilen CRC992 tıbbi epigenetik tarafından TV için fon tarafından desteklenmiştir. Yazarlar Freiburg Galaxy takım destek kabul: Pavankumar Videm, Björn Grüning ve Prof. Dr. Rolf Backofen, Biyoinformatik, Freiburg Üniversitesi, Almanya ortak araştırma merkezi 992 tıbbi epigenetik tarafından (DFG grant SFB 992/1 2012) finanse ve Alman Federal Bakanlığı Eğitim ve araştırma (BMBF grant 031 A538A RBC (de. NBI)).

<ol>
	<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. 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Yazarlar onlar rakip hiçbir mali çıkarları var bildirmek

Histon değişiklikler, transkripsiyon faktörleri, Histon Kodu okuyucular, yazarlar veya silgi genomik doluluk algılamaya Kromatin immunoprecipitation gerçekleştirmek için iki ana yolu vardır. Bir sindirilmiş, nükleaz için immunoprecipitation, yerel Kromatin kullanarak yerel çip yöntem ve diğer içinde oluşturarlar ve diğer DNA bağlı proteinler kovalent DNA'yı bağlı olduğu İngiltere'de yılın sabit, yamultulmuş Kromatin kullanarak sunulan yöntemdir 39. yerli küçük parça...

Beynin duyusal, motor ve Bilişsel işlevler son derece karmaşık ve fiziksel ve çevresel değişikliklere duyarlı. Beyin üç genel parçalar hind-, orta-ve ön, hangi derin bağlı oluşur. Ön içinde telencephalon dorsal telencephalon (DT) ve ventral telencephalon (VT) bölünmüş olabilir. DT farelerin, E11.5 ve E18.5 arasında bir "Inside-out" şekilde1' de kurulan altı kortikal Katmanlar oluşur. VT ganglionic efendiler gelişmede, daha sonra Bazal gangliyon2,3formu içerir. Çeşitli hücre tiplerinin nöronlar, astrocytes veya oligodendrocytes4, gibi memeli merkez sinir sisteminde temporo-kayma şekilde5' te geliştirmek sınıflandırılabilir. İlk olarak, nöral progenitör hücre (NPCs) nöronlar, interneurons VT ve projeksiyon nöronların farklı türde DT ve daha sonra üzerine gliyal hücreler (örneğin, astrocytes6) ortaya çıkmasına. Sırasında kortikal gelişim, en yüzeysel Katmanı (katman ben), hangi Cajal Retzius hücreleri içeren ilk oluşturulur. O zaman, E12.5 ve E14.5 arasında NPCs derin nöronal katmanları (VI, V) iken üst katmanı (IV-II) nöronlar7,814.5 ve 16.5, ataları vermek yükselişi arasında oluşturmak. Nöronal kimlik farklı morphogen kaynaklı temporo-kayma transkripsiyon program tarafından ve ayrıca epigenetik programları2tarafından belirtilir.
Motor koordinasyon içinde karıştığı, beyincik, arka beyin içinde yer alan ve E10 ve kabaca P20 arasında fare9' geliştirir. Serebellar korteksin ve serebellar çekirdeği10içerir. Yetişkin serebellar korteksin üç kat, en dıştaki moleküler katmanı, Purkinje hücre katmanı ve taneli nöronlar10içeren en içteki taneli katman oluşur. Serebellar granül hücre en küçük nöronlar vardır ve tüm omurgalı beyin11nöronlarda yaklaşık % 80'i temsil eder. Onlar üzerinden dış germinal bölgede bulunan öncüleri geliştirmek ve onların hedef12Purkinje hücre katmanı üzerinden göç. Telencephalon, beyincik geliştirilmesi birkaç önemli morfojenlerdeki tarafından düzenlenmiştir gibi hangi-si olmak belirli zaman ve uzay bağımlı görev ve başlatmak transkripsiyon programlar10tanımlı.
Kortikal ve serebellar katmanları gelişimi belirli morfojenlerdeki transkripsiyon ifadesi tarafından ve böylece, DNA'ın Kromatin devlet tarafından kontrol edilir. Basitleştirilmiş bir görünümde, Kromatin Birleşik transcriptionally etkin olarak euchromatin ve heterochromatin transcriptionally sessiz bölgeleri olarak ayrılabilir. Kromatin temel birim olarak nucleosome her çekirdek Histon H2A, H2B, H3 ve H4, DNA13147 baz çifti tarafından çevrili iki kopyasını içerir. Histon metilasyonu, asetilasyon, fosforilasyon, ubiquitination, sumoylation, ADP-ribosylation, Deaminasyon ve prolin isomerization14,15tarafından son derece post-translationally değiştirilir. Histon lizin metilasyonu transkripsiyon, çoğaltma, Rekombinasyon16, DNA hasarı yanıt17ve genomik sürecinden18denetler en istikrarlı Histon değişiklik olarak kabul edilir. Lysines mono-, di- veya üç metillenmiş19 olması ve sadece erişilebilir Histon kuyrukları üzerinde aynı zamanda Histon20küresel etki alanı içinde görünür. H3K4 ve H3K36 belirli methylations euchromatin ile ilişkili, belirli methylations H3K9, H3K27 veya H4K20-esas olarak bulundu heterochromatic bölgelerde, her ne kadar tüm artıkları Histon kuyruk14içinde, yer alan 19,21. H3K79 metilasyonu Histon küresel etki alanı içinde yer alan ve transkripsiyon etkinlik, ama aynı zamanda transcriptionally etkisiz genomik bölgeleri22ile ile ilişkili olduğu düşünülmektedir. Maya, buzağı timus, tavuk ve insan23gözlenmiştir beri değişiklik örümceklerle korunmuş. H3K79 mono, di ve trimethylation (H3K79me1, me2, me3), Histon methyltransferases DOT1L24,25 ve26nükleer SET etki alanını içeren Protein 2 (Nsd2) tarafından katalize. DOT1L nükleer silahların yayılmasına karşı DNA tamiri ve27REPROGRAMING hücresel karıştığı. Dot1l kaybı farelerde çevresinde gelişimsel sahne E10.528,29doğum öncesi bir ölüme yol açar. Kalp Geliştirme sırasında ve myocardiocyte ayrımında DOT1L gen ifade Yönetmeliği30için esastır. Merkezi sinir sistemi DOT1L işlevi Nöral tüp geliştirme31' karıştığı, Tbr1bastırmak ilgilenmektedir-ön geliştirme32sırasında ifade ve ER-stres yönetmelikte çalışmayabilir Yanıt genler33. İçerik bağımlı etkinleştirme veya özellikle merkezi sinir sistemi gelişimi gibi vivo içinde durumlar ile H3K79me, bastırmak eylem için kısmen32anladım sadece tarihidir. H3K79 metilasyonu Histon 3 küresel etki alanında bulunduğundan, bu sterically daha az esnek Histon kuyrukları23değişiklikler ile karşılaştırıldığında erişilemez. H3K79 metilasyonu işlevini anlamak için güvenilir ve tekrarlanabilir analiz yöntemleri konumu ve genomik çevre belirlemek için gereklidir. Bu yöntemleri yazıda, biz izolasyon yöntemleri farklı sinirsel ataları (korteks için TBM'ler) ve CGNPs için beyincik, etkili DOT1L inhibitörü tedavisi ve bir çip yöntem H3K79 metilasyonu yolu ile qPCR ya da sıralama farklı zamanda analiz etmek için mevcut Puan kortikal ve serebellar geliştirme sırasında. Protokol ve olanakların genel bakış için bkz: şekil 1.

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

Beyin gelişimi temporo-kayma bir şekilde morfojenlerdeki ve farklı transkripsiyon programları gradyanlar tarafından kontrol edilir karmaşık bir süreçtir. Ayrıca, gibi Histon metilasyonu, epigenetik Kromatin değişiklikler kurulması ve belirli hücre kaderi bu işlemde sürdürmek için önemli bir role sahiptir. Histon metilasyonu büyük çoğunluğu Histon değiştiriciler, silgi ve Histon okuyucu proteinlerin erişilebilen esnek Histon kuyruk üzerinde oluşur. Buna ek olarak, H3K79 metilasyonu Histon 3 küresel etki alanında bulunan ve farklı gelişim işlevlerinde karıştığı. H3K79 metilasyonu örümceklerle korunmuş ve türlerin geniş bir Homo sapiens Saccharomyces cerevisiaeiçin bulunabilir. Farklı hücre popülasyonlarının sinirsel ataları da dahil içinde değişiklik oluşur. Histon 3 küresel etki alanında H3K79 metilasyonu konumunu değerlendirmek zorlaştırır. Burada, biz izole etmek için yöntemler mevcut ve embriyonik kortikal beyin dokusu (E11.5-E14.5) veya serebellar taneli nöron ataları (CGNPs) Doğum sonrası doku (P5-P7) ve verimli bir şekilde immunoprecipitate (TBM) kültür kortikal progenitör hücre Kantitatif PCR (qPCR) ve genom çapındaki sıralama için H3K79me2.

Nöral progenitör yalıtım, ekimi, H3K79me2 ChIP ve çipi analiz yöntemleri genel düzeni: H3K79me2 çip kortikal progenitör hücre embriyonik beyin gelişimi sırasında farklı zaman noktalarda veya serebellar taneli nöron ataları, postnatal aşamalarında gerçekleştirmek için bir akış şeması şekil 1 gösterir. Bir ilk adım olarak beyin izole olmak zorunda ve telencephalon (E11.5-E14.5 arası) veya beyincik (P5-P7) alınması...

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

Yalıtım ve sinirsel ataları Kromatin-Immunoprecipitation Histon 3 lizin 79 Dimethylation Mark tarafından takip tarımı

Biz yalıtmak ve nöral progenitör hücreler embriyonik ve postnatal beyin dokusundan Kromatin immunoprecipitation (ChIP) Histon 3 lizin 79 dimethylation (H3K79me2) - içinde yer alan bir Histon işareti için kültür için etkili ve tekrarlanabilir bir yöntem mevcut Histon 3 küresel etki alanı.

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

isolation-cultivation-neural-progenitors-followed-chromatin

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7.4K Görüntüleme.  University of Freiburg. Biz yalıtmak ve nöral progenitör hücreler embriyonik ve postnatal beyin dokusundan Kromatin immunoprecipitation (ChIP) Histon 3 lizin 79 dimethylation (H3K79me2) - içinde yer alan bir Histon işareti için kültür için etkili ve tekrarlanabilir bir yöntem mevcut Histon 3 küresel etki alanı.

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

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

Periventriküler Yetişkin Rat Bölgesinde ve İnsan Omurilik gelen Nöral Kök / Progenitör Hücreler izolasyonu

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

Gelişim Biyolojisi

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

2D ve 3D Ortamlarda Sinir Progenitör Hücreleri Elektrik Alanı kontrollü Yönetmen Göç

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

Biyomühendislik

Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres

Epigenetic modifications may be involved in the development and progression of glioma. Changes in methylation and acetylation of promoters and regulatory regions of oncogenes and tumor suppressors can lead to changes in gene expression and play an important role in the pathogenesis of brain tumors. Native chromatin immunoprecipitation (ChIP) is a popular technique that allows the detection of modifications or other proteins tightly bound to DNA. In contrast to cross-linked ChIP, in native ChIP, cells are not treated with formaldehyde to covalently link protein to DNA. This is advantageous because sometimes crosslinking may fix proteins that only transiently interact with DNA and do not have functional significance in gene regulation. In addition, antibodies are generally raised against unfixed peptides. Therefore, antibody specificity is increased in native ChIP. However, it is important to keep in mind that native ChIP is only applicable to study histones or other proteins that bind tightly to DNA. This protocol describes the native chromatin immunoprecipitation on murine brain tumor neurospheres.

Epigenetic mechanisms are frequently altered in glioma. Chromatin immunoprecipitation could be used to study the consequences of genetic alterations in glioma that result from changes in histone modifications which regulate chromatin structure and gene transcription. This protocol describes native chromatin immunoprecipitation on murine brain tumor neurospheres.

Fare Beyin tümör Neurospheres kullanarak yerel Kromatin Immunoprecipitation

Epigenetic modifications may be involved in the development and progression of glioma. Changes in methylation and acetylation of promoters and regulatory ...

Kanser Araştırmaları

Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to study. When dissociated from embryonic or post-natal brain, single NPCs will proliferate in suspension to form neurospheres. Daughter cells within these cultures spontaneously adopt distinct developmental lineages (neurons, oligodendrocytes, and astrocytes) over the course of expansion despite being exposed to the same extracellular milieu. This progression recapitulates many of the stages observed over the course of neurogenesis and gliogenesis in post-natal brain and is often used to study basic NPC biology within a controlled environment. Assessing the full impact of 3D topography and cellular positioning within these cultures on NPC fate is, however, difficult. To localize target proteins and identify NPC lineages by immunocytochemistry, free-floating neurospheres must be plated on a substrate or serially sectioned. This processing is required to ensure equivalent cell permeabilization and antibody access throughout the sphere. As a result, 2D epifluorescent images of cryosections or confocal reconstructions of 3D Z-stacks can only provide spatial information about cell position within discrete physical or digital 3D slices and do not visualize cellular position in the intact sphere. Here, to reiterate the topography of the neurosphere culture and permit spatial analysis of protein expression throughout the entire culture, we present a protocol for isolation, expansion, and serial sectioning of post-natal hippocampal neurospheres suitable for epifluorescent or confocal immunodetection of target proteins. Connexin29 (Cx29) is analyzed as an example. Next, using a hybrid of graphic editing and 3D modelling softwares rigorously applied to maintain biological detail, we describe how to re-assemble the 3D structural positioning of these images and digitally map labelled cells within the complete neurosphere. This methodology enables visualization and analysis of the cellular position of target proteins and cells throughout the entire 3D culture topography and will facilitate a more detailed analysis of the spatial relationships between cells over the course of neurogenesis and gliogenesis in vitro.
Both Imbeault and Valenzuela contributed equally and should be considered joint first authors.

Here, we describe how to produce, expand, and immunolabel postnatal hippocampal neural progenitor cells (NPCs) in three-dimensional (3D) culture. Next, using hybrid visualization technologies, we demonstrate how digital images of immunolabelled cryosections can be used to reconstruct and map the spatial position of immunopositive cells throughout the entire 3D neurosphere.

3D Nöral Kök Hücre Kültürü yerelleştirme Protein: Hybrid Görselleştirme Metodolojisi

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to ...

Nörobilim

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

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

Embriyonik Mürin Nöral Tüp çıkan sinirsel Crest Hücre İzolasyonu ve Kültür

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

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.

Yalıtım ve kültür embriyonik fare nöral kök hücrelerin

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

Native Chromatin Immunoprecipitation of Neurosphere Cells

<a href='https://app.jove.com/v/57016/native-chromatin-immunoprecipitation-using-murine-brain-tumor'>Source: Mendez, F. M. et al., Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres.&#160;J. Vis. Exp.&#160; (2018).</a>This video demonstrates the isolation of protein-DNA complexes from neurosphere cells derived from a mouse brain tumor using native chromatin immunoprecipitation. It details the steps for chromatin preparation, immunoprecipitation, washing, and protein digestion to release DNA for downstream analysis.

Source: Mendez, F. M. et al., Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres.&#160;J. Vis. Exp.&#160; (2018).This video ...

JoVE Encyclopedia of Experiments

Neuropathology

Tumors

Isolating Induced Neural Progenitor Cell Colonies Derived From Adult Human Fibroblasts

Source: Meyer, S. et al., <a href="https://app.jove.com/v/52831">Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells.</a>&#160;J. Vis. Exp. (2015).
This video demonstrates a technique for isolating induced neural progenitor cells (iNPCs) derived from adult human fibroblasts. It outlines the steps involved in isolating the iNPC colonies and culturing them in laminin-coated multi-well plate wells in the presence of suitable culture media that facilitate the continued proliferation and expansion of the iNPC population.

Yetişkin İnsan Fibroblastlarından Türetilen İndüklenmiş Nöral Progenitör Hücre Kolonilerinin İzole Edilmesi

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Isolation of Putative Induced ...

Nöronal Kültür Teknikleri

Birincil Nöronal Kültür

Isolating and Culturing Murine Cerebellar Granular Neuron Progenitors

Source: Bovio, P., et al., <a href="https://app.jove.com/v/56631">Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark.</a> J. Vis. Exp. (2018)
This video demonstrates the isolation and cultivation of cerebellar neural progenitor cells from a mouse pup&#39;s cerebellum. Cerebellar granule neuron progenitors are isolated and purified through differential adhesion, effectively removing astrocytes. These cultured progenitors subsequently differentiate into cerebellar granule neurons.

Murin serebellar granüler nöron progenitörlerinin izole edilmesi ve kültürlenmesi

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1.&#160;Preparations

	Preparations ...

Isolation of Neural Stem Progenitor Cells from the Spinal Cord Periventricular Region of an Adult Rat

Source: Mothe, A., et al., <a href="https://app.jove.com/v/52732">Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord.</a> J. Vis. Exp. (2015).
This video presents a method for isolating neural stem/progenitor cells from the periventricular tissue of the spinal cord in adult rats. The technique includes incubating the tissue fragments in a solution containing proteolytic enzymes to disrupt the extracellular matrix and release the cells. Subsequently, the released cells are plated in a medium containing specific growth factors that enhance neural stem/progenitor proliferation and promote aggregation into three-dimensional neurospheres.

Erişkin Bir Sıçanın Omurilik Periventriküler Bölgesinden Nöral Kök Progenitör Hücrelerin İzolasyonu

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 
1. Preparation of Dissection ...