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

We bedanken Henriette Bertemes voor het helpen om het protocol van de teelt van CGN in het lab. Deze methode papier werd gesteund door de Medische epigenetica DFG-gefinancierde CRC992 door financiering van TV. De auteurs erkennen de steun van het Team van de Melkweg Freiburg: Pavankumar Videm, Björn Grüning en Prof. Rolf Backofen, bio-informatica, Universiteit van Freiburg, Duitsland gefinancierd door Collaborative Research Centrum 992 Medische epigenetica (DFG grant SFB 992/1-2012) en het Duitse federale ministerie van onderwijs en onderzoek (goedgekeurd subsidie 031 A538A RBC (de. NBI)).

Dierenwelzijn commissies van de Universiteit van Freiburg en de plaatselijke instanties goedgekeurd alle dierproeven (G12/13, G16/11) vermeld in het volgende protocol.
1. voorbereiding
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Voorbereiding van de CPC's isolatie
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<li>Instellen getimede paring om te verkrijgen van embryo's in de verschillende stadia van ontwikkeling van de cortex (tussen E11.5 en E14.5). Gebruik de muizen van de stam NMRI (Naval Medical Research Institute), die ten minste 8 oud weken. N...

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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. 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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. 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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. 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Er zijn twee belangrijke manieren voor het uitvoeren van de chromatine immunoprecipitation om te ontdekken de genomic bezetting van Histon modificaties, transcriptiefactoren, histone code lezers, schrijvers of gummen. Een is de inheemse ChIP methode met behulp van de nuclease verteerd, inheemse chromatine voor immunoprecipitation, en anderzijds de onderhavige methode met PFA-vaste, sheared chromatine, waarin de nucleosomes en andere DNA-ingeschrevenen eiwitten wijze aan het DNA covalente 39. nativ...

De sensorische, motorische en cognitieve functies van de hersenen zijn zeer complex en gevoelig voor fysieke en ecologische veranderingen. De hersenen bestaat uit drie algemene delen de hind-, midden-, en reukkolf, die diep met elkaar zijn verbonden. Binnen de reukkolf, kan de telencephalon worden onderverdeeld in een dorsale telencephalon (DT) en een ventrale telencephalon (VT). De DT van muizen bestaat uit zes corticale lagen die worden gevormd tussen E11.5 en E18.5 in een "binnenstebuiten" manier1. De VT bevat de ganglionaire eminenties in ontwikkeling, die later het vormen van de basale ganglia2,3. Verschillende soorten cellen kunnen worden ingedeeld in de zoogdieren centrale zenuwstelsel zoals neuronen, astrocyten, oligodendrocyten4, of die zich in een temporo-ruimtelijke wijze5 ontwikkelen. Ten eerste, de neurale voorlopercellen (NPC) aanleiding geven tot verschillende soorten neuronen, interneuronen in de VT en projectie neuronen in het DT, en later naar gliale cellen (bijvoorbeeld, astrocyten6). Tijdens de corticale ontwikkeling, de meest oppervlakkige laag (layer ik), die bevat Cajal-Retzius cellen, eerst wordt gevormd. Vervolgens, tussen E12.5 en E14.5, NPC's genereren diepere neuronale lagen (VI, V) terwijl tussen 14,5 en 16,5, progenitoren aanleiding geven tot7,8van de neuronen van de bovenste laag (IV-II). Neuronale identiteit is opgegeven door de verschillende morphogen-geïnduceerde temporo-ruimtelijke transcriptionele programma's, en daarnaast door epigenetische programma's2.
Het cerebellum, die is betrokken bij de coördinatie van de motor, is gelegen in de hindbrain en ontwikkelt tussen E10 en ongeveer P20 in muizen9. Het bevat de cerebellaire cortex en de cerebellaire kernen10. De volwassen cerebellaire cortex bestaat uit drie lagen, de buitenste laag van de moleculaire, het Purkinje cellaag en de binnenste granulaire laag met granulaire neuronen10. De cerebellaire submodule cellen zijn de kleinste neuronen en vertegenwoordigen ongeveer 80% van alle neuronen in de hersenen van de gewervelde11. Ze migreren door het Purkinje cellaag naar hun bestemming12en ontwikkelen van precursoren in de externe germinal zone bevindt. Zoals in de telencephalon, de ontwikkeling van het cerebellum wordt geregeld door verschillende belangrijke morphogens, die hebben specifieke tijd - en ruimte-afhankelijke functies en initiëren gedefinieerd transcriptionele programma's10.
De ontwikkeling van corticale en cerebellaire lagen wordt gecontroleerd door transcriptionele expressie van specifieke morphogens en, dus, door de staat van de chromatine van het DNA. In een vereenvoudigde weergave, kunnen chromatine-Staten worden onderverdeeld in euchromatine als transcriptionally actief en heterochromatin als transcriptionally stille gebieden. De nucleosoom als de basiseenheid van de chromatine bevat twee kopieën van elke kern Histon H2A, H2B, H3 en H4, omringd door 147 basenparen van DNA13. Histonen zijn zeer post-translationally gewijzigd door methylering, acetylation, fosforylatie, ubiquitination, sumoylation, ADP-ribosylation, deamination en proline isomerisatie14,15. Histon lysine methylering wordt beschouwd als de meest stabiele Histon wijziging die transcriptie, replicatie, recombinatie16, DNA-schade antwoord17en genomische inprenting18 besturingselementen. Lysines worden mono-, di- en tri-gemethyleerd19 en verschijnen niet alleen op de staarten toegankelijk Histon, maar ook binnen het bolvormige domein van histones20. Specifieke methyleringen op H3K4 en H3K36 worden vooral geassocieerd met euchromatine, specifieke methyleringen op H3K9, H3K27 of H4K20 zijn voornamelijk te vinden in de hétérochromatique regio's, hoewel alle residuen bevinden zich binnen de Histon staart14, 19,21. Methylation van H3K79 is bevindt zich in het bolvormige domein van Histon en geassocieerd met een transcriptionele activiteit, maar ook met transcriptionally inerte genomic regio22. De wijziging is evolutionair bewaard aangezien geconstateerd in gist, kalf zwezerik, kip en menselijke23. H3K79 mono-, di- en trimethylation (H3K79me1, me2, me3) worden gekatalyseerd door de Histon methyltransferases DOT1L24,25 en de nucleaire SET Domain-bevattende eiwitten 2 (Nsd2)26. DOT1L is betrokken bij de proliferatie, DNA-reparatie en mobiele reprograming27. Verlies van Dot1l in muizen leidt tot een prenatale dood rond de ontwikkelingsstadium E10.528,29. Tijdens de ontwikkeling van het hart en bij myocardiocyte differentiatie is DOT1L essentieel voor gen expressie verordening30. In het centrale zenuwstelsel, DOT1L functie kan worden betrokken bij de neurale buis ontwikkeling31, het is betrokken bij het onderdrukken van Tbr1-expressie tijdens reukkolf ontwikkeling32, en kan functioneren in de regulering van ER-stress reactie genen33. De context-afhankelijke activeren of de actie van de bestrijding van H3K79me, vooral met in vivo situaties zoals de ontwikkeling van het centrale zenuwstelsel, is tot nu toe slechts gedeeltelijk begrepen32. Aangezien H3K79 methylering zich in het bolvormige gebied van Histon 3 bevindt, is het sterically minder toegankelijk in vergelijking met wijzigingen op de flexibele Histon staarten23. Om te begrijpen van de functie van H3K79 methylering, zijn betrouwbare en reproduceerbare analysemethoden ter bepaling van de locatie en de genomische omgeving nodig. In deze paper methoden presenteren wij isolatie methoden van verschillende neurale progenitoren (CPC voor de cortex) en CGNPs voor het cerebellum, effectieve DOT1L remmer behandeling en een ChIP-methode voor het analyseren van methylation van H3K79 via qPCR of rangschikken op ander moment punten tijdens corticale en Cerebellaire ontwikkeling. Voor een overzicht van het protocol en de mogelijkheden, Zie Figuur 1.

<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

Ontwikkeling van de hersenen is een complex proces, dat wordt gecontroleerd in een temporo-ruimtelijke wijze door gradiënten van morphogens en verschillende transcriptionele programma's. Epigenetische chromatine wijzigingen, zoals Histon methylering, hebben bovendien een belangrijke rol voor de totstandbrenging en het onderhoud van specifieke cel lot binnen dit proces. De overgrote meerderheid van Histon methylatie optreedt op de flexibele Histon staart, die toegankelijk is voor Histon modifiers, gummen en Histon lezer eiwitten. In tegenstelling, H3K79 methylation bevindt zich in het bolvormige gebied van Histon 3 en is betrokken bij verschillende ontwikkelings functies. H3K79 methylation is evolutionair bewaard en kan worden gevonden in een breed scala van soorten van Homo sapiens naar Saccharomyces cerevisiae. De wijziging treedt op in verschillende cel populaties binnen organismen, waaronder neurale progenitorcellen. De locatie van methylering van de H3K79 in het bolvormige domein van Histon 3 maakt het moeilijk in te schatten. Hier presenteren we methoden om te isoleren en cultuur corticale voorlopercellen cellen (CPC) van embryonale corticale hersenweefsel (E11.5-E14.5) of cerebellaire granulaire neuron progenitoren (CGNPs) van postnatale weefsel (P5-P7), en naar efficiënt immunoprecipitate H3K79me2 voor kwantitatieve PCR (qPCR) en sequentiebepaling van het genoom-brede.

Opzet van neurale voorlopercellen isolatie, teelt, H3K79me2 ChIP en ChIP analysemethoden: Figuur 1 toont een stroomdiagram voor het uitvoeren van H3K79me2 ChIP van corticale voorlopercellen op verschillende tijdstippen tijdens de embryonale hersenontwikkeling of cerebellaire granulaire neuron progenitoren in postnatale stadia. Als een eerste stap, de hersenen moet worden geïsoleerd en de telencephalon (tussen E11.5 en E14.5) of het cerebellu...

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

Isolatie en teelt van neurale progenitoren gevolgd door chromatine-Immunoprecipitation van Histone 3 Lysine 79 Dimethylation Mark

Presenteren we een effectieve en reproduceerbare methode om te isoleren en cultuur van neurale voorlopercellen van embryonale en postnatale hersenweefsel voor chromatine immunoprecipitation (ChIP) van Histon 3 lysine 79 dimethylation (H3K79me2) - een Histon mark gelegen binnen de bolvormige domein van Histon 3.

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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Analyzing Gene Expression and Function

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Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells

Productie van Chick Embryo Extract voor de teelt van Muizen neurale lijst Stem Cells

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

Fysiologische, morfologische en neurochemische Karakterisering van neuronen gemoduleerd door Movement

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

Chromatine immunoprecipitatie van achterwortelganglia Tissue volgende axonale schade

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

Detectie van Microregional Hypoxie in Mouse Cerebral Cortex door twee-foton Imaging van endogene NADH fluorescentie

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

Reproduceerbare Mouse heupzenuw Crush en de daaropvolgende beoordeling van Regeneratie door Whole Mount Muscle Analyse

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

Isolatie en Cultuur van Rat Embryonale neurale cellen: een Quick-protocol

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

Isolatie en Cultuur van neurale lijst cellen van embryonale 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 ...

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

Een geoptimaliseerde Procedure voor fluorescentie-geactiveerde celsortering (FACS) Isolatie van autonome neurale voorlopercellen uit viscerale organen van de foetus Muizen

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

Bevordering van Survival en differentiatie van neurale stamcellen met fibrine en groeifactor Cocktails na Ernstige Spinal Cord Injury

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

Een kwantitatieve cel-migratie test voor Muriene Enterisch neurale voorlopercellen

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