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

Vi takker Henriette Bertemes for at hjælpe med at etablere CGN dyrkning protokol inden for laboratoriet. Denne metode papiret blev støttet af DFG-finansierede CRC992 medicinsk Epigenetik af finansieringen til TV. Forfatterne anerkender støtten fra Freiburg Galaxy Team: Pavankumar Videm, Björn Grüning og Prof. Rolf Backofen, bioinformatik, universitetet i Freiburg, Tyskland finansieret af Collaborative Research Center 992 medicinsk Epigenetik (DFG grant SFB 992/1 2012) og tyske føderale ministerium for uddannelse og forskning (BMBF grant 031 A538A RBC (de. NBI)).

Dyrevelfærd udvalg af universitetet i Freiburg og lokale myndigheder godkendt alle dyreforsøg (G12/13, G16/11), nævnt i følgende protokol.
1. præparater
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Forberedelserne til CPCs isolation
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<li>Oprette timet parring for at opnå embryoner på forskellige stadier af cortex udvikling (mellem E11.5 og E14.5). Bruge mus af stammen NMRI (Naval Medical Research Institute), som er mindst 8 uger gamle. Efter parring, overveje en positiv vaginal stik på E0.5.</li>
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<ol>
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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. 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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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Der er to vigtige måder at udføre kromatin immunoprecipitation til påvisning af genomisk belægning af Histon ændringer, transkriptionsfaktorer, Histon kode læsere, forfattere eller viskelædere. Ene er metoden indfødte ChIP bruger nukleasen fordøjet, native kromatin for immunoprecipitation, og den anden er metoden præsenteres ved hjælp af PFA-fast, afrevne kromatin, hvor nucleosomes og andre DNA-attached proteiner er kovalent bundet til DNA 39. indfødte ChIP med sin høje antistof opdag...

De sensoriske, motoriske og kognitive funktioner i hjernen er meget komplekse og modtagelige for fysiske og miljømæssige ændringer. Hjernen består af tre almindelige dele den hind-, midt-, og forhjernen, som er dybt forbundet. Inden for forhjernen, kan telencephalon opdeles i en dorsal telencephalon (DT) og en ventral telencephalon (VT). DT af mus består af seks kortikale lag, som er dannet mellem E11.5 og E18.5 i en "inside-out" måde1. VT omfatter de ganglionære eminences i udvikling, som senere danner de basale ganglier2,3. Flere celletyper kan klassificeres i pattedyr centralnervesystemet som neuroner, astrocytter, og oligodendrocytes4, der udvikler sig i en temporo-rumlige måde5. Først, de neurale stamceller (NPC) give anledning til forskellige former for neuroner, interneurons i VT og projektion neuroner i DT, og senere videre til glial celler (f.eks., astrocytter6). Under kortikale udvikling, den mest overfladiske lag (lag jeg), som indeholder Cajal-Retzius celler, dannes først. Derefter, mellem E12.5 og E14.5, NPCs generere dybere neuronal lag (VI, V) mens mellem 14,5 og 16,5, stamfaderen giver anledning til øverste lag (IV-II) neuroner7,8. Neuronal identitet er angivet ved forskellige morphogen-induceret temporo-rumlige transcriptional programmer og desuden epigenetiske programmer2.
Lillehjernen, som er impliceret i motor koordinering, ligger i baghjernen og udvikler mellem E10 og groft P20 i mus9. Det indeholder den cerebellare cortex og de cerebellare kerner10. Voksen cerebellare cortex består af tre lag, den yderste molekylære lag, Purkinje cellelag og den inderste granulerende lag indeholdende granulerede neuroner10. De cerebellare granula celler er de mindste neuroner og udgør omkring 80% af alle neuroner i hvirveldyr hjernen11. De udvikle sig fra prækursorer placeret i den ydre germinal zone og vandrer gennem Purkinje cellelag til deres destination12. Ligesom i telencephalon, udvikling af lillehjernen er reguleret af flere vigtige morphogens, defineret som har specifikke tid - og rum-afhængige funktioner og indlede transcriptional programmer10.
Udviklingen af kortikale og cerebellare lag kontrolleres af transcriptional udtryk for specifikke morphogens og således af kromatin staten af DNA. I en forenklet visning, kan kromatin stater opdeles i euchromatin som transcriptionally aktive og heterochromatin som transcriptionally tavs regioner. Nucleosome som den grundlæggende enhed af kromatin indeholder to kopier af hver kerne Histon H2A, H2B, H3 og H4, omgivet af 147 basepar af DNA13. Histoner er stærkt post-translationally ændret ved methylering, acetylation, fosforylering, ubiquitination, sumoylation, ADP-ribosylation, deamination og prolin isomerisation14,15. Histon lysin methylering er anset for at være den mest stabile Histon ændring, der styrer transskription, replikering, rekombination16, DNA-skade svar17og genomisk prægning18. Lysines kan være mono-, di- eller tri-methylerede19 og vises ikke kun på de tilgængelige Histon haler, men også inden for domænet kugleformede histoner20. Specifikke methylations på H3K4 og H3K36 er især forbundet med euchromatin, specifikke methylations på H3K9, H3K27 eller H4K20 er hovedsageligt findes i heterochromatic regioner, selv om alle rester er placeret inden for Histon hale14, 19,21. H3K79 methylering er placeret inden for domænet Histon kugleformede og har været forbundet med transcriptional aktivitet, men også med transcriptionally inert genomisk regioner22. Ændringen er evolutionært bevaret, da det er blevet observeret i gær, kalv thymus, kylling og menneskelige23. H3K79 mono-, di- og trimethylation (H3K79me1, me2, me3) er katalyseret af Histon methyltransferases DOT1L24,25 og nukleare sæt domæne indeholdende Protein 2 (Nsd2)26. DOT1L er impliceret i spredning, DNA reparation og cellulære reprograming27. Tab af Dot1l i mus fører til en prænatal død omkring udviklingsstadiet E10.528,29. Under udvikling af hjertet og myocardiocyte differentiering er DOT1L afgørende for gen expression forordning30. I det centrale nervesystem, DOT1L funktion kan være impliceret i neuralrøret udvikling31, det er involveret i undertrykke Tbr1-udtryk under forhjernen udvikling32, og kan fungere i reguleringen af ER-stress svar gener33. Kontekst-afhængige aktivering eller undertrykke handling af H3K79me, især med i vivo situationer som udvikling af det centrale nervesystem, er til dato kun delvist forstået32. Da H3K79 methylering er placeret i domænet kugleformede af Histon 3, er det sterically mindre tilgængelige i forhold til ændringer på fleksible Histon haler23. For at forstå funktionen af H3K79 methylering, er pålidelig og reproducerbar analysemetoder til at bestemme dens placering og genomisk miljø nødvendige. I denne metoder papir præsenterer vi isolation metoder til forskellige neurale stamceller (CPC'er for cortex) og CGNPs til lillehjernen, effektiv DOT1L-hæmmer behandling, og en ChIP metode til at analysere H3K79 methylering via qPCR eller sekvensering på andet tidspunkt punkter i løbet af kortikale og cerebellare udvikling. En oversigt over protokollen og dens muligheder, se figur 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

Hjernens udvikling er en kompleks proces, der styres i et temporo-rumlige måde af forløb af morphogens og forskellige transcriptional programmer. Derudover har epigenetiske kromatin ændringer, ligesom Histon methylering, en vigtig rolle for etablering og vedligeholdelse af specifikke celle skæbner i denne proces. Langt størstedelen af Histon methylering opstår på fleksible Histon hale, som er tilgængelige for Histon modifikatorer, viskelædere og Histon læseren proteiner. Derimod H3K79 methylering er placeret i den kugleformede domæne af Histon 3 og er involveret i forskellige udviklingsmæssige funktioner. H3K79 methylering er evolutionært bevaret og kan findes i en bred vifte af arter fra Homo sapiens at Saccharomyces cerevisiae. Ændringen indtræder i forskellige cellepopulationer i organismer, herunder neurale stamceller. Placeringen af H3K79 methylering i domænet kugleformede af Histon 3 gør det vanskeligt at vurdere. Her præsenterer vi metoder til at isolere og kultur kortikale stamfader celler (CPCs) fra embryonale kortikale hjernevæv (E11.5-E14.5) eller cerebellare kornede neuron stamfaderen (CGNPs) fra postnatal væv (P5-P7), og effektivt immunoprecipitate H3K79me2 for kvantitativ PCR (qPCR) og genome-wide sekventering.

Generelle ordning af neurale stamceller isoleret, dyrkning, H3K79me2 ChIP og ChIP analysemetoder: Figur 1 viser et rutediagram til at udføre H3K79me2 ChIP af kortikale stamceller på forskellige tidspunkter i løbet af embryonale hjernens udvikling eller af cerebellare kornede neuron ophav i postnatal faser. Som et første skridt, hjernen skal isoleres og telencephalon (mellem E11.5 og E14.5) eller lillehjernen (P5-P7) har at blive genfundet...

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

Isolering og dyrkning af neurale stamceller efterfulgt af kromatin-Immunoprecipitation af Histon 3 lysin 79 Dimethylation Mark

Vi præsenterer en effektiv og reproducerbar metode til at isolere og kultur neurale stamceller fra embryonale og postnatal hjernevæv for kromatin immunoprecipitation (ChIP) af Histon 3 lysin 79 dimethylation (H3K79me2) - en Histon mark ligger inden for den kugleformede domæne af 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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Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest 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

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

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

Påvisning af Microregional Hypoxia i mus hjernebarken ved to-foton Imaging af endogent NADH Fluorescens

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

Reproducerbar Mouse iskiasnerven Crush og efterfølgende vurdering af Regeneration af Whole Mount Muskel 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

Isolering og dyrkning af Rotte Embryonale Neurale Celler: En hurtig protokol

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

Isolering og dyrkning af neurale kamceller fra embryonale Murin 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

En optimeret Fremgangsmåde til fluorescens-aktiveret cellesortering (FACS) Isolering af autonome Neurale stamceller fra indre organer af føtale Mus

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

Fremme af overlevelse og differentiering af neurale stamceller med fibrin og Growth Factor Cocktails efter Svær Rygmarvsskader

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

En kvantitativ Cell Migration Assay for Murine Enteriske Neural Progenitorer

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