Name: chromatin immunoprecipitation (chip) to assay dynamic histone modification in activated gene expression in human cells
Uploaded: 2010-07-29T17:00:00
Description: Watch this Scientific Journal Video about Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells at JoVE.com

Dit werk wordt ondersteund door de Universiteit van Virginia, de Universiteit van Virginia Cancer Center en The Thomas F. en Kate Miller Jeffress Memorial Trust. Wij danken de James E. Darnell Lab (Rockefeller University) de Robert Ross Lab (Fordham University) voor cellijnen.

PLANNING De dag voor een ChIP experiment is gepland, moeten cellen worden gekweekt, zodat een voldoende aantal beschikbaar is. Ga ervan uit dat elke chip test zal ~ 1-1,5 x 10 7 cellen vereisen. Altijd van plan om zowel de negatieve (IgG) en positieve (pan histone antilichaam) controles en dubbele experimenten te doen. Tip - Voor 2FTGH cellen, elke chip test duurt ongeveer een 15 cm weefselkweek schotel bij 80% confluentie. DEEL 1: ...

<ol>
	<li>Levy, D., Darnell, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signalling:+Stats:+transcriptional+control+and+biological+impact.">Signalling: Stats: transcriptional control and biological impact.</a> Nature Reviews Molecular Cell Biology. 3, 651-662 (2002).</li><li>Bromberg, J., Darnell, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+STATs+in+transcriptional+control+and+their+impact+on+cellular+function.">The role of STATs in transcriptional control and their impact on cellular function.</a> Oncogene. 19, 2468-2473 (2000).</li><li>Hu, X., Ivashkiv, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-regulation+of+signaling+pathways+by+interferon-gamma:+implications+for+immune+responses+and+autoimmune+diseases.">Cross-regulation of signaling pathways by interferon-gamma: implications for immune responses and autoimmune diseases.</a> Immunity. 31, 539-550 (2009).</li><li>Campos, E. I., Reinberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histones:+annotating+chromatin.">Histones: annotating chromatin.</a> Annu Rev Genet. 43, 559-599 (2009).</li><li>Kouzarides, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+Modifications+and+Their+Function.">Chromatin Modifications and Their Function.</a> Cell. 128, 693-705 (2007).</li><li>Jenuwein, T., Allis, 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=Translating+the+histone+code.">Translating the histone code.</a> Science. 293, 1074-1080 (2001).</li><li>Wittwer, C. T., Herrmann, M. G., Moss, A. A., Rasmussen, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+fluorescence+monitoring+of+rapid+cycle+DNA+amplification.">Continuous fluorescence monitoring of rapid cycle DNA amplification.</a> Biotechniques. 22 (130-131), 134-138 (1997).</li><li>Higuchi, R., Dollinger, G., Walsh, P. S., Griffith, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+amplification+and+detection+of+specific+DNA+sequences.">Simultaneous amplification and detection of specific DNA sequences.</a> Biotechnology (N Y). 10, 413-417 (1992).</li><li>Kroger, A., Koster, M., Schroeder, K., Hauser, H., Mueller, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activities+of+IRF-1.">Activities of IRF-1.</a> J Interferon Cytokine Res. 22, 5-14 (2002).</li><li>Ross, R. A., Spengler, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+neuroblastoma+stem+cells.">Human neuroblastoma stem cells.</a> Seminars in cancer biology. 17, 241-247 (2007).</li></ol>

De geschetste ChIP protocol is met succes gebruikt in het lab voor het testen van meer dan twintig verschillende histon-modificaties. Daarnaast hebben we gekarakteriseerd veranderingen in de bezetting van transcriptiefactoren, histon-modificerende enzymen, eiwitten die histon modificaties en het RNA polymerase II de transcriptie machinerie te herkennen, op een aantal IFN-γ geïnduceerde genen. We hebben ook gebruik gemaakt van de procedure om veranderingen in histon modificaties karakteriseren tijdens de differentiatie...

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<td>SH30087.04N</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
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<td>&nbsp;</td>
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<td>D0632</td>
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<td>&nbsp;</td>
<td>Fisher</td>
<td>BP118-500</td>
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<td>Pharmco</td>
<td>zp1110EP</td>
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<td>&nbsp;</td>
<td>Roche</td>
<td>100149</td>
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<td>Roche</td>
<td>901393</td>
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<td>HEPES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3784</td>
<td>&nbsp;</td>
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<td>IFN-gamma</td>
<td>&nbsp;</td>
<td>R&amp;D Systems</td>
<td>285-IF-100</td>
<td>&nbsp;</td>
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<td>IgG</td>
<td>&nbsp;</td>
<td>Jackson Immunoresearch</td>
<td> 109-005-003</td>
<td>&nbsp;</td>
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<td>KCl</td>
<td>&nbsp;</td>
<td>MP Biologicals</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>Fisher</td>
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<td>Deoxycholic Acid Sodium Salt</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP349-100</td>
<td>&nbsp;</td>
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<td>NaCl</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>7647-14-5</td>
<td>&nbsp;</td>
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<td>NP40</td>
<td>&nbsp;</td>
<td>US Biological</td>
<td>N3500</td>
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<td>Anti-Histone H3, CT, pan</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>07-690</td>
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<td>Phenol/chloroform/isoamyl</td>
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<td>Fisher</td>
<td>108-95-2</td>
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<td>Phosphate Buffered Saline</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>7647-14-5</td>
<td>&nbsp;</td>
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<td>PMSF</td>
<td>&nbsp;</td>
<td>EMD</td>
<td>50-230-0316</td>
<td>&nbsp;</td>
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<td>Proteinase K</td>
<td>&nbsp;</td>
<td>5-Prime</td>
<td>2500140</td>
<td>&nbsp;</td>
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<td>RNAse A</td>
<td>&nbsp;</td>
<td>5-Prime</td>
<td>2500130</td>
<td>&nbsp;</td>
<tr>
<td>Salmon Sperm DNA/Protein A Agarose</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>16-157</td>
<td>&nbsp;</td>
<tr>
<td>Salmon Sperm DNA/Protein G Agaorse</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>16-201</td>
<td>&nbsp;</td>
<tr>
<td>SDS</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP166-500</td>
<td>&nbsp;</td>
<tr>
<td>Tris-Cl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T5941</td>
<td>&nbsp;</td>
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<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Acros</td>
<td>327372500</td>
<td>&nbsp;</td>
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<td>Anti-K36me3</td>
<td>&nbsp;</td>
<td>Abcam</td>
<td>ab9050</td>
<td>&nbsp;</td>
<tr>
<td>Anti-STAT1</td>
<td>&nbsp;</td>
<td>Santa Cruz</td>
<td>sc-345X</td>
<td>&nbsp;</td>
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<td>Anti-RNA Polymerase II</td>
<td>&nbsp;</td>
<td>Santa Cruz</td>
<td>sc-899</td>
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chromatin immunoprecipitation (chip) to assay dynamic histone modification in activated gene expression in human cells

In antwoord op een verscheidenheid van extracellulaire liganden, het STAT (signaal transducer en activator van transcriptie) transcriptiefactoren snel gerekruteerd uit hun latente toestand in het cytoplasma naar receptoren op het celoppervlak, waar ze worden door fosforylatie geactiveerd op een tyrosine residu 1. Vervolgens dimeriseren en verplaatsen naar de kern om de transcriptie van doelwitgenen rijden, gevolgen hebben voor groei, differentiatie, homeostase en de immuunrespons. Niet verrassend, gezien hun brede betrokkenheid bij normale cel processen, ontregeling van de STAT functie draagt ​​bij aan menselijke ziekten, met name om vormen van kanker en auto-immuunziekten 2 3. Het is bekend dat transcriptie wordt gereguleerd door veranderingen in het chromatine template 4,5. Deze veranderingen omvatten de activiteiten van de ATP-afhankelijke complexen, evenals covalente histon-modificaties en DNA-methylatie 6. Vanwege STAT activatie van gen-expressie is zowel snel als van voorbijgaande aard, het vereist specifieke mechanismen voor het moduleren van de chromatine template op STAT-afhankelijke genloci. Te definiëren deze mechanismen, we karakteriseren de histon modificaties en de enzymatische activiteiten die ze genereren op genloci die reageren op STAT-signalering. Dit protocol beschrijft chromatine immunoprecipitatie, een methode die is waardevol voor de studie van STAT signalering naar chromatine in actief genexpressie.

Watch this Scientific Journal Video about Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells at JoVE.com

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

Dit protocol beschrijft hoe chromatine immunoprecipitatie (ChIP) wordt gebruikt om de dynamische wijzigingen studie naar de chromatine sjabloon die transcriptie veroorzaakt door een signaal transductie route te reguleren.

Chromatine Immunoprecipitatie (ChIP) om Assay Dynamic histon-eiwitten in Actieve genexpressie in menselijke cellen

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

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