Bu çalışma, Virginia Üniversitesi, Virginia Üniversitesi Kanser Merkezi ve Thomas F. ve Kate Miller Jeffress Memorial Trust tarafından desteklenmektedir. James E. Darnell Lab (Rockefeller Üniversitesi) Robert Ross Lab (Fordham Üniversitesi), hücre hatları için teşekkür ederiz.

PLANLAMA ChIP deney planlanan gün önce, hücrelerin yeterli sayıda kullanılabilir, böylece kültüre olmalıdır. Her ChIP saflık ~ 1-1.5 x 10 7 hücre gerektirdiğini varsayalım. Her zaman negatif (IgG) ve pozitif (pan histon antikor) denetimleri ve yinelenen deneyler yapmak için plan. İpucu - 2FTGH hücreler için, her ChIP tahlil% 80 confluency yaklaşık 15 cm doku kültürü çanak gerektirir. BÖLÜM 1: Kromatin HAZIRLANMALI VE UYGULAMAYA ChIP <ol><li> Kültür ortamı çıkarın ve 20 ml% 10 kozmik buzağı serumu (CCS) / 5 ng / ml IFN-gamma içeren DMEM gerekli kez hücrelerinin neden. CCS / DMEM yanı sıra% 10 20 ml uninduced plakaları medya değiştirin. </li><li> Davlumbaz, 15 cm çanak medya 20 ml (% 1 final konsantrasyon) 540 mcL% 37 formaldehit ekleyin. İpucu - Tilt plaka ve sonra ortam havuzu formaldehit - kaya levhası eşit bir şekilde yayılması için. </li><li> Crosslinking oda sıcaklığında 10 dakika boyunca devam etmek için izin ver. </li><li> Crosslinking durdurmak için 0.125 M final konsantrasyon glisin ekleyin. </li><li> Medya aspire Vakum ve 10 ml buz gibi fosfat ile hücreler bir kez yıkayın tamponlu salin (PBS). </li><li> ~ 7 mL PBS içinde hücre kazıyıcı ile hücreleri toplamak ve buz üzerinde 50 ml konik tüp içinde aynı tedavi edildi (6 plakalardan) hücreleri birleştirmek. </li><li> 4 1800 devirde santrifüj (Beckman Coutler Allegra 12-R Santrifüj) ° C ve 5 dakika PBS aspirat. İpucu - Snap donma hücre pelletini buraya durdurma ve -80 saklamak ° C. </li><li> 4 mL Şişme Buffer (25 mM HEPES pH 7.8, 1.5 mM MgCl 2, 10 mM KCl,% 0.1 NP40 1 mM DTT, Komple proteaz inhibitörleri ve kullanmadan önce buz üzerinde soğuk ekle) hücre pelletini tekrar ve 15 transfer ml konik tüp. İpucu - Bu şişme tampon hacmi 6 plaka havuzu (~ 6-9 x 10 7 hücre) için uygun. Daha fazla veya daha az tampon douncing verimliliği değişebilir. </li><li> 10 dakika boyunca buz üzerinde hücre süspansiyonu inkübe edin. </li><li> Önceden soğutulmuş 15 mL douncer (Wheaton) süspansiyon transferi ve havaneli A. ile 20 vuruş Dounce </li><li> Transfer buz üzerinde 15 ml konik tüp geri hücreleri dounced. </li><li> 2500 rpm&#39;de 5 dakika Santrifüj 4 ° C ve süpernatantı atmak. </li><li> Sonikasyon Tampon 3 mL nükleer pelet (6 plakaları ~ 200 mcL) (15 pelet hacimleri) (50 mM HEPES pH 7.9, 140 mM NaCl, 1 mM EDTA,% 1 Triton X-100,% 0.1 Sodyum deoksikolatın, yeniden süspanse % 0.1 SDS) kullanımdan hemen önce buz üzerinde komple proteaz inhibitörleri ve soğuk ekleyin. İpucu - Bu sonication tampon hacmi 6 plaka havuzu (~ 6-9 x 10 7 hücreleri) için uygun. Daha fazla veya daha az tampon sonication verimliliğini değişebilir. </li><li> Mikrotip ile donatılmış bir Misonix sonikatör 3000 ile 10 döngü (20 saniye (8) ayarı on/40 saniye açık / kapalı genlik) buz çekirdekleri süspansiyon sonikasyon. Bu genellikle boyutu 200-1000 bps ortalama kromatin parçaları sonuçlanır. İpucu - sonicators değişir Çünkü, sonication etkinliği deneysel olarak tespit edilmelidir. Bunu yapmak için, her sonication döngüsü sonication önce ve sonra 20 mcL hacimde. Bu örneklerin, 4 saat boyunca 65 ° C&#39;de ısıtın. DNA yükleme tamponu ekleyin ve DNA parçalarının görselleştirmek için bir standart% 1 agaroz / TBE jel çalıştırın.) </li><li> Transferi 4, 20 dakika boyunca bir SS34 santrifüj tüpü ve santrifüj malzeme sonicated ° genellikle küçük bir miktar hücre enkaz ne bir SS34 rotor 13000 rpm (Sorvall RC6 Plus, Santrifüj) pelet C. </li><li> Süpernatantı buz üzerinde dikkatlice 15 ml konik tüp transferi. </li><li> 360 mcL Somon Sperm DNA / Protein A veya G agaroz boncuk bulamaç ekleyerek ve en az 30 dakika süreyle 4 ° C&#39;de sallanan Öncesi temizleyin. İpucu - Bu adım, non-spesifik bağlayıcı olaylar ortaya çıkar tayininde gürültü azalır . </li><li> 4 ° C&#39;de 1000 rpm&#39;de 5 dakika pelet agaroz boncuk santrifüjleyin. </li><li> Buz üzerinde başka bir 15 ml konik tüp supernatant önceden temizlenmiş aktarın. </li><li> Önceden temizlenmiş kromatin A260 birimleri ölçün ve dört (4) A260 birimleri 1 A260 birim = 0,050 mg / ml (0.2 mg / ml varsayarak) Sonikasyon Tampon ile ses seviyesini ayarlamak 190 mcL DDW 10 mcL sonication tamponu ile boş spektrofotometre - İpucu A260 birimleri ölçmek için, 190 mcL DDW önceden temizlenmiş kromatin 10 mcL sulandırmak. </li><li> Kromatin 75 mcL (4 A260 birimleri) kaydedin Giriş Örnek ve mağaza gibi 20 ° C çapraz bağları tersine çevirmek için hazır olana kadar. İpucu - Bu adım, veri analizi için çok önemli. Giriş kısım almak için ihmal etmeyin. </li><li> I kısım 1.7 ml mikrofuge&#39;de tüpler için hazırlanan kromatince, 1 ml alikotları olarak. İpucu - burada durma, -80, kuru buz ve mağaza hazırlanan kromatin dondurmak ek bileşenini ° C Not: Eğer mümkünse depolanmış kromatin optimum sonuçlar daha az üreten düşürebilir, bu yüzden önlemek. </li><li> Kaynaklı ve uninduced örnekleri ChIP dereceli antikorlar çift ayarlar. Negatif ve pozitif kontrol olarak tetiklenen ve uninduced örnekleri setleri IgG (2 mg) ve pan Histon H3 antikor (15 mcL) ekleyin. İpucu - antikor miktarı deneysel olarak belirlenen veya önerilen miktarlar için tedarikçinin ürün sayfasına bakınız olmalıdır. Genellikle 1-2 mg ChIP dereceli antikor iyi çalışır. Uygun hacmi, serum olarak verilir antikorları için ampirik olarak tespit edilmelidir. </li><li> Rock tüm tüpler gecede 4 ° C </li></ol> BÖLÜM 2: immün TOPLA <ol><li> Tüm tüpler Somon Sperm DNA / Protein veya protein G agaroz boncuk bulamaç 60 mcL ekleyin. İpucu Kullanım Protein fare monoklonal antikorlar için tavşan poliklonal antikorlar ve Protein G. </li><li> 4 ° C&#39;de en az 60 dakika süreyle Rock. </li><li> 2000 rpm&#39;de 4 hafifçe microfuging Pelet immün (agaroz boncuk) ° C, 3 dakika. </li><li> Süpernatant aspire. </li><li> 1 ml, 10 dakika için aşağıdaki Yıkama Tamponlar her ile 4 ° C&#39;de sallanan immün yıkayın 2000 rpm&#39;de 3 dakika mikrofuge&#39;de 4 ° C, her yıkama ve aspirat süpernatant arasında. Buz üzerinde soğutulmuş örnekleri tutun. Kullanımdan hemen önce PMSF ekleyin. <ul><li> Düşük Tuz Yıkama -% 0.1 SDS,% 1 Triton X-100, 2 mM EDTA, 20 mM Tris-Cl, pH 8.0, 150 mM NaCl, 1 mM PMSF </li><li> Yüksek Tuz Yıkama -% 0.1 SDS,% 1 Triton X-100, 2 mM EDTA, 20 mM Tris-Cl, pH 8.0, 500 mM NaCl, 1 mM PMSF </li><li> LiCl Yıkama - 0.25 M LiCl,% 1 NP-40,% 1 Sodyum deoksikolatın, 1 mM EDTA, 10 mM Tris-Cl, pH 8.0, 1 mM PMSF </li><li> TE Yıkama - TE Tampon -10 mM Tris-Cl, pH 8.0, 1 mM EDTA, 1 mM PMSF İKİ yıkar </li></ul></li><li> Son TE Tampon yıkama çıkarın ve boncuklar, 500 mcL Elüsyon Buffer (50 mM Tris-Cl, pH 8.0, 10 mM EDTA,% 1 SDS) ekleyin. İpucu - Elüsyon Tampon taze hazırlanmış olmalıdır. </li><li> 65 az 30 dakika süreyle Elüsyon Tampon ve ısı agaroz boncuk karıştırmak için Vortex ° C İpucu - Bu 30 dakika boyunca her tüp, en azından her 10 dakikada bir girdap . </li><li> 2000 devirde 30 saniye mikrofuge&#39;de ve yeni bir mikrofuge&#39;de boru Eluent transfer. İpucu - yürütülmesi sırasında, giriş örnekleri Çözülme ve her biri için 500 mcL Elüsyon Tampon ekleyin. Ileriye dönük tüm adımları bu örnekler içerir. </li><li> Tüm örnekler ve vorteks 200 mM nihai konsantrasyonu 5 M NaCl ekleyin. </li><li> 65 ° C&#39;de gece (ya da en az 4 saat). </li></ol> BÖLÜM 3: ISLAH ChIP&#39;d VE GİRİŞ DNA <ol><li> Kapakları üzerinde yoğunlaşma toplamak için 30 saniye için tüm örnekler mikrofuge&#39;de. </li><li> 15 dakika oda sıcaklığında inkübe her tüp ve 1μL RNAse A (10 U / ml) ekleyin. </li><li> 10 mcL 0,5 M EDTA, 40 mcL 0.5 M Tris-Cl, pH 6.5, 2 mcL Proteinaz K (10 mg / ml) ekleyin ve 45 ° C&#39;de 60 dakika inkübe edin. İpucu Bu reaktiflerin bir ana karışımı hazırlayın ve her bir tüpe 52 mcL ekleyin. </li><li> Fenol / kloroform / izoamil alkol özü ve kloroform / izoamil alkol sonra numunelerin ayıklayın. </li><li> 2 mcL glikojen (25 mg / ml) ve 3M Na asetat, pH 5.2 ve vorteks 0.1 hacimleri ekleyin. </li><li> 2.5 hacim% 100 etanol ve vorteks ekleyin. </li><li> 20 ° C&#39;de bir gece inkübe edin. </li></ol> BÖLÜM 4: ISLAH ChIP&#39;d VE GİRİŞ DNA DEVAM <ol><li> 4 13000 rpm&#39;de 20 dakika en az mikrofuge&#39;de örnekleri ° C DNA / glikojen pelet çöktürmek için. </li><li> 1 ml% 70 Etanol ile DNA / glikojen pelet ile yıkayın. </li><li> Hava DNA Savant hız vakum santrifüj veya hızlı kuru pelet kurulayın. İpucu - DNA üzerinde kuru değil. </li><li> 200 mcL TE Tampon DNA / glikojen pelet tekrar </li><li> Real-time PCR ile DNA (1 mcL) ölçümü için hazır. 20 Mağaza kalan DNA ° C İpucu - 1 mcL fazla DNA kullanarak gerçek zamanlı PCR reaksiyonları inhibe edebilir, bu yüzden dikkatli büyütmek. </li></ol> Bölüm 5: TEMSİLCİSİ SONUÇLAR: ChIP ve Giriş DNA örnekleri ilgi gen lokusu için spesifik primerler kullanılarak Real-Time PCR 7,8 (Uygulamalı Biyosistem 7500 Hızlı Real-Time PCR Sistemi) ile sayısal. Hedef genom dizisi bilinen zenginleştirilmiş veya test olan histon modifikasyonları eksik o Astarlar, pozitif ve negatif kontrollerin yanı sıra kullanılabilir. Veri Giriş yüzdesi olarak sunulmaktadır. ChIP prosedürü tamamen olmalıdır rüç epeated biyolojik histon modifikasyonları bildirilen değişiklikler istatistiksel olarak anlamlı sağlamak için çoğaltır. DNA, astar tasarım, PCR verimlilik ve giriş ve normalizasyon işlemleri yüzde hesaplamak için uygun bir yol da dahil olmak üzere ölçmek için gerçek zamanlı PCR kullanarak birçok önemli sorunlar vardır. Histon değişiklik bir gen üzerinde doluluk profil, PCR verimliliği tüm primer çiftleri arasında tutarlı olmalıdır. Gerçek zamanlı PCR sistemi üreticisi, gerekli bilgi ve eğitim sağlayabilir. Şekil 1 kromatin temsilcisi sonuçlar IFN-γ tarafından tetiklenen IRF1 gen 9 STAT1 etkinleştirme sırasında immunoprecipitated gösterir. <img src="/files/ftp_upload/2053/2053fig1.jpg" alt="Şekil 1" /> Şekil 1. STAT1, RNA Polimeraz II ve H3K36me3 doluluk IRF1 gen STAT1 etkinleştirme sırasında dinamiktir. (A, B, C), α-H3K36me3, α-STAT1 ve kromatin α-Pol II 2FTGH hücreleri toplanan ChIP, IFN-γ (kırmızı kareler) 30 dakika, 1.5 saat (mavi daireler), 5 saat ( ile indüklenen yeşil üçgenler) veya uninduced (siyah elmas). IgG negatif kontrol (gri çarpılar) (D) Pan H3 pozitif kontrol ve bağlı (mavi daireler) lokus üzerinde histon doluluk ve uninduced koşulları (siyah elmas) gösterir. -5 Bp etrafında dalış transkripsiyon başlangıç ​​siteleri bulunan nucleosome tükenmesi nedeniyle.

<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>

Ana hatlarıyla ChIP protokol laboratuarda tahlil yirmiden fazla farklı histon modifikasyonları başarıyla kullanılmaktadır. Buna ek olarak, transkripsiyon faktörleri doluluk değişiklikler karakterize, histon değiştirerek birkaç IFN-γ kaynaklı genlerin, histon modifikasyonları ve RNA Polimeraz II transkripsiyon makine kabul enzimler, proteinler. Ayrıca, kanser kök hücre 10 farklılaşması sırasında histon modifikasyonları değişiklikleri karakterize etmek için prosedürü kullandık . <...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>37% Formaldehyde</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP531-500</td>
<td>&nbsp;</td>
<tr>
<td>Chloroform/Isoamyl alcohol</td>
<td>&nbsp;</td>
<td>Acros</td>
<td>AC32715-5000</td>
<td>&nbsp;</td>
<tr>
<td>Complete Mini Protease Inhibitors</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>1836153</td>
<td>&nbsp;</td>
<tr>
<td>Cosmic Calf Serum</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30087.04N</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30243</td>
<td>&nbsp;</td>
<tr>
<td>DTT</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D0632</td>
<td>&nbsp;</td>
<tr>
<td>EDTA</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP118-500</td>
<td>&nbsp;</td>
<tr>
<td>Ethanol</td>
<td>&nbsp;</td>
<td>Pharmco</td>
<td>zp1110EP</td>
<td>&nbsp;</td>
<tr>
<td>Glycine</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>100149</td>
<td>&nbsp;</td>
<tr>
<td>Glycogen</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>901393</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3784</td>
<td>&nbsp;</td>
<tr>
<td>IFN-gamma</td>
<td>&nbsp;</td>
<td>R&amp;D Systems</td>
<td>285-IF-100</td>
<td>&nbsp;</td>
<tr>
<td>IgG</td>
<td>&nbsp;</td>
<td>Jackson Immunoresearch</td>
<td> 109-005-003</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>&nbsp;</td>
<td>MP Biologicals</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>LiCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L4408</td>
<td>&nbsp;</td>
<tr>
<td>MgCl2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M8266</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Acetate</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP333-500</td>
<td>&nbsp;</td>
<tr>
<td>Deoxycholic Acid Sodium Salt</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP349-100</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>7647-14-5</td>
<td>&nbsp;</td>
<tr>
<td>NP40</td>
<td>&nbsp;</td>
<td>US Biological</td>
<td>N3500</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Histone H3, CT, pan</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>07-690</td>
<td>&nbsp;</td>
<tr>
<td>Phenol/chloroform/isoamyl</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>108-95-2</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate Buffered Saline</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>7647-14-5</td>
<td>&nbsp;</td>
<tr>
<td>PMSF</td>
<td>&nbsp;</td>
<td>EMD</td>
<td>50-230-0316</td>
<td>&nbsp;</td>
<tr>
<td>Proteinase K</td>
<td>&nbsp;</td>
<td>5-Prime</td>
<td>2500140</td>
<td>&nbsp;</td>
<tr>
<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>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Acros</td>
<td>327372500</td>
<td>&nbsp;</td>
<tr>
<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>
<tr>
<td>Anti-RNA Polymerase II</td>
<td>&nbsp;</td>
<td>Santa Cruz</td>
<td>sc-899</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

chromatin immunoprecipitation (chip) to assay dynamic histone modification in activated gene expression in human cells

STAT (sinyal dönüştürücü ve transkripsiyon aktivatörü) ekstrasellüler ligandların çeşitli yanıt olarak, transkripsiyon faktörleri hızla onlar tek bir tirozin kalıntısı 1 fosforilasyon ile aktive olan hücre yüzey reseptörleri sitoplazmada gizli devlet işe alınırlar. Daha sonra hedef genlerin transkripsiyon sürücü, büyüme, farklılaşma, homeostazı ve immün yanıtı etkileyen çekirdeği dimerize ve yerini değiştirmek. Beklendiği gibi, normal hücre süreçlerini yaygın tutulum verilen, İST fonksiyonu disregülasyonu özellikle 2 ve otoimmün hastalıklar 3 kanserleri, insan hastalığına katkıda bulunur. Bu transkripsiyon 4,5 kromatin şablonunda değişiklik tarafından düzenlenir kurulmuştur. Bu değişiklikler, ATP-bağımlı komplekslerinin yanı sıra kovalent histon modifikasyonları ve DNA metilasyon 6 faaliyetleri içerir . STAT aktivasyon gen ekspresyonu hem hızlı ve hem de geçici olduğundan, İST-bağımlı gen lokuslar modüle kromatin şablon için özel mekanizmalar gerektirir. Bu mekanizmalar tanımlamak için, histon modifikasyonları ve enzimatik faaliyetleri STAT sinyalizasyon cevap gen lokuslar onları oluşturmak karakterize eder. Bu protokol kromatin immunoprecipitation, İST aktif gen ekspresyonu kromatin sinyalizasyon çalışması için değerli bir yöntem açıklanmıştır.

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

Bu protokol, kromatin immunoprecipitation (ChIP) bir sinyal iletim yolu ile indüklenen transkripsiyonu düzenleyen kromatin şablon dinamik değişiklikleri incelemek için nasıl kullanıldığını açıklar.

Kromatin Immunoprecipitation İnsan Hücrelerinde Aktif Gen İfadesi Testi Dinamik Histon Modifikasyon (ChIP)

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

chromatin-immunoprecipitation-chip-to-assay-dynamic-histone

Research

JoVE Journal

Biology

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

28.5K Görüntüleme.  University of Virginia. Bu protokol, kromatin immunoprecipitation (ChIP) bir sinyal iletim yolu ile indüklenen transkripsiyonu düzenleyen kromatin şablon dinamik değişiklikleri incelemek için nasıl kullanıldığını açıklar.

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

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

İnsan Embriyonik Kök Hücre Kromatin Immunoprecipitation

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Biyoloji

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Hazırlık Elektroporasyon murin Kemik İliği itibaren İlköğretim Hematopoetik Hücre Kültürleri

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Yüksek Verimli Primer Hücre Transfect Gen Verici MXcell Elektroporasyon Sistemi

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

Bitkisel Dokular Gen İfade Araştırmalar Robotik ve Dinamik Görüntü Analizi

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

Gen İfadesi Çalışmaları basitleştirin Otomatik Hücre Sayıcı kullanma: Namalwa Hücreler IL-4 Bağımlı Gen İfadesi siRNA demonte

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits1.
This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants)2,3. Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells.
DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers4. The primer extension assay is carried out using the MassARRAY (Sequenom)5 platform according to the manufacturer's specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results.

Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.

Genetik Dernekleri Fonksiyonel Dayanak Test bir Allel özel Gen İfadesi Testi

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

Erken evre Fare Embriyolar kullanarak Doku özel Genler Kromatin Immunoprecipitation Testi

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Active T. in vitro Sulandırma castaneum Telomeraz

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at different developmental stages. Epigenetic regulation contributes to a broad spectrum of biological processes, including cellular differentiation during embryonic development and homeostasis in adulthood. A critical strategy in epigenetic studies is to examine how various histone modifications and chromatin factors regulate gene expression. To address this, Chromatin Immunoprecipitation (ChIP) is used widely to obtain a snapshot of the association of particular factors with DNA in the cells of interest.
ChIP technique commonly uses cultured cells as starting material, which can be obtained in abundance and homogeneity to generate reproducible data. However, there are several caveats: First, the environment to grow cells in Petri dish is different from that in vivo, thus may not reflect the endogenous chromatin state of cells in a living organism. Second, not all types of cells can be cultured ex vivo. There are only a limited number of cell lines, from which people can obtain enough material for ChIP assay.
Here we describe a method to do ChIP experiment using Drosophila tissues. The starting material is dissected tissue from a living animal, thus can accurately reflect the endogenous chromatin state. The adaptability of this method with many different types of tissue will allow researchers to address a lot more biologically relevant questions regarding epigenetic regulation in vivo1, 2. Combining this method with high-throughput sequencing (ChIP-seq) will further allow researchers to obtain an epigenomic landscape.

Chromatin Immunoprecipitation ChIP using Drosophila tissue

Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system.

Kullanarak Kromatin immünopresipitasyon (ChIP) Drosophila Doku

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

Yüksek verim Sıralama için Yüksek Kalite Kromatin Immunoprecipitation DNA Şablon üretimi (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...