Yazarlar sıralama sonuçları sağlayarak yardım ettikleri için Dr Keji Zhao&#39;nun laboratuarı (NIH / NHLBI) teşekkür etmek istiyorum. Biz de eşlenen sıralama okur görselleştirmek için Genom Tarayıcı kullanımı için UCSC genom projesi teşekkür etmek istiyorum. Bu çalışma start-up XC fon NICHD Lucile Parkard Vakfı ve Johns Hopkins Üniversitesi&#39;nden Bağımsızlık Ödülü ve R01HD065816 için R00HD055052 NIH Pathway tarafından desteklenmektedir

(Tüm ChIP prosedürü yaklaşık iki gün sürer. Yüksek verimlilik sıralaması için ChIP kütüphaneler hazırlanması 2-3 gün sürer.) 1. Inceleyin ve ChIP Experiment (~ 1 milyon hücre) için doku hazırlanması <ol><li> Soğuk PBS + 1x proteaz inhibitörleri (7x stok solüsyonu elde etmek için 1.5 ml 1x PBS içinde proteaz inhibitörü kokteyl 1 pelet erimesi) de (Drosophila testislerin örneğin 200 çift) ilgi doku teşrih + PMSF (son konsantrasyonu 100 mikrogram / ​​ml). Iki doku durulama ve inhibitörleri ile aynı PBS çözeltisinden 200 uL doku içinde yeniden süspanse. </li><li> Örnek düzeltmek için, 5.5 uL% 37 formaldehit (37 kullanmadan önce 1 dakika ° C su banyosunda sıcak formaldehit) ekleyin. 15 dakika, vorteks arasındaki her 5 dakika boyunca 37 ° C&#39;de inkübe edilir. </li><li> 2 dakika yerleşmek için doku için buz üzerinde örnek yerleştirin. 450 uL PBS (proteaz inhibitörleri ve PMSF ile) 2x durulayın. Örnek şimdi deposu olabilir-20 ° C&#39;de d ChIP için yeterli malzeme elde etmek diseksiyon birkaç kez tekrarlayın. </li></ol> 2. ChIP Assay için protein-DNA Konjugasyon ile Süpernatant hazırlayın <ol><li> Bir 1.75 ml Eppendorf tüp adım 1.3 yeterince örnek birleştirin. </li><li> Doku örneği PBS çıkarın. 200 lizis tamponu uL (50 mM Tris-HCl, pH 7.6, 1 mM CaCl2,% 0.2 Triton X-100 veya NP-40, 5 mM butirat, ve 1x proteinaz inhibitörü kokteyl). Ekleme Lizis tamponu (0.5 mM PMSF final konsantrasyon) taze PMSF stok çözeltisi ekleyin. Dokular 10 dakika RT (Oda sıcaklığı) &#39;de inkübe herhangi bir toplama olmadan, tamamen ayırmak kadar mavi homojenizör (Fisher Kedi # 749521-1590) ile homojenize edilir. </li><li> Sonikasyon: güç 20 kullanım mikrotip sonikatör (Misonix HS-XL200). 50 sn için buz üzerinde 10 sn ve dinlenme sonikasyon. (Uzun sonication küçük parçalara verecektir optimal sonication zaman, ampirik olarak belirlenmelidir. Histon karşı antikorlar kullanılarak ChIP için 4-5 kez tekrarlayınE değişiklikler, normalde yaklaşık 200-300 bp kromatin ses dalgalarına maruz; transkripsiyon faktörü veya kromatin regülatörü (örneğin Polycomb proteinler) için, biz normalde 200-1000 bp kromatin ses dalgalarına maruz. Bununla birlikte, uygun parçası boyutu ampirik olarak test edilmelidir. Not: HER ZAMAN DAHA YUKARI ISITMA DAN ÖRNEK ÖNLEMEK İÇİN SONICATING SIRASINDA BUZ ÜZERİNDE TÜP TUTUN! </li><li> 1,8 mL RIPA tamponu (ekleyerek Örnek 10 seyreltilir tarif edildiği gibi mM Tris-HCl, pH 7,6, aynı konsantrasyonda proteaz inhibitörleri ve PMSF ile 1 mM EDTA,% 0.1 SDS,% 0.1 Na-deoksikolat,% 1 Triton X-100, Daha önce). 65 2 uL 5M NaCl ve inkübe ekleyerek giriş kontrol olarak 40 uL kaydet ° C kroslink tersine çevirmek için (O / N) gece. </li><li> Boncuk antikor eşlenik için, 1.5 ml Eppendorf tüpüne 40 uL Protein A boncuk ekleyin, daha sonra, 2 dakika süreyle 4 ° C&#39;de 600 uL PBS ve kaya eklemek mıknatıs boncuk uygulamak ve supernatant çıkarın. 100 PBS uL ve ilgi antikoru ekleme (antikor miktarını empir tespit edilmelidirically). 4 saat için 1 saat ya da 4 ° C için oda sıcaklığında inkübe edilir. </li><li> Mıknatıs tarafından boncuklar süpernatantı ve adım 2.4 boncuk 1mL kromatin ekstresi ekleyin. 4 at ° çevirin CO / N </li><li> Mıknatısa ChIP örnek uygulayın ve supernatant çıkarın. 10 dakika her biri için şu da 4 ° C tampon ile yıkanır boncuklar: RIPA tamponu 1 mL 2x [1,89 mL &#39;RIPA tamponu&#39; + ​​315 uL 7x proteaz inhibitörleri + 20 uL PMSF]; RIPA tamponu 1 mL 2x + 0.3 M NaCl [1,89 mL RIPA tamponu + 220 uL 3 M NaCl]; LiCI tamponu 1 mL (0.25 M LiCI,% 0.5 NP40,% 0.5 NaDOC) ile 2x; 1x 1x TE 1 mL +% 0.2 Triton X-100; 1x TE 1 mL ile 1x. </li><li> Çapraz bağ tersine çevirmek, 100 uL TE tampon içinde yeniden süspanse boncuklar + 3 uL% 10 SDS + proteinaz K (20 mg / mL) içindeki 5 ul. 65 inkübe ° CO / N </li><li> Mıknatıs boncuk uygulayın ve yeni bir tüpe süpernatant aktarın. Süpernatant SİZİN DNA ÖRNEĞİ CONTAINS. 100 uL TE + 0.5 M ile boncuk yıkayınNaCl ve iki süpernatan birleştirmektedir. </li><li> Fenol-kloroform DNA örneği ayıklayın. Kloroform: 200 ul Fenol ekle IAA (25:24:1) karışımı ve girdap. RT 5 dakika 14k döndürme. Alternatif olarak, DNA Qiagen PCR saflaştırma kiti kullanılarak elde edilebilir. </li><li> Yeni bir tüp içine süpernatan / sulu katman transferi ve 20 ug / mL (Alternatif olarak 20 mg / süpernatan her 1 mL için mL &#39;de glikojen 1 ul&#39;lik bir son konsantrasyon elde etmek lineer akrilamid eklemek de kullanılabilir. Glikojen için kullanılır Doğrusal akrilamid deneyleri sekanslama için kullanılır ise sonraki qPCR veya PCR analizi.) 3M NaOAc 20 uL ve 500 uL% 100 EtOH ekleyin. İyice karıştırın ve 80 inkübe ° C&#39;de 10 dakika. 4 azından 20 dakika için maksimum hızda dönerler ° C. </li><li> Süpernatantı ve 300 uL% 70 EtOH ile yıkayın. 50 uL TE tamponunda hava kuru ve Pastör pipetiyle örnek. Örnek şimdi -20 ° C&#39;de saklanabilir Örnek kantitatif PCR (qPCR) testi (Şekil 1) için kullanılabilir. </li></ol> 3a. QPCR kullanarak ChIP-ed DNA analiz <ol><li> Seyreltilmemiş, 1/10, 1/100, 1/1000, 1/5000: Bir standart eğri yapmak için aşağıdaki konsantrasyonda genomik DNA sulandırınız. </li><li> Her primer çifti için, adım 3a.1, giriş kontrol ve ChIP-ed DNA genomik DNA ile 96-plaka olarak iki nüsha halinde qPCR kurmak: 10 uL 2x SYBR PCR karışımı (Fermentas, K0222); 1 ileri uL 10 uM astar; 1 uL 10 uM ters astar; x uL ChIP-ed DNA (giriş kontrolü, ya da genomik DNA); y uL nükleaz içermeyen H 2 O 20 uL için tepki seviyesini ayarlamak için. </li><li> Mini plaka spinner (Labnet) ile 96-plaka aşağı Spin. </li><li> Aşağıdaki koşul kullanarak ABI 7300 gerçek zamanlı PCR sistemi (veya diğer gerçek zamanlı PCR sistemi) ile qPCR gerçekleştirin: Aşama 1: 50 ° 2 dakika, 1 döngüsü için C; Aşama 2: 95 10 dakika, 1 döngüsü için ° C; Aşama 3: 95 ° 15 s, 60 ° C&#39;de 1 dakika süreyle C, 40 ° Cycles; 4. Aşama (ayrılma aşaması): 95 ° 15 s, 60 C ° 1 dakika, 95 C ° 15 s C. </li><li> Disosiasyon eğrisi kontrol edin: termal ayrışma arsa az bir tepe PCR reaksiyonu tek bir amplikon göstermektedir. Birden fazla pik primer çifti ile spesifik olmayan ürün gösterir, qPCR veri kullanılmamalıdır. </li><li> 3a seyreltilmiş genomik DNA dizisi kullanılarak standart eğri dayanmaktadır Ct (döngüsü eşik) değerine göre DNA miktarı gideren bir formül hesaplanarak, her bir primer seti için PCR reaksiyon üstel amplifikasyon doğrusal faz belirler. 1. </li><li> ChIP-ed DNA ve giriş kontrol Ct değeri Ct değeri lineer aralığında ise, ChIP-ed DNA ve 2.4 giriş kontrol sulandırma faktörüne göre, ChIP-ed DNA karşı giriş zenginleştirme hesaplayabilirsiniz. </li></ol> 3b. Yüksek verimlilik sıralaması için ChIP-ed DNA&#39;yı çoğaltmak. <ol><li> ChIP-ed DNA karışımı sonu-onarım (kullanınBuz üzerinde kurmak Epicentre DNA SON tamir kiti): Adım 2.12 (0,1-5 ug) 1-34 ul genomik DNA 5 ul 10x Sona onarım tampon 5 ul 2.5 mM dNTP karışımı 5 ul 10 mM ATP x ul H 2 O 49 ul için tepki seviyesini ayarlamak için 1 ul End-Onarım enzim karışımı (T4 DNA Polimeraz + T4 Polinükleotid Kinaz) Oda sıcaklığında 45 dakika inkübe edin. Qiagen MinElute Reaksiyon kullanarak reaksiyon arındırın Temizleme kiti. 30 ul Elüsyon Buffer (EB) in Zehir. </li><li> 3 &#39;ucuna A-çıkıntılar ekleme: Adım 3b.1 gelen Elüsyonlanan DNA ürün 30 ul 5 ul 10x NEB Tampon # 2 10 ul 1 mM dATP 2 ul dH 2 O 3 ul 5 U / ml Klenow Fragment (3 &#39;→ 5&#39; exo-) 37 de 30 dakika inkübe ° C. MinElute ReactionCleanup kiti kullanılarak reaksiyon arındırın. 10 ul EB Zehir. </li><li> Solexa bağlayıcı ligasyonu: Adım 3b.2 gelen Elüsyonlanan DNA 10 ul 10 ul GKD 2 O 2.5 &amp;mu; l 10x T4 DNA ligaz tamponu Illumina 1 ul Adaptör oligo karışımı (stoktan 1/10-diluted) 2,5 ul T4 DNA ligaz (400 ünite / ul) RT az 1 saat inkübe edin. MinElute ReactionCleanup kiti kullanılarak reaksiyon arındırın. 20 ul EB Zehir. Örnek şimdi -20 ° C&#39;de saklanabilir </li><li> E-jel aparat kullanarak adım 3b.3 gelen Yıkanan DNA örneği çalıştırın. Jel 300-500 bp bant yalıtmak ve Qiagen Jel Ekstraksiyon kiti (bu adım adaptörü oligo ligasyonu ile ~ 125 bp kendine bağlanan linkerler ortadan kaldırır) kullanarak arındırmak. 12 ul EB Zehir. </li><li> Illumina kütüphane eşleştirilmiş uç kullanarak (PE) primerleri Amplify: Adım 3b.4 gelen Elüsyonlanan DNA 10.5 ul Master miks 12.5 ul (2X Phusion HF, Finnzymes) PE1 PCR primer 1 ile 1 ul PE2 PCR primer 2 arasında 1 ul PCR koşulları: 98 ° C&#39;de 30 sn (98 ° C&#39;de 10 saniye, 65 ° C&#39;de 30 saniye, 72 ° C&#39;de 30 sn), tekrar 20 döngü 5 dakika süreyle 72 ° C. </li><li> Adım 3b.5 Örnekler 2 standardı% agaroz jel kullanılarak uygun büyüklükte DNA (300-500 bp) seçildikten sonra ChIP-SEQ için de kullanılabilir. Örnek şimdi -20 ° C&#39;de saklanabilir Bu kirliliği önlemek için tek bir jel her ChIP örnek izole etmek en iyisidir. Örnekler yüksek verimlilik sıralaması için sunulabilir. </li></ol> 3c. Solexa boru hattı analizi <ol><li> 25-bp sıralama okur Illumina Genome Analyzer (GA) boru hattından elde edilmiştir. </li><li> Tüm okuma referans dizi ile iki uyumsuzlukları kadar izin Eland (Nükleotid veri Verimli Yerel Hizalama) yazılımını kullanarak Drosophila genomu (dm3) hizalanmış edildi. </li><li> Eşsiz eşlenen okuma aşağı analiz için muhafaza edildi. Birden fazla özdeş okuma için, üç adede kadar kopya PCR gelen önyargıları olasılığını azaltmak için muhafaza edildi. </li><li> GA Boru Hattı&#39;nın çıkış tarayıcı genişletilebilir veri (BED) dosyalarını dönüştürüldü. </li><li> Üretmek içingörüntülenmesi için UCSC tarayıcısına yüklemek için wig dosyaları, bu pencere boyutu olarak 4 bp ve DNA fragmanı boyut olarak 160 bp&#39;lik bir önceki yayın 3 tarif edilmiştir python kese kullanılır. </li><li> Sessiz ve dile getirdikleri genlerine Drosophila genlerin sınıflandırmak için, RPKM adında bir dijital sayı (kilobaz başına / exonic bölge birleşti / milyonda eşlenmiş okur okur) kullanılır. RPKM = 0 ile Genler RPKM ≥ 1, düşük orta ve yüksek düzeyleri ile genler olmak üzere üç gruba ayrılabilir ifade gruplar ayrıldı ile sessiz grubu ve genler olarak sınıflandırılmış ve her bir grup yaklaşık aynı sayıda edildi genler. </li><li> Histon modifikasyonu düzeyi ve gen ekspresyonu karşılaştırmak için, bir önceki yayının 3 tarif edilmiş bir python komut dosyası kullanılır. </li></ol> 4. Temsilcisi Sonuçlar Bam kullanarak ChIP-qPCR sonuçları örnekleri (mermer torba) mutant<html> Şekil 4 testisler 1 &#39;de gösterilmiştir. Bam testislerde proliferatif spermatogonia gelen spermatositler 5, 6 ayırt geçişte bir başarısızlık var. Bu doku tipi bakımından zengindir ve indiferansiye germ hücreleri için bir kaynak olarak bam testisler kullanın. Böyle erkek özgü transkripsiyon faktörü 87 (mst87F), don juan (dj), ve bulanık soğan (FZO) sperm farklılaşması için gerekli Farklılaştırma genler, bam testis ifade değildir. Bu genler çok baskıcı H3K27me3 histon modifikasyonu 7 (Şekil 1A) ile zenginleştirilmiş, ancak aktif H3K4me3 histon modifikasyonu 8 (Şekil 1B), biz &#39;monovalan&#39; 7 adlı bir kromatin imza yoksun edilir. H3K27me3 ya da H3K4me3 zenginleştirilmesi, bir yapısal olarak ifade siklin A (CycA) gen 4 ila normalizasyon tarafından belirlenir. bam mutant testisler (Şekil 2) Şekil 1&#39;de gösterildiği qPCR sonuçlar doğrulanmıştır. üç test terminal farklılaşma genler için mst87F, dj ve FZO ile antikor aynı set (yani baskıcı H3K27me3 ve aktif H3K4me3) kullanarak içerik &quot;&gt; ChIP-seq analizi , bunların genomik bölgeleri yüksek H3K27me3 ancak H3K4me3 (Şekil 2A-2C) zenginleştirilmiş. Bunun aksine, yapısal olarak ifade CycA geni önemli H3K4me3 ancak transkripsiyon başlama sitesi (TAD) (Şekil 2B) yakınında az H3K27me3 bağlayıcı sahiptir. Ayrıca, gen hariç dört sınıfın trafik ayırım şeritleri yakın H3K27me3 ve H3K4me3 ve ChIP profilleri, her histon modifikasyonu işlevi ile uyumludur. Şekil 3A&#39;da gösterildiği gibi, trafik ayırım şeritleri arasında H3K27me3 mansap zenginleşmesi ile ters gen ifadesi seviyesi ile ilişkilidir. Sessiz genler ise en yüksek H3K27me3 varyüksek genlerin hiçbir H3K27me3 bağlayıcı var. Bu veriler, gen ekspresyonu H3K27me3 baskıcı rolü ile tutarlıdır. Bunun aksine, trafik ayırım şeritleri etrafında H3K4me3 zenginleştirilmesi gen ifadesi üzerindeki H3K4me3 aktif rolü ile tutarlıdır gen ifadesi seviyesi (Şekil 3B) ile ters korelasyon gösterdi. <img alt="Şekil 1" src="/files/ftp_upload/3745/3745fig1.jpg" /> Ya da baskıcı H3K27me3 histon modifikasyon (A) veya (B) bam hücre-zenginleştirilmiş andiferansiye mutant testisler aktif H3K4me3 histon modifikasyonu karşı antikor kullanılarak ChIP-ed DNA Şekil 1. QPCR analizi. (A) bam ise testisler, farklılaşma genler (Mst87F, dj, FZO) H3K27me3 baskıcı histon modifikasyonu ile zenginleştirilmiştir. (B) Farklılaştırma genler H3K4me3 aktif işareti ile tükenmiş. Hedef gen (Mst87F, dj ya FZO) at ChIP DNA düzeyi (ChIP DNA / girdi) ilk lev normalize edildi<html> Kontrol CycA geninde ChIP DNA el. Hata çubukları üç bağımsız biyolojik, standart sapma çoğaltır göstermektedir. <img alt="Şekil 2" src="/files/ftp_upload/3745/3745fig2.jpg" /> Şekil 2. UCSC genom tarayıcı H3K27me3 anlık ve (A) Mst87F (B) DJ ve (C) FZO, (D) CycA genler. (D) &#39;in H3K27me3 zenginleştirilmiş bölgenin bütün çember genomik bölgeler arasında H3K4me3 zenginleşme kromatin durumunu gösterir bam da asılıyor ifade edilir örtüşen bir gen CG7264, testis (RPKM = 1) ama son derece vahşi tip testis ifade (RPKM = 130) 8 (7 ChIP-seq veri). Şekil 1&#39;de ChIP sonuç kantitatif PCR analizinde kullanılan sondalar her bir parsel altında etiketlenmiştir. <a href="http://www.jove.com/files/ftp_upload/3745/3745fig2large.jpg" target="_blank">büyük rakam görmek için buraya tıklayın</a> . tp_upload/3745/3745fig3.jpg &quot;/&gt; Şekil 3. ChIP-seq profilleri testislerde 7 bam yılında H3K27me3 ve H3K4me3 kullanarak. Dört gen grupları (7,509 genler) RNA-seq sonuçları 8 dayanarak gen ekspresyon düzeylerine göre sınıflandırıldı. Genlerin temsilcisi sınıflar transkripsiyon başlangıç ​​siteleri (trafik ayırım şeritleri) ile 3kb memba için 3kb aşağı gelen dizileri kullanarak, belirli bir histon modifikasyonu zenginleştirilmesi için çizilmiştir. Bu (A) H3K27me3 (K27) ve her bir grupta (B) H3K4me3 (K4) ChIP-seq analizleri. Zenginleştirilmesi bir profil üreten <a href="http://www.jove.com/files/ftp_upload/3745/3745fig3large.jpg" target="_blank">büyük rakamı görmek için buraya tıklayın</a> .

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Biz ifşa hiçbir şey yok.

Bu protokol tartışılan ChIP analizlerin çok yönlülüğü, biyolojik ilgili sistemde kromatin devletin bir çalışma olanağı sağlar, farklı dokular üzerindeki kullanılabilir. Kültüre sistemlerinden hücreleri kullanılarak ChIP deneyler hücrelerinin büyük miktarda kolayca elde edilebilir çünkü gerçekleştirmek için uygundur. Bununla birlikte, kültürlenmiş hücrelerde mutlaka bir çoklu-hücresel ortamda hücreleri göstermemektedir. Canlı hayvan disseke doku kullanarak bu tekniği geliştirerek...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> Komple Mini proteaz inhibitörü kokteyl </td><td> Roche </td><td> 11836153001 </td><td></td></tr><tr><td> Formaldehit (% 37) </td><td> Supelco </td><td> 47083-U </td><td></td></tr><tr><td> PMSF </td><td> Sigma </td><td> 78830 </td><td></td></tr><tr><td> Kontes pelet havaneli </td><td> Fischer Bilimsel </td><td> K749521-1590 </td><td></td></tr><tr><td> PCR izolasyon kiti </td><td> Qiagen </td><td> 28104 </td><td></td></tr><tr><td> Doğrusal poliakrilamid </td><td> Sigma </td><td> 56.575-1mL </td><td></td></tr><tr><td> Glikojen </td><td> Qiagen </td><td> 158930 </td><td></td></tr><tr><td> SYBR yeşil / ROX qPCR Master Mix </td><td> Fermentas </td><td> K0223 </td><td></td></tr><tr><td> Mini plaka spinner </td><td> Labnet </td><td> Z723533 </td><td></td></tr><tr><td> Real Time PCR sistemi </td><td> Applied Biosystems </td><td> 4351101 </td><td></td></tr><tr><td> Küçük Hacim Ultrasonik İşlemci </td><td> Misonix </td><td> HS-XL2000 </td><td> Model kesildi </td></tr><tr><td> Dynabeads, Protein A </td><td> Invitrogen </td><td> 100-01D </td><td></td></tr><tr><td> Mıknatıs Dynamag </td><td> Invitrogen </td><td> 123-21D </td><td></td></tr><tr><td> Fenol: kloroform: IAA </td><td> Invitrogen </td><td> 15593-049 </td><td></td></tr><tr><td> Epicentre DNA SON Tamir Takımı </td><td> Epicentre Biyoteknoloji </td><td> ER0720 </td><td></td></tr><tr><td> MinElute Reaction Temizleme Kiti </td><td> Qiagen </td><td> 28204 </td><td></td></tr><tr><td> Klenow parçacığı (3 &#39;→ 5&#39; ekso-) </td><td> New England Biolabs </td><td> M0212S </td><td></td></tr><tr><td> T4 DNA ligaz </td><td> Promega Şirketi </td><td> M1794 </td><td></td></tr><tr><td> Adaptör oligonükleotidler </td><td> Illumina </td><td> PE-400-1001 </td><td></td></tr><tr><td> Eşleştirilmiş-End Primer 1.0 ve 2.0 </td><td> Illumina </td><td> 1001783 1001 784 </td><td></td></tr><tr><td> E-Jel Electorphoresis sistemi </td><td> Invitrogen </td><td> G6512ST </td><td></td></tr><tr><td> 2X Phusion HF Mastermix </td><td> Finnzymes </td><td> F-531 </td><td></td></tr></tbody></table>

chromatin immunoprecipitation (chip) using drosophila tissue

Epigenetik hızla gelişen bir alan kalır kromatin devletin farklı gelişim aşamalarında farklı hücre tiplerinde farklı gen ifadesini nasıl katkıda çalışmaları. Epigenetik düzenleme yetişkinlikte embriyonik gelişimi ve homeostazı sırasında hücresel farklılaşma da dahil olmak üzere biyolojik süreçleri, geniş bir yelpazede katkıda bulunur. Epigenetik çalışmalar eleştirel bir strateji çeşitli histon modifikasyonları ve kromatin faktörleri gen ifadesinin düzenlenmesine nasıl incelemektir. Bu adres için, Kromatin immünopresipitasyon (ChIP) ilgi hücrelerde DNA ile belirli faktörlerin dernek bir enstantane elde etmek için yaygın olarak kullanılmaktadır. ChIP tekniği yaygın olarak tekrarlanabilir verileri oluşturmak için bolluk ve homojenlik elde edilebilir malzeme, başlangıç ​​olarak kültür hücreleri kullanır. Bununla birlikte, birçok uyarılar vardır: İlk olarak, Petri tabağına hücrelerinin büyümesini ortamı, böylece, in vivo olarak farklı olan olabiliryaşayan bir organizma hücrelerin endojen kromatin devlet yansıtmamaktadır. İkinci olarak, hücreler değil, her türlü kültürü ex vivo olabilir. Insan ChIP tayini için yeterince malzeme edinebilecekleri hücre dizileri sadece sınırlı sayıda vardır. Burada Drosophila dokuları kullanarak ChIP deney yapmak için bir yöntem tarif. Başlangıç ​​malzemesi böylece doğru endojen kromatin devlet yansıtabilir, yaşayan bir hayvan dokusu diseke edilir. Dokusu çok farklı tipleri ile bu yöntemin uyum araştırmacılar biyolojik olarak daha bir çok in vivo 1, 2, epigenetik düzenleme konusunda ilgili soruları sağlayacaktır. Yüksek verimlilik sıralama (ChIP-seq) ile bu yöntemi birleştirerek daha araştırmacı bir epigenomic manzara elde sağlayacaktır.

Watch this Scientific Journal Video about Chromatin Immunoprecipitation ChIP using Drosophila tissue at JoVE.com

Chromatin Immunoprecipitation ChIP using Drosophila tissue

Son zamanlarda yüksek verimlilik sıralama teknolojisi büyük ölçüde Kromatin immünopresipitasyon (ChIP) deney duyarlılığı artmış ve saflaştırılmış hücreleri veya disseke doku kullanarak uygulama açtı. İşte biz ChIP tekniği kullanılacak bir yöntem tasvir<em&gt; Drosophila</emIyi karakterize biyolojik sisteminde endojen kromatin devleti ele alabilir&gt; doku.

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 ...

chromatin-immunoprecipitation-chip-using-drosophila-tissue

Research

JoVE Journal

Biology

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

25.0K Görüntüleme.  Johns Hopkins University. Son zamanlarda yüksek verimlilik sıralama teknolojisi büyük ölçüde Kromatin immünopresipitasyon (ChIP) deney duyarlılığı artmış ve saflaştırılmış hücreleri veya disseke doku kullanarak uygulama açtı. İşte biz ChIP tekniği kullanılacak bir yöntem tasvir Drosophila doku.

Video: Chromatin Immunoprecipitation ChIP using Drosophila tissue

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such as tumor growth, stem cell differentiation, and stem cell self-renewal. A number of methods currently exist to modulate cell signaling in vitro. One method of modulating the total amount of diffusible signaling is to vary the cell seeding density during culture. Due to the random nature of cell seeding, this results in considerable variation in the actual cell-cell spacing and amount of cell-cell contact, and cannot prescribe the local environment. A more specific approach for modulating cell signaling is to use molecular inhibitors or genetic approaches to knock down specific signaling proteins, but both of these methods are best suited to manipulating small numbers of molecules. Here, we demonstrate a new approach to modulating cell-cell signaling that modulates the local environment of a cluster of cells by placing different numbers of cells at desired locations on a substrate. This method provides a complementary way to control the local diffusible and juxtacrine signaling between cells. Our method makes use of the Bio Flip Chip (BFC), a microfabricated silicone chip containing hundreds-to-thousands of microwells, each sized to hold either a single cell or small numbers of cells. We load the chip with cells simply by pipetting them onto the array of wells and washing unloaded cells off the array. The chip is then flipped onto a substrate, whereby the cells fall out of the wells and onto the substrate, maintaining their patterning. After the cells have attached, the chip can be removed (or left on). This approach to cell patterning is unique in that it: 1) doesn't alter the chemistry of the substrate, thus allowing cells to proliferate and migrate; 2) allows patterning onto any substrate, including tissue-culture polystyrene, glass, matrigel, and even feeder cell layers; and 3) is compatible with traditional microcontact printing, allowing the creation of extracellular matrix islands with cells placed inside those islands. In this video, we demonstrate the patterning of mouse embryonic stem cells onto tissue-culture polystyrene using the BFC.

We demonstrate a simple method for placing cells at desired locations on a substrate. This method patterns cells by flipping a silicone chip containing microwells filled with cells onto the substrate. This method provides a new way to modulate diffusible and juxtacrine signaling between cells.

Bio Flip-Chip kullanarak Embriyonik Kök Hücreler, Desenlendirme

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such ...

Biyoloji

A Chromatin Assay for Human Brain Tissue

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental factors that might result in epigenetic alterations of gene expression and other molecular pathology. Traditionally, however, expression studies in postmortem brain were confined to quantification of mRNA or protein. The limitations encountered in postmortem brain research   such as variabilities in autolysis time and tissue integrities   are also likely to impact any studies of higher order chromatin structures. However, the nucleosomal organization of genomic DNA   including DNA:core histone binding - appears to be largely preserved in representative samples provided by various brain banks. Therefore, it is possible to study the methylation pattern and other covalent modifications of the core histones at defined genomic loci in postmortem brain. Here, we present a simplified native chromatin immunoprecipitation (NChIP) protocol for frozen (never-fixed) human brain specimens. Starting with micrococcal nuclease digestion of brain homogenates, NChIP followed by qPCR can be completed within three days. The methodology presented here should be useful to elucidate epigenetic mechanisms of gene expression in normal and diseased human brain.

Until recently, expression studies on human brain were limited to quantification of RNA or protein. With the chromatin immunoprecipitation techniques described in this paper, it will be possible to map histone methylation and other epigenetic regulators of gene expression in postmortem brain.

İnsan Beyninin Doku Kromatin Testi

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental ...

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 ...

Methylated DNA Immunoprecipitation

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on gene expression, and is involved with normal development as well as disease 1-4. Thus, the ability to discriminate between methylated DNA and non-methylated DNA is essential for generating methylation profiles for such studies. Methylated DNA immunoprecipitation (MeDIP) is an efficient technique for the extraction of methylated DNA from a sample of interest 5-7. A sample of as little as 200 ng of DNA is sufficient for the antibody, or immunoprecipitation (IP), reaction. DNA is sonicated into fragments ranging in size from 300-1000 bp, and is divided into immunoprecipitated (IP) and input (IN) portions. IP DNA is subsequently heat denatured and then incubated with anti-5'mC, allowing the monoclonal antibody to bind methylated DNA. After this, magnetic beads containing a secondary antibody with affinity for the primary antibody are added, and incubated. These bead-linked antibodies will bind the monoclonal antibody used in the first step. DNA bound to the antibody complex (methylated DNA) is separated from the rest of the DNA by using a magnet to pull the complexes out of solution. Several washes using IP buffer are then performed to remove the unbound, non-methylated DNA. The methylated DNA/antibody complexes are then digested with Proteinase K to digest the antibodies leaving only the methylated DNA intact. The enriched DNA is purified by phenol:chloroform extraction to remove the protein matter and then precipitated and resuspended in water for later use. PCR techniques can be used to validate the efficiency of the MeDIP procedure by analyzing the amplification products of IP and IN DNA for regions known to lack and known to contain methylated sequences. The purified methylated DNA can then be used for locus-specific (PCR) or genome-wide (microarray and sequencing) methylation studies, and is particularly useful when applied in conjunction with other research tools such as gene expression profiling and array comparative genome hybridization (CGH) 8. Further investigation into DNA methylation will lead to the discovery of new epigenetic targets, which in turn, may be useful in developing new therapeutic or prognostic research tools for diseases such as cancer that are characterized by aberrantly methylated DNA 2, 4, 9-11.

This video demonstrates the protocol for methylated DNA immunoprecipitation (MeDIP). MeDIP is a two day procedure that selectively extracts methylated DNA fragments from a genomic DNA sample using antibodies with specificity for 5 -methylcytosine (anti-5 mC).

Metillenmiş DNA Immunoprecipitation

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on ...

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

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

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

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

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 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 ...

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE possesses an arrangement of binding sites for several transcription factor proteins that confer a regulatory logic specifying when, where, and at what level the regulated gene(s) is expressed. The full set of CREs within an animal genome encodes the organism&prime;s program for development1, and empirical as well as theoretical studies indicate that mutations in CREs played a prominent role in morphological evolution2-4. Moreover, human genome wide association studies indicate that genetic variation in CREs contribute substantially to phenotypic variation5,6. Thus, understanding regulatory logic and how mutations affect such logic is a central goal of genetics. 
Reporter transgenes provide a powerful method to study the in vivo function of CREs. Here a known or suspected CRE sequence is coupled to heterologous promoter and coding sequences for a reporter gene encoding an easily observable protein product. When a reporter transgene is inserted into a host organism, the CRE&prime;s activity becomes visible in the form of the encoded reporter protein. P-element mediated transgenesis in the fruit fly species Drosophila (D.) melanogaster7 has been used for decades to introduce reporter transgenes into this model organism, though the genomic placement of transgenes is random. Hence, reporter gene activity is strongly influenced by the local chromatin and gene environment, limiting CRE comparisons to being qualitative. In recent years, the phiC31 based integration system was adapted for use in D. melanogaster to insert transgenes into specific genome landing sites8-10. This capability has made the quantitative measurement of gene and, relevant here, CRE activity11-13 feasible. The production of transgenic fruit flies can be outsourced, including phiC31-based integration, eliminating the need to purchase expensive equipment and/or have proficiency at specialized transgene injection protocols. 
Here, we present a general protocol to quantitatively evaluate a CRE&prime;s activity, and show how this approach can be used to measure the effects of an introduced mutation on a CRE&prime;s activity and to compare the activities of orthologous CREs. Although the examples given are for a CRE active during fruit fly metamorphosis, the approach can be applied to other developmental stages, fruit fly species, or model organisms. Ultimately, a more widespread use of this approach to study CREs should advance an understanding of regulatory logic and how logic can vary and evolve.

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

Phenotypic variation for traits can result from mutations in cis-regulatory element (CRE) sequences that control gene expression patterns. Methods derived for use in Drosophila melanogaster can quantitatively compare the levels of spatial and temporal patterns of gene expression mediated by modified or naturally occurring CRE variants.

Uygulamalarda Kantitatif Karşılaştırılması Cis</emTransgenik>-Regulatory Element (CRE) Etkinlikler Drosophila melanogaster</em

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass

Chromatin immunoprecipitation (ChIP) is a widely-used method for determining the interactions of different proteins with DNA in chromatin of living cells. Examples include sequence-specific DNA binding transcription factors, histones and their different modification states, enzymes such as RNA polymerases and ancillary factors, and DNA repair components. Despite its ubiquity, there is a lack of up-to-date, detailed methodologies for both bench preparation of material and for accurate analysis allowing quantitative metrics of interaction. Due to this lack of information, and also because, like any immunoprecipitation, conditions must be re-optimized for new sets of experimental conditions, the ChIP assay is susceptible to inaccurate or poorly quantitative results.
Our protocol is ultimately derived from seminal work on transcription factor:DNA interactions1,2 , but incorporates a number of improvements to sensitivity and reproducibility for difficult-to-obtain cell types. The protocol has been used successfully3,4 , both using qPCR to quantify DNA enrichment, or using a semi-quantitative variant of the below protocol.
This quantitative analysis of PCR-amplified material is performed computationally, and represents a limiting factor in the assay. Important controls and other considerations include the use of an isotype-matched antibody, as well as evaluation of a control region of genomic DNA, such as an intergenic region predicted not to be bound by the protein under study (or anticipated not to show changes under the experimental conditions). In addition, a standard curve of input material for every ChIP sample is used to derive absolute levels of enrichment in the experimental material. Use of standard curves helps to take into account differences between primer sets, regardless of how carefully they are designed, and also efficiency differences throughout the range of template concentrations for a single primer set. Our protocol is different from others that are available5-8 in that we extensively cover the later, analysis phase.

We describe a robust method for chromatin immunoprecipitation using primary T cells. The method is founded on standard approaches, but uses a specific set of conditions and reagents that improve efficiency for limited a quantities of cells. Importantly, a detailed description of the data analysis phase is presented.

Biyokütle Limit Tutarlar kullanarak Verimli Kromatin Immunoprecipitation

Chromatin immunoprecipitation (ChIP) is a  widely-used method for determining the interactions of different proteins with  DNA in chromatin of living ...

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 ...

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.

By combining native and crosslinking chromatin immunoprecipitation with high-resolution Mass Spectrometry, ChroP approach enables to dissect the composite proteomic architecture of histone modifications, variants and non-histonic proteins synergizing at functionally distinct chromatin domains.

ChroP Yaklaşım Kromatin Odağı özgü Proteomic Manzaralar teşrih Chip ve Kütle Spektrometre birleştirir

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and ...

Methods Article

Kullanarak Kromatin immünopresipitasyon (ChIP) Drosophila Doku