This work was supported by the Natural Sciences and Engineering Council of Canada and the Ontario Ministry of Research and Innovation.

Hücre Kültürü 1. Hazırlıklar <ol><li> insan hücrelerinin ticari olarak temin stoklarının al. NOT: Bu protokol, HCT116 kolon karsinomu ve birincil insan renal proksimal tübüler epitelyel hücrelerin (HRPTEC) kullanır. </li><li> Dulbecco Modifiye Edilmiş Eagle Ortamı (DMEM)% 7.5 fetal sığır serumu (FBS) ve% 1 Penisilin / Streptomisin (P / S) ile desteklenmiş / yüksek glukoz ortamı: HCT116 kültürü için 500 mi tanesidir. </li><li> % 5 FBS,% 1 epitel hücre büyüme takviyesi ile takviye Epitelyal hücre ortamı, ve% 1 P / S: 500 mi HRPTEC kültürü için tanesidir. </li><li> 140 mM NaCI, 3 mM KCI, 10 mM Na-2 HPO 4, 15 mM KH 2 PO 4: 1x Fosfat Tamponlu Salin (PBS) 500 ml hazırlayın. 7.4 pH ayarlamak ve 121 ° C&#39;de 40 dakika süre ile otoklavlama sureti ile sterilize. </li></ol> Hücre kültürü 2. Başlatma <ol><li> Dikkatle t bozmadan% 80-90 konfluent çanak orta aspireO hücreleri. </li><li> kültür çanak başına 1x PBS Kısım 3-5 ml yavaşça kaplamak çanak yüzey alanını her şişeyi sallayın ve dikkatle 1x PBS aspire. İkinci 1x PBS yıkama için tekrarlayın. </li><li> 3, her bir kültür tabağı içine% 0.05 tripsin etilendiamintetraasetik asit (EDTA) ile yıkanır ve hafifçe eşit şekilde kaplamak tripsin-EDTA ile hücrelere kaya kısım. 2-3 dakika boyunca, 37 ° C&#39;de inkübe edin. </li><li> 90 sn için 4000 xg, 15 ml&#39;lik bir santrifüj tüpü ve santrifüje müstakil hücreleri aktarın. </li><li> tam DMEM 1 ml tekrar süspansiyon hücre pelet. </li><li> bir hemositometre kullanarak hücreleri sayın. Cm2 başına yaklaşık 2,500-5,000 hücre ilk önce tohum ekme yoğunluğu elde etmek için gereken kaç pirojenik olmayan ve polistiren 100 mm&#39;lik bir hücre kültür kaplarına hesaplayın. </li><li> adım 30-45 dakika boyunca 37 ° C su banyosu içinde 1.2 veya 1.3 yapılan ön sıcak tam hücre kültürü ortamı. </li><li> % 70 etanol ile orta şişe ve hücre şişesini dekontamine. ileriye bu noktadan itibarenişlemler aseptik olarak yapılmalıdır. </li><li> Kısım adım 2.6 belirtilen tohum ekme yoğunluğu olan dondurulmuş hücre, tüm birimi kullanmak, çok 100 mm hücre kültür kaplarına tam DMEM 6 mi. </li><li> 100 mm kültür çanakları her adım 2.6 hesaplanan hücre süspansiyonu hacmi aktarın. eşit plaka yüzeyi üzerinde hücreleri dağıtmak için elle her plakayı sallayın. </li><li> % 5 CO2 atmosferinde 37 ° C&#39;de inkübatörde tohumlanmış 100 mm hücre kültür çanağı yerleştirin. Hücreler sonraki adımlarda önce en az 24 saat inkübe izin verin. </li></ol> 3. besi yerinden <ol><li> Hücre konfluent yüzdesini (çanak alanı kapsayan% hücreleri) belirlemek için bir mikroskop altında her bir hücre kültür çanağı gör. kuvöz ve öncesi sıcak tam orta ve 1x PBS hücreleri dönün. Hücreler% 80-100 konfluent iseniz bir sonraki adıma geçin. </li><li> Dikkatlice bozmadan harcanan orta aspireHer bir kültür kabı hücreler. </li><li> kültür çanak başına 1x PBS Kısım 3-5 ml yavaşça kaplamak çanak yüzey alanını her şişeyi sallayın ve dikkatle 1x PBS aspire. İkinci 1x PBS yıkama için tekrarlayın. </li><li> Kısım 3, her bir kültür tabağı içine% 0.05 tripsin-EDTA ve yavaşça kaya eşit şekilde kaplamak tripsin-EDTA ile hücrelerine temin etmektedir. 2-3 dakika boyunca, 37 ° C&#39;de inkübe edin. </li><li> Bu kuluçka sırasında, kısım kadar kültür tabaklarında tam ortam, 10 ml cm2 başına 2,500-5,000 hücre hücre yoğunluğu elde etmek için gereklidir. </li><li> Hücreler plakasından ayrılmış olduğunda, hızlı bir şekilde 1 x tanımlanan tripsin inhibitörü 3 ml ilave edilir ve yavaşça tripsin-EDTA tüm nötralize edilmiş olmasını sağlamak için 1 dakika için çanak girdap. </li><li> steril bir 15 ml&#39;lik santrifüj tüpüne ayrılmış hücreleri aktarın ve kenara. Kalan hücreler bir araya ve aynı 15 mi santrifüj borusuna bu transfer çanak PBS 1X 3 ml ekleyin. </li><li> 150 xg santrifüj edilerek pelet hücreleri5 dakika karıştırıldı. </li><li> Süpernatant aspire ve tam ortam 3 ml hücre pelletini. </li><li> Bir hemositometre kullanılarak hücrelerin sayısı ve cm2 başına 2,500-5,000 hücre, bir hücre yoğunluğu elde etmek için tam ortam 15 ml ihtiva eden 150 mm kültür kaplarına tohum. Her bir kap-bağlanma tahlili için iki 150 mm kullanın. Not: sıvı ortam ile hücrelere oksijen difüzyon Cilt 36 bağlıdır. Süspansiyon içindeki yapışan hücreler veya hücre kullanılıp kullanılmadığını deneyler arasında tutarlı medya hacmini tutulması tavsiye edilir. Medya yayılmasında bu değişkenliğe önlemek için (Tartışma tartışıldığı gibi 24 saat iş istasyonu inkübe) önceden klimalı edilebilir. </li><li> % 5 CO2 atmosferinde 37 ° C&#39;de inkübatör tohumlanmış kültür yemekler yerleştirin. Hücreler sonraki adımlara geçmeden önce en az 24 saat inkübe izin verin. </li></ol> 4. Physioxic Pozlama <ol><li> mikroskop altında hücre gör. bes içint sonuçları, hücreler 24 saat pozlama için% 70-80 konfluent, 48 saat pozlama için% 50-60 konfluent ve 72 saat pozlama için% 40-50 konfluent olduğundan emin olun. </li><li> İstenen süre (24, 48, ya da deneye bağlı olarak 72 saat) hipoksiyaya iş istasyonuna hücreleri yerleştirin. </li><li> Uygun physioxia için iş istasyonu ayarlayın. NOT: Örneğin,% 3 O 2 ayar% 5 CO 2 ve% 92 N2 eşlik ediyorum. NOT: Dinamik aralık içinde oksijen dalgalanmaları aracı haline belirli bir zaman çizelgesi, program takvimi üzerinde istenen veya manuel olarak istenen aralıklarla oksijen ayarlarını varsa. </li><li> % 60 nem ayarlayın. iş istasyonu atmosfer yine de çok kuru ve medya belirgin uzun deneyler (&gt; 48 saat) boyunca buharlaşır. (Ortam iş istasyonu bir atmosfer ile dengelenir böylece) hücre kültürü DIS ortamı doldurmak için iş istasyonu olan bir hücre kültür şişesi içinde ortam rezervini muhafazahes. Not: çözünmüş oksijen, atmosferik oksijen 36 dengelenir, böylece (örneğin, PBS ve tripsin-EDTA gibi) liziz önce hücreler ile etkileşim herhangi bir çözüm hipoksi iş istasyonu olan, kullanımdan önce, 24 saat için önceden şartlandınlmalıdır. </li><li> lizis kadar iş istasyonu içinde hücreler tutun. </li></ol> Cap-bağlama deneyi 5. hazırlanması Tamponlar <ol><li> 1x Tris-tamponlu tuzlu su (TBS) hazırlayın. <ol><li> DH 2 O 800 ml, 8.76 g, NaCl ve 6.05 g Tris baz çözülür. </li><li> 1 M HCI kullanılarak 7.4&#39;luk bir nihai pH&#39;a çözüm sağlamaktadır. </li><li> DH 2 O. kullanarak 1 L son hacim kazandırın </li></ol></li><li> denatüre lizis tamponu hazırlayın. Cap-bağlama analizinin yapılacağı güne liziz tamponunda hazırlanır. Boş agaroz boncuk ve γ-aminofenil-m 7 GTP agaroz C10-bağlantılı boncuk (7,9 ve 7.13 adımlar) yıkamak için ekstra lizis tamponu yapın. <ol><li> DH 2 O 7 ml, 160 ul 5 M NAC eklemeL, 160 ul 1 M Tris-HCI pH 7.4, 40 ul 200 mM NaF, 40 ul 1 M MgCl2, 40 ul 1 mM sodyum ortovanadat. </li><li> 7 mi çözeltisine Igepal 40 ul ekle. son derece viskoz olduğu gibi Igepal ™ almak yardımcı olmak için pipet ucu kesin. </li><li> Igepal tüm çözünmüş kadar, aşağı yukarı pipetleme veya vorteks 7 ml solüsyon karıştırın. </li><li> 8 ml çözeltinin son hacmi kaldırın ve buz üzerinde tutun. </li></ol></li><li> 4x sodyum dodesil sülfat (SDS) -PAGE Örnek Tamponu hazırlayın. <ol><li> Bir cam kaba, 16 ml 1 M Tris-HCI pH 6.8, 12.64 ml gliserol, ve 8 ml ditiotreitol (DTT) birleştirir. </li><li> SDS 3.2 g ve bromofenol mavisi, 0.16 g ekleyin. Toz tamamen manyetik karıştırıcı ile karıştırılarak erimesine izin verir. </li><li> -20 ° C&#39;de 1.5 ml mikrosantrifüj tüpleri içine kısım ve mağaza. </li></ol></li></ol> 6. Hücre Liziz <ol><li> denatüre edici olmayan lizis tamponu hazırlayın ve buz üzerinde th günlük tutmakE Cap-bağlanma tahlili. </li><li> 30 dakika boyunca 37 ° C su banyosu içinde 1 x PBS öncesi sıcak ve tripsin. </li><li> Her bir kap-bağlanma tahlili için, 1.5 ml mikrosantrifüj tüpü içine lizis tamponu kısım 980 ul yapılmaktadır. </li><li> liziz tamponu 980 ul 4- (2-aminoetil) benzensülfonil fluorür hidroklorür 10 ul ve 100x proteaz önleyici kokteyl 10 ul ekle. buz üzerinde tutun. </li><li> hipoksi iş istasyonu içinde bir çöp konteynırına her 150 mm çanak medya atın. </li><li> Sıcak 1x PBS 3-4 ml hücreleri yıkayın ve tüm sıvı atın. </li><li> Her bir tabak sıcak% 0.05 tripsin-EDTA 1 ml ilave edilir ve 2 dakika boyunca ya da hücreleri artık plakaya tutunur kadar bekletin. </li><li> Bir pipet kullanarak, 1.5 ml mikrosantrifüj tüpü, bir plaka hücreleri transferi. hücrelerin her plaka ayrı bir 1.5 ml mikrosantrifüj tüpüne aktarılır, böylece gerektiğinde tekrar edin. </li><li> 90 saniye t 6,000 xg&#39;de 1.5 ml mikrosantrifüj tüpleri santrifüjO pelet hücreleri. </li><li> pelet bozmadan pipet kullanarak tripsin aspire. pelet 90 saniye boyunca 6.000 xg, yeniden santrifüj 1,5 ml&#39;lik ependorf tüp rahatsız edilirse. </li><li> nazikçe sadece pelet yukarıda tüpe PBS 200 ul pipetleme sıcak 1x PBS ile hücreleri yıkayın. Yeniden santrifüj pelet rahatsız olup olmadığını. </li><li> pelet bozmadan PBS aspire. </li><li> 1 ml Pipet 500 ul birinci hücresel pelet üzerine liziz tampon çözeltisi hazırlandı. Yukarı ve aşağı pipetleme tamamen pelletini. </li><li> İkinci hücre pelet ile yeniden süspanse hücreleri birleştirin ve yukarı ve aşağı pipetleme ikinci pelletini. Tüm topaklar birleştirildi ve her bir örnek için yeniden süspanse edilene kadar bu işlemi tekrarlayın. </li><li> 1.5 ml mikrosantrifüj tüpü içinde liziz tamponunda kalan 500 ul lizat birleştirin. </li><li> hipoksi iş istasyonundan örnekleri çıkarın. Not: liziz tamponu ilave edildikten sonra, birII Daha sonraki aşamalar, hipoksi, iş istasyonu, 37 ° C olarak ayarlanmış ve aşağıdaki adımlar soğuk (4 ° C veya buz üzerinde) gerçekleştirilir edilecek olduğu gibi çevre havası ifa edilecek olan vardır. </li><li> Lyse 1.5-2 saat boyunca 4 ° C&#39;de örnekleri çevirerek nazik ajitasyon kullanılarak hücreler. </li><li> selüler artığın ayrılması için 4 ° C&#39;de 15 dakika boyunca 12,000 x g&#39;de lize hücreleri santrifüjleyin. </li><li> Yeni 1.5 ml mikrosantrifüj tüp lizat aktarın ve pelet atın. </li><li> Süpernatantın bir kısmı (% 5-10), gelecekte Western blot analizi için bir bütün hücre lizatı giriş kontrol olarak kullanılmak üzere ayırın. </li></ol> 7. Cap-bağlanma deneyi <ol><li> Her numune için, 1.5 ml mikrosantrifüj tüpleri ayırmak için boş agaroz boncuk kontrol bulamacın 50 ul ve γ-aminofenil m 7 GTP agaroz C10-bağlanmış boncuk çamuru 50 ul transfer. boncuk bulamaç toplanmasını kolaylaştırmak için pipet ucu çıkarmak için makas kullanın. </li><li> boncuk b peletY 30 sn boyunca 500 xg&#39;de bulamaç santrifüj. </li><li> dikkatlice Süpernatantı ve TBS 500 ul boncuklar tekrar süspansiyon. </li><li> Tekrarlayın 7.2 ve 7.3 adımları tekrarlayın. 30 saniye boyunca 500 xg&#39;de boncuk Pelet ve süpernatant kaldırmak. </li><li> Boş agaroz boncuklar içeren 1.5 ml mikrosantrifüj tüp adım 6.19 lizat içeren süpernatant aktarın. NOT: Boş agaroz boncuklar non-spesifik boncuk ile etkileşime lizat proteinleri kaldırmak için bir ön temizleme adımı olarak hareket ederler. </li><li> yavaşça sallanarak 4 ° C&#39;de 10 dakika süreyle inkübe edin. </li><li> 30 saniye boyunca 500 xg&#39;de santrifüj edilerek boş agaroz boncuk pelet. </li><li> Γ-aminofenil-m 7 GTP agaroz C10-bağlantılı boncuklar içeren 1.5 ml mikrosantrifüj tüpüne lizat aktarın. </li><li> Liziz tamponu 500 ul boncuk yeniden süspanse 30 sn boyunca 500 xg&#39;de santrifüjleme ile boncuk tane haline ve Superna ayıklayarak boş agaroz boncuk yıkayıntant. Beş yıkar toplam bu dört kez tekrarlayın. </li><li> 1x SDS-PAGE numune tampon maddesi içinde boş bir agaroz boncuklar tekrar süspansiyon ve 95 ° C&#39;de 90 sn için boncuk kaynatın. gelecekteki Western blot analizi için -20 ° C&#39;de saklayın ilgili protein boncuk olmayan spesifik olarak bağlanan bakmaktır. </li><li> Cap bağlayıcı proteinler yakalamak için 4 ° C sıcaklıkta 1 saat boyunca yavaşça sallanarak γ-aminofenil m 7 GTP agaroz C10-bağlı boncuklar ile lizat inkübe edin. </li><li> 30 saniye için 500 x g&#39;de santrifüj γ-aminofenil m 7 GTP agaroz C10-bağlı boncuklar Pelet. süpernatant atın. </li><li> Adım 7.9 yineleyerek γ-aminofenil-m 7 GTP agaroz C10-bağlantılı boncuk yıkayın. </li><li> liziz tamponu 600 ul boncuk tekrar. </li><li> 1 mM&#39;lik nihai bir konsantrasyona kadar GTP ekleyin. </li><li> 4 ° C sıcaklıkta 1 saat boyunca yavaşça sallanarak γ-aminofenil m 7 GTP agaroz C10-bağlı boncuklar + 1 mM GTP inkübe edin. Not: Bunon-spesifik m 7 GTP ile etkileşim proteinleri ilişkisini (yani, non-metile GTP ile etkileşim). </li><li> 30 saniye için 500 x g&#39;de santrifüj γ-aminofenil m 7 GTP agaroz C10-bağlı boncuklar Pelet. </li><li> yeni bir 1.5 ml mikrosantrifüj tüpü süpernatantı aktarın ve 4x SDS-PAGE örnek tamponu (50 mM Tris-HCI, 100 mM DTT,% 2 SDS, mavi,% 0.1 bromofenol ve% 10 gliserol) 200 ul ilave edin. Gelecekteki western blot analizi 37 -20 ° C &#39;de GTP kontrol örneği saklayın. adım 7.9 tekrarlayarak boncuk yıkayın. </li><li> 90 sn için 95 ° C &#39;de 1 x SDS-PAGE örnek tamponu (50 mM Tris-HCI, 100 mM DTT,% 2 SDS, mavi,% 0.1 bromofenol ve% 10 gliserol) ve kaynama boncuk tekrar. Gelecekteki western blot analizi 37 -20 ° C &#39;de 7 m GTP&#39;ye bağlı fraksiyonu saklayın. </li></ol>

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The authors declare that they have no competing financial interests.

Fizyolojik oksijen koşullarına maruz insan hücrelerinde kap bağlayıcı proteinlerin analizi, yeni oksijen regüle çeviri başlatma faktörlerinin tanımlanması için izin verir. MRNA veya başka bir kap ile ilişkili proteinlerin hem 5 &#39;başlık için bu faktörlerin afinite m 7&#39;ye olan ilişkilerine gücü ölçülebilir agaroz boncuklar GTP-bağlı. Bu tekniğin bir uyarı proteinlerin post-lisis kapağı bağlama potansiyelini ölçer, ancak, protein-protein etkileşimleri ve post-translasyo...

Çeviri kontrol özellikle hücresel stres 1 dönemlerde, gen ifadesinin transkripsiyonel düzenlemeye eşit derecede önemli bir adım olarak ortaya çıkmaktadır. Çeviri kontrolünün odak noktası protein sentezi ilk adımları 7-methylguanosine ökaryotik başlatma faktörü 4E (eIF4E) bağlanma içeren başlatılması hız kısıtlayıcı adım (m 7 GTP) mRNA 2 5 &#39;kapak olan . eIF4E eIF4A, bir RNA helikaz ve eIF4G, diğer çeviri faktörleri ve 40S 3 ribozom alımı için gerekli bir iskele protein içerir eIF4F adında bir trimerik kompleksinin bir parçasıdır. Normal fizyolojik şartlar altında, mRNA büyük çoğunluğu bir kapak-bağımlı mekanizma yoluyla çevrilir, hücresel stres dönemlerinde, insan mRNA yaklaşık% 10 1,4 intiation kap bağımsız çeviri izin verebilir 5 &#39;UTRs içerir. Cap-bağımlı çeviri tarihsel eşanlamlı olmuşturous eIF4F ile, ancak, eIF4F stres özgü varyasyonlar eğilimi konu 5-8 haline gelmiştir. Çeşitli hücresel stresler eIF4E aktivite rapamisin kompleksi 1 (mTORC1) memeli hedefin üzerinden bastırılmış neden olur. Bu kinaz hedefleri birinin aktivite artışı ile sonuçlanır stres altında güçleşir, (4E-BP) proteini 4E bağlayıcı değildir. Fosforilatlanmamış 4E-BP eIF4E ve bloklar eIF4G kap bağımlı çeviri 9,10 baskı neden ile etkileşim yeteneğini bağlanır. İlginç bir şekilde, eIF4E2 (veya 4EHP) adı eIF4E bir homologu, belki de stres kaynaklı baskı kaçmasına izin 4E-BP 11 için daha düşük bir afiniteye sahiptir. Gerçekten de, ilk bağlı eIF4G 12 ile etkileşim olmayışı nedeniyle çevrilmesinin bir bastıncısı olarak, özelliği, eIF4E2 hipoksik strese 6,13 sırasında 3 &#39;UTR RNA hipoksiya tepki unsurları içeren mRNA yüzlerce çeviri başlatır. Bu aktivasyon, IeIF4G3, RNA hipoksik eIF4F kompleksi veya eIF4F H 6,13 teşkil protein motifi 4, ve hipoksi indüklenebilir faktör (HIF) 2a bağlayıcı etkileşimler yoluyla elde s. Normal koşullar altında, bir bastıncısı olarak eIF4E2 GIGYF2 ve ZNF598 14 ile bağlanmaktadır. Bu kompleksler, kısmen, Agaroz bağlanmış m 7 GTP afinite reçinesi ile tespit edilmiştir. Bu klasik yöntem 15 çeviri alanında standart ve en iyi ve en yaygın aşağı çekme de kap-bağlama kompleksleri izole etmek için tekniği kullanılmıştır ve in vitro bağlanma deneyleri 16-19 olduğunu. Kap-bağımlı çeviri makine arası değişen parçalar 6-8,13 gibi esnek ve uyarlanabilir ortaya çıkmaktadır olarak, bu yöntem hızla stres yanıtı katılan yeni kap-bağlayıcı proteinlerin tanımlamak için güçlü bir araçtır. Birkaç ökaryot model sistemler stres yanıtları gibi bir eIF4E2 homolog kullanmak göründükleri gibi Dahası, eIF4F değişimleri geniş etkileri olabilirC. thaliana 20 gibi, S. Pombe 21, D 22 melanogaster ve C 23 elegans. Kanıt eIF4F değişiklikler sıkı koşullar stres, ancak normal fizyolojisi 24 dahil olmak sınırlandırılamaz düşündürmektedir. (Mikroelektronlar üzerinden ölçülen) (kılcal uçlarında) ya da dokular içinde dokulara oksijen kaynağı beyindeki 25% 2-6 arasında değişmektedir, akciğerlerde 26% 3-12, bağırsak 27,% 4 in 3,5-6% karaciğer 28, böbrek 29% 7-12, kas 30 içinde% 4 ve kemik iliği 31% 6-7. Hücreler ve mitokondri% 1,3 oksijen 32 daha az içerirler. Bu değerler hücrelerinin rutin olarak kültürlendiği olan çevre havası daha hipoksiye daha yakındırlar. Bu ne daha önce hipoksi özgü hücresel süreçleri fizyolojik bir ortamda ilgili olabileceği gibi düşündüm olduğunu göstermektedir. İlginç bir şekilde, eIF4F ve eIF4F H </sup&gt; aktif fizyolojik oksijen ya da &quot;physioxia&quot; 24 maruz çeşitli insan hücre çizgileri belirgin havuzlar ya da mRNA sınıfları için başlatma katılabilir. Düşük oksijen, aynı zamanda uygun fetal gelişim 33 sürücüler ve hücreler genellikle daha yüksek çoğalma oranları, uzun ömürleri, daha az DNA hasarını ve physioxia 34 daha az genel stres yanıtları var. Bu nedenle, eIF4F lH olasılıkla fizyolojik koşullar altında seçme genin ifadesinde önemli bir faktördür. Burada, sabit fizyolojik oksijen koşullarında veya doku mikroçevrelerde büyük olasılıkla daha fazla temsilcisi olan dinamik bir dalgalanma aralığı kültür hücreleri bir protokol sağlar. Bu yöntemin bir avantajı, hücre hipoksi iş istasyonu içinde lize olmasıdır. Hücre lizizi hipoksik hücre kültüründen geçiş diğer protokollere yapılan ne sıklıkta açık değildir. Hücreler genellikle ilk olarak küçük bir hipoksi inkübatör kaldırılır olmakoksijene hücresel yanıtı (bir ya da iki dakika) 35 hızlı olduğu ön parçalama, ancak oksijen bu poz biyokimyasal yollar etkileyebilir. Bazı kap-bağlayıcı proteinler ikinci bir baz ile etkileşimleri gerektiren veya bu nedenle bazı kapak interaktörler arıtma sürecinde gözden kaçabilir m 7 GTP, hidrolize olabilir. Agaroz bağlanmış enzimatik dayanıklı kap analoglarına Bu protokolde ikame edilebilir. Burada anlatılan yöntemi ile aktivite ve eIF4F H ve eIF4F diğer varyasyonları kompozisyonunu keşfetmek hücreler fizyolojik koşullar ya da stres yanıtları sırasında kullanmak karmaşık gen ifadesi makine ışık tutacaktır.

<table border="0" cellpadding="0" cellspacing="0" width="901">
	<tbody>
		<tr height="26">
			<td height="26" style="height:26px;width:380px;">&#947;-aminophenyl-m7GTP agarose C10-linked beads</td>
			<td style="width:151px;">Jena Bioscience</td>
			<td style="width:165px;">AC-1555</td>
			<td style="width:205px;">Agarose-linked m7GTP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 mm culture dish</td>
			<td>Corning</td>
			<td>877222</td>
			<td>10-cm culture dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">150 mm culture dish</td>
			<td>Thermofisher</td>
			<td>130183</td>
			<td>15-cm culture dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AEBSF Hydrochloride</td>
			<td>ACROS Organics</td>
			<td>A0356829</td>
			<td>AEBSF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose Beads</td>
			<td>Jena Bioscience&#160;</td>
			<td>AC-0015</td>
			<td>Agarose bead control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bromophenol Blue</td>
			<td>Fisher</td>
			<td>BP112-25</td>
			<td>Component of SDS-PAGE loading buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5 mL Centrifuge Tubes</td>
			<td>FroggaBio</td>
			<td>1210-00S</td>
			<td>Used to centrifuge small volumes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 mL Conical Centrifuge Tubes</td>
			<td>Fisher</td>
			<td>1495970C</td>
			<td>Used in culturing primary cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Defined trypsin inhibitor</td>
			<td>Fisher</td>
			<td>R007100</td>
			<td>DTI</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dithiothreital</td>
			<td>Fisher</td>
			<td>BP172-25</td>
			<td>DTT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Epithelial cell medium (complete kit)</td>
			<td>ScienCell</td>
			<td>4101</td>
			<td>Includes serum and growth factor supplements)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Fisher</td>
			<td>BP229-1</td>
			<td>Component of SDS-PAGE loading buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 mM Guanosine 5&#39;-triphosphate, 1 mL</td>
			<td>Jena Bioscience</td>
			<td>272076-0251M</td>
			<td>GTP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HCT116 colorectal carcinoma</td>
			<td>ATCC</td>
			<td>CCL-247</td>
			<td>Human cancer cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human renal proximal tubular epithelial cells</td>
			<td>ATCC</td>
			<td>PCS-400-010</td>
			<td>HRPTEC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hyclone DMEM/High Glucose</td>
			<td>GE Life Sciences</td>
			<td>SH30022.01</td>
			<td>Standard media for human cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hyclone Penicillin-Streptomycin solution</td>
			<td>GE Life Sciences</td>
			<td>SV30010</td>
			<td>Antibiotic component of DMEM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">H35 HypOxystation</td>
			<td>Hypoxygen</td>
			<td>N/A</td>
			<td>Hypoxia workstation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Igepal CA-630</td>
			<td>MP Biomedicals</td>
			<td>2198596</td>
			<td>Detergent component of lysis buffer</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Monopotassium phosphate</td>
			<td>Fisher</td>
			<td>P288-500</td>
			<td>KH2PO4</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Potassium chloride</td>
			<td>Fisher</td>
			<td>P217-500</td>
			<td>KCl</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Magnesium chloride</td>
			<td>Fisher</td>
			<td>M33-500</td>
			<td>MgCl2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride</td>
			<td>Fisher</td>
			<td>BP358-10</td>
			<td>NaCl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium fluoride</td>
			<td>Fisher</td>
			<td>5299-100</td>
			<td>NaF (phosphatase inhibitor component of lysis buffer)</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Disodium phosphate</td>
			<td>Fisher</td>
			<td>5369-500</td>
			<td>Na2HPO4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Premium Grade Fetal Bovine Serum</td>
			<td>Seradigm</td>
			<td>1500-500</td>
			<td>FBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease Inhibitor Cocktail (100 x)</td>
			<td>Cell Signalling</td>
			<td>58715</td>
			<td>Component of lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Dodecyl Sulfate</td>
			<td>Fisher</td>
			<td>BP166-100</td>
			<td>SDS</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Sodium Orthovanadate</td>
			<td>Sigma</td>
			<td>56508</td>
			<td>Na3VO4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris Base</td>
			<td>Fisher</td>
			<td>BP152-5</td>
			<td>Component of buffers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.05% Trypsin-EDTA (1x)</td>
			<td>Life Technologies</td>
			<td>2500-067</td>
			<td>Trypsin used to detach adherent cells</td>
		</tr>
	</tbody>
</table>

analysis of cap-binding proteins in human cells exposed to physiological oxygen conditions

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex is by far the most common pathway to initiate protein synthesis in eukaryotic cells, but stress-specific variations of this complex are now emerging. Purifying cap-binding proteins with an affinity resin composed of Agarose-linked m7GTP (a 5' mRNA cap analog) is a useful tool to identify factors involved in the regulation of translation initiation. Hypoxia (low oxygen) is a cellular stress encountered during fetal development and tumor progression, and is highly dependent on translation regulation. Furthermore, it was recently reported that human adult organs have a lower oxygen content (physioxia 1-9% oxygen) that is closer to hypoxia than the ambient air where cells are routinely cultured. With the ongoing characterization of a hypoxic eIF4F complex (eIF4FH), there is increasing interest in understanding oxygen-dependent translation initiation through the 5' mRNA cap. We have recently developed a human cell culture method to analyze cap-binding proteins that are regulated by oxygen availability. This protocol emphasizes that cell culture and lysis be performed in a hypoxia workstation to eliminate exposure to oxygen. Cells must be incubated for at least 24 hr for the liquid media to equilibrate with the atmosphere within the workstation. To avoid this limitation, pre-conditioned media (de-oxygenated) can be added to cells if shorter time points are required. Certain cap-binding proteins require interactions with a second base or can hydrolyze the m7GTP, therefore some cap interactors may be missed in the purification process. Agarose-linked to enzymatically resistant cap analogs may be substituted in this protocol. This method allows the user to identify novel oxygen-regulated translation factors involved in cap-dependent translation.

Bir m 7 GTP Affinity Sütun içinde eIF4E ve eIF4E2 ve Oksijen Cevabı Cap-bağlama yeteneği analizi 1. Şekil 2, iki insan hücre çizgileri oksijen dalgalanmalara tepki olarak iki ana kap bağlayıcı proteinler tipik m 7 GTP afinite saflaştırma western lekelerinin temsil etmektedir: F ŞEKIL 1 ve kolorektal karsinom primer insan renal proksimal tübül...

Watch this Scientific Journal Video about Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions at JoVE.com

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

Here, we present human cell culture protocols to analyze translation initiation factors that bind the 5' cap of mRNA during physiological oxygen conditions. This method utilizes an Agarose-linked m7GTP cap analog and is suitable to investigate cap-binding factors and their interacting partners.

Fizyolojik Oksijen Koşullarına Maruz İnsan hücrelerinde Cap-bağlayıcı proteinlerin analizi

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex ...

analysis-cap-binding-proteins-human-cells-exposed-to-physiological

Research

JoVE Journal

Genetics

7.7K Görüntüleme.  University of Guelph. Here, we present human cell culture protocols to analyze translation initiation factors that bind the 5' cap of mRNA during physiological oxygen conditions. This method utilizes an Agarose-linked m7GTP cap analog and is suitable to investigate cap-binding factors and their interacting partners.

Video: Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend diseases related to pregnancy. In this protocol, human primary villous cytotrophoblasts freshly isolated from placentas through a standard DNase/trypsin protocol are microporated with small interfering RNA (siRNA). This approach provided greater efficiency for siRNA transfection when compared to a lipofection-based method. Transfected cells can subsequently be analyzed by standard Western blot within a time frame of 3-4 days post-transfection. In addition, using cultured primary villous cytotrophoblasts, Electrophoretic Mobility Shift Assay (EMSA) analysis was optimized and performed on extracts from days 1 to 4. The use of these cultured primary cells and the protocol described allow for an evaluation of the implication of specific genes and transcription factors in the process of villous cytotrophoblast differentiation into a syncytiotrophoblast-like cell layer. However, the limited time span allowable in culture precludes the use of methods requiring more time, such as generation of a stable cell population. Therefore testing of this cell population requires highly optimized gene transfer protocols.

This protocol describes a method for efficiently transfecting siRNA in freshly isolated human villous cytotrophoblasts using microporation and identifying DNA-protein complexes in these cells. Transfected cells can be monitored by Western blot and EMSA analyses during the 4-day culture time.

Taze izole edilmiş insan Villöz sitotrofoblastlar ilgili siRNA transfeksiyonu ve EMSA analizi

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

Genetik

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

Bakteriyel Konjugasyon ilgilendim Transferi Proteinlerin Dizi özgü Analiz konjugatif Çiftleşme Tahliller

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which specifically binds the pre-mRNA target and an effector domain. Previously, we have taken advantage of the unique RNA binding mode of the PUF domain in human Pumilio 1 to generate a programmable RNA binding scaffold, which was used to engineer various artificial RBPs to manipulate RNA metabolism. Here, a detailed protocol is described to construct Engineered Splicing Factors (ESFs) that are specifically designed to modulate the alternative splicing of target genes. The protocol includes how to design and construct a customized PUF scaffold for a specific RNA target, how to construct an ESF expression plasmid by fusing a designer PUF domain and an effector domain, and how to use ESFs to manipulate the splicing of target genes. In the representative results of this method, we have also described the common assays of ESF activities using splicing reporters, the application of ESF in cultured human cells, and the subsequent effect of splicing changes. By following the detailed protocols in this report, it is possible to design and generate ESFs for the regulation of different types of Alternative Splicing (AS), providing a new strategy to study splicing regulation and the function of different splicing isoforms. Moreover, by fusing different functional domains with a designed PUF domain, researchers can engineer artificial factors that target specific RNAs to manipulate various steps of RNA processing.

This report describes a bioengineering method to design and construct novel Artificial Splicing Factors (ASFs) that specifically modulate the splicing of target genes in mammalian cells. This method can be further expanded to engineer various artificial factors to manipulate other aspects of RNA metabolism.

Yapay Faktörler Mühendislik Özellikle İnsan Hücreleri alternatif bağlantı işleyin için

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

DNA değişiklikler için Immunostaining: Confocal görüntülerin sayısal analiz

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein interactions in yeast. It allows analysis of a target region of interest without the need for prior knowledge about likely proteins bound to the target region. This, in theory, allows HyCCAPP to be used to analyze any genomic region of interest, and it provides sufficient flexibility to work in different cell systems. This method is not meant to study binding sites of known transcription factors, a task better suited for Chromatin Immunoprecipitation (ChIP) and ChIP-like methods. The strength of HyCCAPP lies in its ability to explore DNA regions for which there is limited or no knowledge about the proteins bound to it. It can also be a convenient method to avoid biases (present in ChIP-like methods) introduced by protein-based chromatin enrichment using antibodies. Potentially, HyCCAPP can be a powerful tool to uncover truly novel DNA-protein interactions. To date, the technology has been predominantly applied to yeast cells or to high copy repeat sequences in mammalian cells. In order to become the powerful tool we envision, HyCCAPP approaches need to be optimized to efficiently capture single-copy loci in mammalian cells. Here, we present our adaptation of the initial yeast HyCCAPP capture protocol to human cell lines, and show that single-copy chromatin regions can be efficiently isolated with this modified protocol.

This is a method to identify novel DNA-interacting proteins at specific target loci, relying on sequence-specific capture of crosslinked chromatin for subsequent proteomic analyses. No prior knowledge about potential binding proteins, nor cell modifications are required. Initially developed for yeast, the technology has now been adapted for mammalian cells.

Hibridizasyon uyarlaması yakalama kromatin ilişkili proteinlerin proteomik memeli hücreleri için

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. Complete gene ablation in HSCs required the generation of knockout mice from which HSCs could be isolated, and gene ablation in primary human HSCs was not possible. Viral transduction could be used for knock-down approaches, but these suffered from variable efficacy. In general, genetic manipulation of human and mouse hematopoietic cells was hampered by low efficiencies and extensive time and cost commitments. Recently, CRISPR/Cas9 has dramatically expanded the ability to engineer the DNA of mammalian cells. However, the application of CRISPR/Cas9 to hematopoietic cells has been challenging, mainly due to their low transfection efficiencies, the toxicity of plasmid-based approaches and the slow turnaround time of virus-based protocols.
A rapid method to perform CRISPR/Cas9-mediated gene editing in murine and human hematopoietic stem and progenitor cells with knockout efficiencies of up to 90% is provided in this article. This approach utilizes a ribonucleoprotein (RNP) delivery strategy with a streamlined three-day workflow. The use of Cas9-sgRNA RNP allows for a hit-and-run approach, introducing no exogenous DNA sequences in the genome of edited cells and reducing off-target effects. The RNP-based method is fast and straightforward: it does not require cloning of sgRNAs, virus preparation or specific sgRNA chemical modification. With this protocol, scientists should be able to successfully generate knockouts of a gene of interest in primary hematopoietic cells within a week, including downtimes for oligonucleotide synthesis. This approach will allow a much broader group of users to adapt this protocol for their needs.

A protocol for fast CRISPR/Cas9-mediated gene disruption in mouse and human primary hematopoietic cells is described in this article. Cas9-sgRNA ribonucleoproteins are introduced via electroporation with sgRNAs generated through in vitro transcription and commercial Cas9. High editing efficiencies are achieved with limited time and financial cost.

Fare ve insan hematopoetik progenitör hücre tarafından CRISPR/Cas9 yüksek verimli Gene bozulma

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations

Although over 200 genetic mutations in the human BEST1 gene have been identified and linked to retinal degenerative diseases, the pathological mechanisms remain elusive mainly due to the lack of a good in vivo model for studying BEST1 and its mutations under physiological conditions. BEST1 encodes an ion channel, namely BESTROPHIN1 (BEST1), which functions in retinal pigment epithelium (RPE); however, the extremely limited accessibility to native human RPE cells represents a major challenge for scientific research. This protocol describes how to generate human RPEs bearing BEST1 disease-causing mutations by induced differentiation from human pluripotent stem cells (hPSCs). As hPSCs are self-renewable, this approach allows researchers to have a steady source of hPSC-RPEs for various experimental analyses, such as immunoblotting, immunofluorescence, and patch clamp, and thus provides a very powerful disease-in-a-dish model for BEST1-associated retinal conditions. Notably, this strategy can be applied to study RPE (patho)physiology and other genes of interest natively expressed in RPE.

Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations

Here we present a protocol to differentiate retinal pigment epithelium (RPE) cells from human pluripotent stem cells bearing patient-derived mutations. The mutant cell lines may be used for functional analyses including immunoblotting, immunofluorescence, and patch clamp. This disease-in-a-dish approach circumvents the difficulty of obtaining native human RPE cells.

Farklılaşma, bakım ve insan Retina Pigment epitel hücre Analizi: BEST1 mutasyonlar için bir tabak içinde hastalık modeli

Although over 200 genetic mutations in the human BEST1 gene have been identified and linked to retinal degenerative diseases, the pathological mechanisms ...

Single-cell RNA Sequencing and Analysis of Human Pancreatic Islets

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to normal and pathological conditions. The goal of this protocol is to generate high-quality, large-scale transcriptome data of each endocrine cell type with the use of a droplet-based microfluidic single-cell RNA sequencing technology. Such data can be utilized to build the gene expression profile of each endocrine cell type in normal or specific conditions. The process requires careful handling, accurate measurement, and rigorous quality control. In this protocol, we describe detailed steps for human pancreatic islets dissociation, sequencing, and data analysis. The representative results of about 20,000 human single islet cells demonstrate the successful application of the protocol.

Here, we present a protocol to generate high-quality, large-scale transcriptome data of single cells from isolated human pancreatic islets using a droplet-based microfluidic single-cell RNA sequencing technology.

Insan pankreatik Izletinin tek hücreli RNA sıralaması ve Analizi

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to ...

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise heterozygous genetic modifications is challenging due to CRISPR-CAS9 mediated indel formation in the second allele. Here, we demonstrate a protocol to help overcome this difficulty by using two repair templates in which only one expresses the desired sequence change, while both templates contain silent mutations to prevent re-cutting and indel formation. This methodology is most advantageous for gene editing coding regions of DNA to generate isogenic control and mutant human stem cell lines for studying human disease and biology. In addition, optimization of transfection and screening methodologies have been performed to reduce labor and cost of a gene editing experiment. Overall, this protocol is widely applicable to many genome editing projects utilizing the human pluripotent stem cell model.

This protocol provides a method to facilitate the generation of defined heterozygous or homozygous nucleotide changes using CRISPR-CAS9 in human pluripotent stem cells.

İnsan Pluripotent Kök Hücrelerinde CRISPR-CAS9 Kullanılarak Tanımlanmış Genomik Modifikasyonların Üretimi

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise ...

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding sites for transcriptional regulators to modulate gene expression. Alterations of CREs have been implicated in various diseases including cancer. While promoters and enhancers have been the primary CREs for studying gene regulation, very little is known about the role of silencer, which is another type of CRE that mediates gene repression. Originally identified as an adaptive immunity system in prokaryotes, CRISPR/Cas9 has been exploited to be a powerful tool for eukaryotic genome editing. Here, we present the use of this technique to delete an intronic silencer in the human RUNX1 gene and investigate the impacts on gene expression in OCI-AML3 leukemic cells. Our approach relies on electroporation-mediated delivery of two preassembled Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complexes to create two double-strand breaks (DSBs) that flank the silencer. Deletions can be readily screened by fragment analysis. Expression analyses of different mRNAs transcribed from alternative promoters help evaluate promoter-dependent effects. This strategy can be used to study other CREs and is particularly suitable for hematopoietic cells, which are often difficult to transfect with plasmid-based methods. The use of a plasmid- and virus-free strategy allows simple and fast assessments of gene regulatory functions.

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

Direct delivery of preassembled Cas9/guide RNA ribonucleoprotein complexes is a fast and efficient means for genome editing in hematopoietic cells. Here, we utilize this approach to delete a RUNX1 intronic silencer and examine the transcriptional responses in OCI-AML3 leukemic cells.

Akut Miyeloid Lösemi Hücrelerinde CRISPR/Cas9 Ribonükleoprotein ile RUNX1 Intronic Susturucunun Transkripsiyonel Rolünün İncelendirin

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding ...