The authors would like to acknowledge Wensheng Liu for invaluable help with cell imaging. This work was supported by grants from the&#160;US National Institutes of Health (R01 CA182467), American Cancer Society (RSG-09-252-01-RMC), Lynn Sage Foundation, and A Sister&#8217;s Hope Foundation.

EMT İndüksiyon 1. Hücre Kültürü Not: EMT TGFβ tedavisi veya epitel hücrelerinde transkripsiyon faktörleri Twist, salyangoz veya Zeb1 / 2 ektopik sentezlenmesi ile indüklenebilir. Ölümsüzleştirilmiş insan meme epitelyum hücreleri (HMLE / döndürme-ER, Dr. J. Yang bir hediye UCSD) 9,11,29 yılında Twist-ER, füzyon proteininin sentezlenmesi yoluyla uyarılabilir bir EMT sistemi bu protokolde tarif edilmektedir. 4-OH (TAM) İşleme üzerine, füzyon proteini Bükülme ER 12-14 gün 9,11,29 tam EMT geçiş transkripsiyon ve sonuçları elde etmek için çekirdeğe doğru yer değiştirir. Bu HMLE / Salyangoz-ER, HMLE / TGFβ ve MCF10A / TGFβ gibi ek EMT uyarılabilir sistemler, önceki yayınlarda 11,30 bulunabilir. <ol><li> % 5 CO2 içeren bir 37 ° C kuluçka makinesi içinde, serumsuz meme epitelyal hücre büyüme ortamında (MEGM) olarak HMLE / Bükülme ER hücreleri korumak. Passage hücreler her 2-3 gün% 80 izdiham ulaştığınızda. % 5 buzağı serumu / DMEM ile tripsin inaktive daha sonra 5-10 dakika boyunca 37 ° C&#39;de% 0.15 tripsin ile inkübe hücreleri, vb. Hücreleri aşağı Spin ve MEGM onları tekrar süspansiyon. 1-4 arasında bir oranda yeni bir doku kültürü çanağı içinde plakası hücreleri. </li><li> 200 uM çözelti yapmak için etanol içinde TAM çözülür. -20 ° C&#39;de ışık ve mağaza TAM koruyun. 20 nM TAM bir son konsantrasyon yapmak için 10,000 seyreltmenin kullanımdan önce yeni bir 1 de MEGM ortamına TAM ekleyin. </li><li> EMT uyarılması için, iki nüsha halinde 10 cm doku kültürü çanak içine 1,6 x 10 6 HMLE / Twist ER hücreleri plaka. 20 nM TAM-ihtiva eden ortam ile MEGM Kültür hücreleri. </li><li> Nüsha halinde 10 cm çanak içine Plaka 1,6 x 10 6 HMLE / Twist ER hücreleri ve olmayan TAM muamele kontrol olarak% 0.01 etanol ile tedavi hücreleri. </li><li> İki gün kuluçkadan sonra, hücrelerin morfolojik değişikliklerini kaydetmek için 10X büyütmede ışık mikroskobu altında fotoğraf çekmek. </li><li> Kontrol veya TAM ile muamele edilmiş bir çanak ortamı basınçla emiliphücreler. RNA izolasyonu ve protein analizi için RIPA tamponunda diğer yarısı için RNA, liziz tamponu içinde plakanın yarısını hurdaya soğuk PBS ile hücreleri ve hasat hücreleri yıkayın. </li><li> Nüsha halinde 10 cm çanak yeniden kaplama 1,6 x 10 6 hücre tarafından kontrol veya TAM-işlenmiş yemekler Passage hücreleri. Sürekli 20 nM TAM veya araç ile hücre tedavisi. </li><li> Tekrar, araçla tedavi edilmiş kontrol hücreleri, parke taşı gibi epitel morfolojisi muhafaza ise, bu sürenin sonunda, TAM ile muamele edilmiş Bükülme ER hücreler bir iğ şeklinde mezenkimal morfolojisini, 14 gün kadar 5 kez her gün ila 7. </li></ol> EMT 2. Karakterizasyonu Not: EMT A tamamlanması ile gösterilir: bir iğ şeklinde fibroblastik gibi görünümü için bir Arnavut kaldırımı gibi epitel morfolojiden (1) Cep morfolojik değişimler; (2) hücre-hücre kavşaklarda e-kaderin lokalizasyon kaybı; kaybıyla tanımlanan EMT belirteçleri ve (3) açık ifadesi,epitel belirteçleri ve mezenkimal belirteçlerinin kazanç. Hücre morfolojik değişiklikler bu bölümde anlatılan hücre kavşaklarda E-kaderin Bölüm 1. immüno-floresans algılama tarif EMT indüksiyon zamanı sırasında 10X büyütmede ışık mikroskobu tarafından yakalanır. EMT markerlerin ifadesi immünolojik işaretleme ile tespit edilir. Ortak epitelial yapıcıları E-kadherin, γ-katenin ve occludin, ve mezenkimal belirteçler, fibronektin, N-kaderin ve vimentin içerir. Imünoblotlarını genel prosedür Bölüm 4&#39;te açıklanmıştır. <ol><li> TAM işlemden geçirilmesi, tohumların, bir 24-yuvalı plakanın bir çukuruna yerleştirilmiş bir 12 mm&#39;lik dairesel bir örtücü cam üzerine 1 x 10 5 hücre bölgesinin 12. günde. </li><li> Önceden ısıtılmış PBS ile tohumlama, aspirat orta ve yıkama hücrelerin 48 saat sonra. </li><li> 15 dakika boyunca ön ısıtılmış% 4 paraformaldehid, hücreler. </li><li> PBS ile hücreler üç kez yıkayın. </li><li> 15 için PBS içinde% 0.2 Triton X-100 ile inkübe hücrelerioda sıcaklığında Min. </li><li> PBS ile hücreler üç kez yıkayın. </li><li> Spesifik olmayan bağlanmayı bloke etmek için oda sıcaklığında 1 saat süreyle% 5 BSA / PBS içinde hücreleri inkübe edin. </li><li> 4 ° C&#39;de 1:50 seyreltme,% 1 BSA / nemli bir oda O PBS / H de birincil antikor E-kaderin hücreleri inkübe edin. Not: Primer antikor seyreltmesi için aralık 1:50 ile 1 arasında, normal olarak: 200, ve her bir antikor için optimal dilüsyon deneysel olarak tespit edilmelidir. </li><li> Birincil antikor süzün ve PBS ile hücreler üç kez yıkayın. </li><li> Oda sıcaklığında 1 saat boyunca 500 oranında seyreltilmiş bir 1: floresan-etiketli sekonder antikor ile inkübe hücreleri. Bu aşama, ışıktan hücreleri korumak. </li><li> İkincil antikor süzün ve üç kez PBS ile yıkayın. </li><li> 10 dakika boyunca 0.1-1 ug / ml DAPI veya Hoechst ile Leke hücreleri. </li><li> PBS ile üç kez hücreleri durulayın. </li><li> Dağı oje ile bir montaj orta damla ve mühür lamelleri ile lamelleri. </li><li> Hücreleri gözlemlemek ve almak63X floresan mikroskop altında görüntüler. </li></ol> Ek antikorlar ile lekeleme EMT fenotipi teyit etmek için yapılabilir. Örneğin, N-kaderin ve α-düz kas aktini mezenkimal fenotip 29,31,32 için boyanmış olabilir. Iskelet yapısı yeniden düzenlenmesi, F-aktin için 11 falloidinle hücrelerin boyanması ile izlenebilir. Buna ek olarak, hücre motilitesi ve hücre ölümü direnç için deneyler, EMT 11 özelliklerini incelemek için gerçekleştirilebilir. Mah-PCR Tahliller kullanma Algılama Splice izoformlannın 3. <ol><li> Primer Tasarım Not: Primer çiftleri dikkatlice nokta bağlantı izoformları amplifiye etmek için tasarlanmış olmalıdır. Atlama ekson, en sık görülen alternatif bağlama olayı, birincil tasarım stratejisini açıklamak için bir örnek olarak gösterilmektedir. Şekil 1A değişken ekson olarak üçüncü ekson, bir dört-ekson ön-mRNA göstermektedir. Ekson İçerme 3 sonuçlarıekson 3&#39;ün atlama ise daha uzun bir bağlantı izoformunun üretimi, daha kısa bir bağlantı izoformunu oluşturur. Primerler, ekson-3 dahil olmak üzere iki izoformu ve ekson-3-atlanan izoformunu ayırt etmek ve bu mRNA&#39;nın toplam transkript tespit etmek için tasarlanmış olması gerekir. <ol><li> Ekson dahil belirlenmesi için, dikkatli bir şekilde değişken ekson dahil izoformunun spesifik amplifikasyonu sağlayan, değişken ekson 3 ve ekson 4 yakın kurucu içinde bir ters primer içinde bir ileri primer tasarımı. Hem ileri hem de tasarım ve genomik DNA veya ön-mRNA &#39;dan çoğaltılmış PCR ürünleri önlemek amacıyla aynı değişken ekson içinde ters primerlerli değil emin olun. </li><li> Özellikle ekson atlama olayları, tasarım amplifiye etmek için, değişken ekson 3 dahil edildiğinde, aksi imha edilecek kurucu ekson 2 ve ekson 4 birleşim yerini kapsayarak tek primer. Bu nedenle yapısal ekson 4&#39;ün içine, diğer birincil tasarım, PCR ürünleri değişken ekson 3 exclud olduğu zaman oluşturulacaked ve ekson 2 ve ekson 4 sonradan birleştirilir kurucu. </li><li> , Genin toplam transkriptlerini algılamak iki kurucu ekson 1 ve 2 içinde primerler Böylece tasarlamak için, PCR ürünleri tüm izoformları amplifiye edilmiştir. </li><li> Emin, tüm primerler için erime sıcaklığı (Tm) yaklaşık olarak 55 ° C arasında, her bir primer uzunluğu yaklaşık 18-22 nükleotid olduğunu olun ve PCR ürünlerinin boyutunu 80 ile 150 baz çiftidir. </li></ol></li><li> RNA ekstraksiyonu: (Malzeme Tabloya bakınız), toplam RNA izolasyon kiti kullanılarak RNA ekstrakte edin. <ol><li> 350 ul RNA lizis tampon hücreleri toplayın. </li><li> Lizat% 70 etanol içinde 350 ul ekleyin ve iyice karıştırın. </li><li> RNA saflaştırma Numuneyi kolona aktarın. </li><li> 1 dakika ve atma flow-through için 10.000 x g&#39;de santrifüj. </li><li> RNA, yıkama tamponu II ile bir kez RNA, yıkama tamponu I 500 ul ile ve iki kez de sütun yıkayın. </li><li> RNA, kalıntı yıkama tamponunu çıkarın2 dakika süreyle maksimum hızda santrifüj ile kolondan II. </li><li> , Yeni bir 1.5 ml&#39;lik bir tüp içine transfer sütun sütun matrisine merkezinde 50 ul nükleaz içermeyen su ilave edin ve en yüksek hızda santrifüj ile RNA aynlır. </li></ol></li><li> Birinci-şerit cDNA sentezi <ol><li> UV absorpsiyon ölçümü ile konsantrasyon ve RNA kalitesini belirler. NOT: Kaliteli RNA yaklaşık 2,0 260 nm / 280 nm absorbans oranı olmalıdır. </li><li> 20 ul nihai hacim içinde 250 ng toplam RNA, 0.05 ug tesadüfi heksamer öncüllerin, 50 pmol MgCl2, 10 pmol dNTP, 2 ul 5 x GoScript tamponu ve 1 ul RT enzimi ters transkriptaz (RT) reaksiyon ayarlayın. </li><li> RT enzimin inaktivasyonu için 5 dakika boyunca 70 ° C &#39;de inkübasyon, ardından 1 saat için 42 ° C RT reaksiyonları gerçekleştirmek. </li></ol></li><li> Gerçek zamanlı PCR <ol><li> 10 ul SYBR yeşil ana karışımı oluşur 20 ul reaksiyonları kurmak,0.1-1.0 ul cDNA şablon ve 5 ileri pmol ve geri primerler. </li><li> Aşağıdaki adımlardan 40 döngü ile gerçek-zamanlı PCR çalıştırın: 95 ° C&#39;de 10 saniye denatürasyon, 10 saniye için 58 ° C tavlama, 30 saniye için 60 ° C uzatma. PCR reaksiyonları, üç kopya halinde gerçekleştirin. </li><li> Ayrışma eğrileri kontrol edin ve bir tek tepe her astar kümesi için gözlenen emin olun. </li><li> Amplifikasyon eğrileri ikinci türev değerleri hesaplanarak ölçüm döngüsü (Cq) değerlerini elde edin. </li><li> &quot;Delta Cq (ΔΔCq &#39;), aşağıdaki formül ile gösterildiği gibi, yöntem kullanılarak indüklenmemiş numunesine kıyasla neden örneklerdeki hedef mRNA nispi miktarı (RQ) hesaplayın. Bir referans olarak, örneğin GAPDH veya TBP olarak temizlik genleri kullanın. </li></ol></li></ol><img alt="Denklem 1" fo:content-width="3in" src="/files/ftp_upload/51845/51845eq1.jpg" /> Daha doğru bağlantı izoformlannın göreli değerlerini hesaplamak için to dikkate alınmalıdır çoklu referans gen kullanır. Sonuçlar Pfaffl ve ark 33,34 tarafından tarif edilen modifiye mutlak değeri, ölçüm yöntemi kullanılarak analiz edilebilir. Protein Düzeyinde Splice izoformlannın 4. incelenmesi <ol><li> 100 ul buz gibi soğuk RIPA tampon maddesi içinde hücreler toplanır. </li><li> Yaklaşık 10 dakika boyunca buz üzerinde lizatları ayarlayın. </li><li> 4 ° C&#39;de 10 dakika boyunca 14,000 x g&#39;de santrifüje ve süpernatan toplamak. </li><li> Bradford protein konsantrasyonu tahlilleri ile ölçülür. </li><li> SDS yükleme tamponu içinde protein örnekleri seyreltin ve 10% SDS-PAGE jelleri içine proteinlerin 20-40 ug yükleyin. </li><li> 1.5 saat için 20 mA&#39;da SDS-PAGE jelleri çalıştırın. </li><li> 85 V, 3 saat boyunca 4 ° C&#39;de nemli bir transfer sistemi olarak PVDF membranlarına transfer proteinleri hedef proteinlerin moleküler ağırlığa göre transfer süresi ayarlayın. </li><li> 30 dakika oda sıcaklığında 1 saat için% 5 yağsız süt içinde Blok membran. </li><li> Membran wi inkübe4 ° CO / K bir birincil antikor th. 200 ve 1: 5000, deneysel olarak her bir antikor için optimum seyreltme oranını belirleyen birincil antikor seyreltmesi için aralık normalde 1 arasındadır. Bu protokolde kullanılan antikorların seyreltme &quot;özel reaktifler ve ekipman Tablo&quot; bulunabilir. <ol><li> Bağlantı izoform tespit etmek için, belirli bir izoformunu tanıyan spesifik antikorlar kullanılır. Seçenek olarak ise, kurucu ekson kodlama bölgesi epitopunu tanıyan bir antikorun kullanılması ve bunların, protein farklı boyutlarına göre eş zamanlı olarak bağlantı izoform algılar. </li><li> Aynı zamanda, EMT belirteçleri için immüno-lekeleme EMT izler. Mezenkimal belirteçler olarak e-kaderin, γ-katenin ve epitel belirteç olarak occludin ve fibronektin, N-kaderin ve vimentin. </li></ol></li><li> 5 dakikalık bir aralıkla TBS-T (50 mM Tris, 150 mM NaCI,% 0.05 Tween 20, pH 7.4) ile yıkayın zar üç kez. </li><li> HRP-konjuge sekonder ant ile membran inkübeoda sıcaklığında 1 saat için 10,000 seyreltmede bir 1: ibody. </li><li> 5 dk aralıklarla TBS-T ile yıkayın membran üç kez. </li><li> Bir kimyasal ışıldama tespit sistemi ile proteinin görsel ve otoradyografi filmine maruz membran. </li></ol>

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The authors declare that they have no competing financial interests.

Burada tarif edilen prosedür, bir uyarılabilir EMT modelinde alternatif bağlantısının belirlenmesini sağlar. Bağlantı izoform ekspresyon örneğin dinamik değişiklik EMT zaman süreci boyunca yakalanabilir gibi. Un-bağlı hücre hatlarının farklı genetik temel gereksiz alternatif eklemeler etkileyebilir, çünkü bu yöntem alternatif splicing karşılaştırılması için farklı epithelial- da mezenkimal-temsil eden hücre çizgilerinin kullanımı üzerinde avantajlara sahiptir. Ancak, EMT başarılı ...

Epitel-mezenkimal geçiş (EMT) Embriyogenesis sırasında organ morfogenetiğine ve doku yenileme sürücüler gelişimsel bir programdır. Anormal aktive edildiğinde, EMT tümör metastazı ve organ fibrozis 1,2 teşvik etmektedir. Zorlayıcı çalışmalar cobble- kaybıyla sonuçlanan adherens birleşme proteini E-kaderin ifadesini bastırmaktadır örneğin Twist, salyangoz ve Zeb gibi çeşitli transkripsiyon faktörleri, tarafından tanımlanan, EMT sürecinde transkripsiyon düzenlemesi önemini tarif etmişlerdir epitel morfolojisi ve iğ biçimli mezenkimal fenotip 3-8 kazancı gibi taş. RNA&#39;lar genom analizi ile yapılan son çalışmalar yapıştırma desenler epitel veya mezenkimal fenotiplerle 9,10 ya ile ilişkili genlerin bir grup var olduğunu ortaya koymuştur. Laboratuarımızda çalışmak işlevsel alternatif eklemeler ve EMT bağlı. Hücre yüzey adezyon molekülü CD44 inceleyerek, CD44 altern gösterdiative yapıştırma sıkı EMT sırasında düzenlenir, ve daha da önemlisi, nedensel anahtarlama bu CD44 ekleme izoformdur 11 EMT katkıda bulunmaktadır. Alternatif 12-14 raptedilmektedir insan çoklu-ekson genlerin% 95 kadar alternatif yapıştırma, gen regülasyonu bir yaygın ve korunmuş modelini temsil eder. Tek bir genin birden fazla protein ürünlerinin üretilmesi ile beraber, alternatif birleştirme insan genomuna karmaşıklık bir katman eklenerek protein çeşitliliği için önemli bir mekanizma oluşturur. Bu nedenle, alternatif uç uca eklenmesi düzensizliği, potansiyel olarak hastalığa neden olan insan derin biyolojik etkileri yol açabilir. Nitekim, hastalıklarda anormal alternatif yapıştırma spliceosom makine kodlayan genlerde mutasyonlar sık miyelodisplastik sendromlarda 26-28 bulunan son bulgular da dahil olmak üzere, bir yılda 15-25 rapor edilmiştir. Bu nedenle, alternatif olarak eklenmiş i tespiti için güvenilir yöntemler geliştirmeksoforms EMT de dahil olmak üzere çeşitli biyolojik süreçlerin çalışmada büyük önem taşımaktadır. Burada bir uyarılabilir EMT modeli kullanılarak alternatif ekleme değişiklikleri algılamak için bir protokol sağlar. Bağlantı izoformları PCR primerlerini tasarlama ve tespit edilmesi için yöntemler, EMT sırasında alternatif splicing çalışma için değil, aynı zamanda diğer biyolojik süreçlerde alternatif splicing çalışma için sadece uygundur. EMT sırasında alternatif eklemeler araştıran iyi böylece kanser metastazı tedavi etmek için etkili stratejilerin geliştirilmesini kolaylaştırmak, EMT ve tümör metastazı mekanizmalarını anlamak için zorunludur.

<table border="0" cellpadding="0" cellspacing="0" width="1167">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Name of the reagent</td>
			<td style="width:183px;">Company</td>
			<td style="width:123px;">Catalogue number</td>
			<td style="width:564px;">Comments (optional)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-hydroxytamoxifen</td>
			<td>Sigma</td>
			<td>H7904</td>
			<td>Make stock solution by dissolving in ethanol to 200&#956;M, and keep at -20&#8451; protected from light.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">E.Z.N.A. Total RNA Isolation kit</td>
			<td>Omega Bio-Tek</td>
			<td>R6731</td>
			<td>Total RNA isolation kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GoScript Reverse Transcription System</td>
			<td>Promega</td>
			<td>A5001</td>
			<td>Reagent for qRT-PCR assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GoTaq qPCR Master Mix</td>
			<td>Promega</td>
			<td>A6002</td>
			<td>Reagent for qRT-PCR assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LightCycler 480 Real-Time PCR System</td>
			<td>Roche</td>
			<td></td>
			<td>Equipment for qRT-PCR assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD44 antibody</td>
			<td>R&#38;D Systems</td>
			<td>BBA10</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">E-cadherin antibody</td>
			<td>Cell Signaling Technology</td>
			<td>4065</td>
			<td>1:2500 dilution for immunoblotting; 1:50 dilution for immunofluorescence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#947;-catenin antibody</td>
			<td>Cell Signaling Technology</td>
			<td>2309</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">occludin antibody</td>
			<td>Santa Cruz Biotechnology Inc.</td>
			<td>sc-5562</td>
			<td>1:500 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fibronectin antibody</td>
			<td>BD Transduction Laboratories</td>
			<td>610077</td>
			<td>1:5000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-cadherin antibody</td>
			<td>BD Transduction Laboratories</td>
			<td>610920</td>
			<td>1:2000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">vimentin antibody</td>
			<td>NeoMarkers</td>
			<td>MS-129-p1</td>
			<td>1:500 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GAPDH antibody</td>
			<td>Millipore Corporation</td>
			<td>MAB374</td>
			<td>1:10000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amasham ECL Western blotting detection reagent</td>
			<td>GE Health Life Science</td>
			<td>RPN2209</td>
			<td>Chemiluminescence system</td>
		</tr>
	</tbody>
</table>

detection of alternative splicing during epithelial-mesenchymal transition

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various physiological and pathological processes. Here we describe a strategy to detect alternative splicing during EMT using an inducible EMT model by expressing the transcription repressor Twist. EMT is monitored by changes in cell morphology, loss of E-cadherin localization at cell-cell junctions, and the switched expression of EMT markers, such as loss of epithelial markers E-cadherin and &#947;-catenin and gain of mesenchymal markers N-cadherin and vimentin. Using isoform-specific primer sets, the alternative splicing of interested mRNAs are analyzed by quantitative RT-PCR. The production of corresponding protein isoforms is validated by immunoblotting assays. The method of detecting splice isoforms described here is also suitable for the study of alternative splicing in other biological processes.

Yukarıda tarif edilen prosedürler sırasında EMT alternatif eklemeler tespit etmek için güçlü bir yöntem sağlar. Twist kaynaklı EMT sırasında CD44 ekleme izoformu anahtarlama Temsilcisi sonuçları bir örnek olarak aşağıda verilmiştir. HMLE / Büküm-ER hücrelerinde Bükülme kaynaklı EMT uzatılmış bir fibroblastik fenotipe epitel fenotip gibi çakıl taşı bir geçiş (Şekil 2A), epitelyal işaretleri E-kadherin, γ-katenin ve occludin ve yokluğu il...

Watch this Scientific Journal Video about Detection of Alternative Splicing During Epithelial-Mesenchymal Transition at JoVE.com

Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Alternative splicing regulation has been shown to contribute to the epithelial-mesenchymal transition (EMT), an essential cellular program in various physiological and pathological processes. Here we describe a method utilizing an inducible EMT model for the detection of alternative splicing during EMT.

Epitel-Mezenkimal Geçiş sırasında alternatif birleştirme Algılama

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various ...

detection-alternative-splicing-during-epithelial-mesenchymal

Research

JoVE Journal

Biology

12.8K Görüntüleme.  Northwestern University Feinberg School of Medicine. Alternative splicing regulation has been shown to contribute to the epithelial-mesenchymal transition (EMT), an essential cellular program in various physiological and pathological processes. Here we describe a method utilizing an inducible EMT model for the detection of alternative splicing during EMT.

Video: Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Light/dark Transition Test for Mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

Fareler için açık / koyu Geçiş Testi

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

Biyoloji

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

Protein Ubikitinasyon Tespiti

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Optimized PCR-based Detection of Mycoplasma

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. Although mycoplasma do not usually kill contaminated cells, they are difficult to detect and can cause a variety of effects on cultured cells, including altered metabolism, slowed proliferation and chromosomal aberrations. In short, mycoplasma contamination compromises the value of those cell lines in providing accurate data for life science research.
The sources of mycoplasma contamination in the laboratory are very challenging to completely control. As certain mycoplasma species are found on human skin, they can be introduced through poor aseptic technique. Additionally, they can come from contaminated supplements such as fetal bovine serum, and most importantly from other contaminated cell cultures. Once mycoplasma contaminates a culture, it can quickly spread to contaminate other areas of the lab. Strict adherence to good laboratory practices such as good aseptic technique are key, and routine testing for mycoplasma is highly recommended for successful control of mycoplasma contamination.
PCR-based detection of mycoplasma has become a very popular method for routine cell line maintenance. PCR-based detection methods are highly sensitive and can provide rapid results, which allows researchers to respond quickly to isolate and eliminate contamination once it is detected in comparison to the time required using microbiological techniques. The LookOut Mycoplasma PCR Detection Kit is highly sensitive, with a detection limit of only 2 genomes per &mu;l. Taking advantage of the highly specific JumpStart Taq DNA Polymerase and a proprietary primer design, false positives are greatly reduced. The convenient 8-tube format, strips pre-coated with dNTPs, and associated primers helps increase the throughput to meet the needs of customers with larger collections of cell lines.
Given the extreme sensitivity of the kit, great care must be taken to prevent inadvertent contamination of samples and reagents. The step-by-step protocol we demonstrate highlights the precautions and practices required for reliable mycoplasma detection. We also show and discuss typical results and their interpretation. Our goal is to ensure the success of researchers using the LookOut Mycoplasma PCR Detection Kit.

The LookOut Mycoplasma PCR Detection Kit utilizes the polymerase chain reaction (PCR), which is established as the method of choice for highest sensitivity in the detection of Mycoplasma, Acholeplasma, and Ureaplasma contamination in cell cultures and other cell culture derived biologicals.

Mycoplasma optimize edilmiş PCR tabanlı Algılama

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

Mitokondri İzole Fare Heart Geçirgenliği Geçiş Gözenek Açma Multi-parametre ölçümü

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

Saç Rejenerasyon sırasında epitelyal-mezenkimal Sinyal Rapid Genetik Analizi

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Mezenkimal Geçiş indüksiyon ve Epitel Analizi

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

İnsan Pluripotent için alternatif Kültürleri Hücre Üretim, Bakım ve Genetik Analizi Kök

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually untranslated &#8212; first exons. Bcrp1 (Abcg2), the murine orthologue of the ABC transporter Breast Cancer Resistance Protein (BCRP, ABCG2), has at least four alternative promoters that are designated by the corresponding four alternative first exons produced: E1U, E1A, E1B, and E1C. Herein, in-silico protocols are presented to predict alternative promoter usage for Bcrp1. Furthermore, reporter assay methods are described to produce reporter constructs for alternative promoters and to determine the functionality of putative promoters upstream of the alternative first exons that are identified.

With the murine ABC transporter Bcrp1 (Abcg2) as an example, in-silico protocols are presented to detect alternative promoter usage in genes expressed in mouse tissues, and to evaluate the functionality of the alternative promoters identified using reporter assays.

Yöntemler Fare Bcrp1 Alternatif Promoter Kullanımı ve Transkripsiyonal Yönetmeliği Discover

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually ...

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, especially cells in suspension such as hematopoietic stem cells (HSCs), remains limited due to the requirement of a high cell number during sample preparation. There are a few cytospin or monolayer approaches for TEM analysis from scarce samples, but these approaches fail to get significant quantitative data from the limited number of cells. Here, an alternative and novel approach for sample preparation in TEM studies is described for rare cell populations that enables quantitative analysis.
A relatively low cell number, i.e., 10,000 HSCs, was successfully used for TEM analysis compared to the millions of cells typically used for TEM studies. In particular, Evans blue staining was performed after paraformaldehyde-glutaraldehyde (PFA-GA) fixation to visualize the tiny cell pellet, which facilitated embedding in agarose. Clusters of numerous cells were observed in ultra-thin sections. The cells had a well preserved morphology, and the ultra-structural details of the Golgi complex and several mitochondria were visible. This efficient, easy and reproducible protocol allows sample preparation from a low cell number and can be used for qualitative and quantitative TEM analysis on rare cell populations from limited biological samples.

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

TEM analizi için düşük Hücre sayısı ile örnek hazırlanması için alternatif bir yaklaşım

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&#946; Signaling

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with organ fibrosis. In these conditions, differentiated endothelial cells acquire mesenchymal-like phenotypes. This process is called endothelial-mesenchymal transition (EndMT) and is characterized by downregulation of endothelial markers, upregulation of mesenchymal markers, and morphological changes. EndMT is induced by several signaling pathways, including transforming growth factor (TGF)-&#946;, Wnt, and Notch, and regulated by molecular mechanisms similar to those of epithelial-mesenchymal transition (EMT) important for gastrulation, tissue fibrosis, and cancer metastasis. Understanding the mechanisms of EndMT is important to develop diagnostic and therapeutic approaches targeting EndMT. Robust induction of EndMT in vitro is useful to characterize common gene expression signatures, identify druggable molecular mechanisms, and screen for modulators of EndMT. Here, we describe an in vitro method for induction of EndMT. MS-1 mouse pancreatic microvascular endothelial cells undergo EndMT after prolonged exposure to TGF-&#946; and show upregulation of mesenchymal markers and morphological changes as well as induction of multiple inflammatory chemokines and cytokines. Methods for the analysis of microRNA (miRNA) modulation are also included. These methods provide a platform to investigate mechanisms underlying EndMT and the contribution of miRNAs to EndMT.

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&amp;#946; Signaling

A protocol for in vitro induction of endothelial-mesenchymal transition (EndMT), which is useful for investigating cellular signaling pathways involved in EndMT, is described. In this experimental model, EndMT is induced by treatment with TGF-&#946; in MS-1 endothelial cells.

Mezenkimal endotel geçiş büyüme faktörü β sinyal dönüşüm tarafından indüklenen moleküler Analizi

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with ...