J. Heijmans is supported by a stipendium from the Dutch cancer foundation (KFW). G.R. van den Brink is supported by funding from the European Research Council under the European Community&#8217;s Seventh Framework Program (FP7/2007-2013)/European Research Council grant agreement number 241344 and by a VIDI grant from the Netherlands Organization for Scientific Research (GvdB).

Transfeksiyon Reaktif olarak polietilenimin (PEI) hazırlanması 1. <ol><li> H2O, 100 ml PEI&#39;nın yaklaşık 150 mg çözülür </li><li> Solüsyon berrak hale gelir ve tam olarak çözülene kadar karıştırın kadar HCI eklenerek pH 7.4 çözeltisi ayarlayın. Bu durum, 10 ila 60 dakika sürer ve 1 mg / ml bir son konsantrasyona kadar su ilave edilebilir. </li><li> Ne zaman açık, 5 ml hacimde ° C derin dondurucuda bir -80 steril 0.22 mikron filtre ve mağaza üzerinden PEI çözüm filtre. </li></ol> Lentiviral Parçacıkların 2. Üretim 1. Gün: <ol><li> 162 cm2 şişeye ya da hücre hattı kültürü ortamında büyük bir petri (% 10 FCS,% 1 penisilin / streptomisin ve 2 mM glutamin takviyesi ile desteklenmiş DMEM) içinde% 60 -80% konfluansa Split HEK293T hücreleri. </li></ol> Gün 2: <ol start="2"><li> Birlikte ekleyerek toplam plazmid DNA 45 ug içeren DNA transfeksiyon çözüm hazırlayınLentiviral ambalaj vektörleri (7 pVSVg ug, pRSV rev 5 ug; 13 pMDL ug) ilgi faiz veya shRNA genini kodlayan lentiviral plazmid ve 20 ug. DMEM ile 1 ml &#39;lik bir hacme kadar ayarlayın. </li><li> DMEM 930 ul 1 mg / ml bir PEI 90 ul ekleyerek PEI transfeksiyon çözelti hazırlanır ve oda sıcaklığında 5 dakika inkübe edilir. </li><li> PEI solüsyonuna DNA transfeksiyon solüsyonu ekleyin. Vorteks birkaç kez ters çevirin ve DNA transfeksiyon çözelti elde etmek için, oda sıcaklığında 5 dakika inkübe veya. </li><li> HEK293T hücrelerine, DNA transfeksiyon solüsyonu 2 ml damla ve 37 ° C nemlendirilmiş bir hücre kültürü kuluçka makinesi içinde, 4 saat boyunca inkübe edilir. </li><li> 4 saat sonra, PEI çıkarmak için kültür ortamı yenileyin. Bu yeni orta eklemeden önce Hücreleri yıkamak için gerekli değildir. NOT: 4 saat daha uzun PEI olan sitotoksik ve inkübasyon süreleri HEK293T hücrelere zarar verebilir. </li></ol> 4. Gün: <ol start="7"><li> Supe değiştirinyeni kültür ortamı ile rnatant. Süpernatant (virüs içeren) tutun; Bu aşamada 2.10 kullanılacaktır. </li><li> 15 ml&#39;lik bir şişe içinde süpernatant koyun. 500 x g&#39;de 5 dakika boyunca ölü hücreleri, santrifüj kaldırın. </li><li> Büyük bir 60 ml şırınga kullanılarak 0.45 um filtre içinden yüzer itin. 4 ° C &#39;de bir gece boyunca muhafaza ediniz. </li></ol> 5. Gün: <ol start="10"><li> Süpernatant ikinci parti toplayın; adımlarda 2.8-2.9 gibi santrifüj ve filtre. </li><li> 90 dakika boyunca bir ultrasantrifüjdeki 50.000 x g&#39;de adımlar 2.9 ve ultrasantrifüjdeki tüpleri 2.10 ve santrifüj süpernatantlar Havuz. </li><li> Çok dikkatli ultrasantrifüjdeki tüpleri içeren kapsüller çıkarın ve santrifüj içinde tüp yönünü hatırlayarak, bir laminer akış kaputu koymak. </li><li> Topak, tüp üst tarafında olmasını sağlayacak bir şekilde, dikkatli bir ultra santrifüj tüpüne ve dökme sıvı ortam tutan açık kapsül. Viral pelet görselleştirmek için zor olabilir çünkü, üzerinde ne hatırlıyorumtüpün yan bir pelet oluşmuş olacaktır. Bir mikropipet alın ve ultrasantrifüjdeki tüpün alt tarafında görünür opak kahverengi pelet kışkırtmak dikkat çekerken orta son bit çıkarın. </li><li> 10 mM nikotinamid, 10 uM Chir99021, 10 uM Y27632 ve 8 ug / ml polibren ile takviye organoid kültür ortamında 500 ul bu pelletini. Yüksek titre virüs doğrudan organoids Transduct için kullanılan bu yana bu ortamda tabanda önemlidir. </li><li> İsteğe bağlı olarak: -80 ° C&#39;de 250 ul&#39;lik iki seri olarak alınmıştır, bu ortamda virüs dondurma. </li></ol> Organoids 3. lentiviral İletimi Gün 0: <ol><li> Yaklaşık 50 küçük organoids elde etmek amacıyla, yeni bir kuyunun içine transdüksiyon için iki gün önce organoids tam 0.95 cm 2 kuyu bölün. Daha önce yayınlanan bir protokolün 1 (Şekil 3A, B) işlemine göre organoids Böl. </li><li> Kistik aşırı çoğalma crypts (Şekil 3C) elde etmek için 10 uM Chir99021 ve 10 mM nikotinamid ile kültür ortamı Organoid Supplement. Not: taze bölme organoids Chir99021 varlığında büyütülür kistik kript iyi gelişir. </li></ol> Gün 2: <ol start="3"><li> Böylece P1000 mikropipet ile karışımı bozarak, yukarı pipetleme ve matrigel ve orta aşağı Hasat organoids. 15 ml&#39;lik bir tüp içinde karışımın yerleştirin. </li><li> Distal açıklık eritilerek azalmış olan Pasteur pipeti kullanılarak daha bozabilir. Santrifüj organoids 100 x g&#39;de 5 dakika boyunca pelet için. NOT: santrifüj büyüklüğüne bağlı olarak, farklı bir santrifüj hızı kullanın. Bu organoids karışımdan ayrılır bozulduğu olan tam hız bulmak için gereklidir. </li><li> Süpernatantı ve (% 0.25 tripsin benzeri) önceden ısıtılmış 1x tripsin 500 ul ekleyin. Tripsin içinde organoids yeniden süspanse ve inkübe37 ° C&#39;lik bir su banyosu içerisinde 3 dakika. </li><li> Hücre hattı kültürü ortamı içinde 3.5 ml tripsin inaktive (DMEM,% 10 FCS,% 1 penisilin / streptomisin ve glutamin ile takviye edilmiş). 500 x g&#39;de 5 dakika boyunca santrifüje. </li><li> Orta yaklaşık 20 ul pelet bırakmak süpernatantı. Bir 48-kuyu plaka üzerinde kuyuya ortamının bu son küçük miktarda organoids aktarın. </li><li> Adım 2.14, tekrar süspansiyon tarif edilir ve 3.10: adım olarak iletim ortamı içinde yüksek titre lentivirüs ekleyin. Düşük transdüksiyon etkinliğini karşılaşmak, 3.9 eklenebilir adım. Not: etkin iletimi için, tipik olarak organoids bir oyuğuna yüksek titre lentivirüs 250 ul bir kısım ilave edin. Bu protokolün adım 2&#39;de tarif edildiği gibi, tek bir üretim Hasat lentivirüs% 50&#39;sini oluşturur. </li><li> İsteğe bağlı olarak: 32 ile ilgili, bir önceden ısıtılmış bir santrifüj içinde 48 gözenekli plaka ihtiva organoids koyarak spinoculation yerine, transdüksiyon etkinliğinin arttırılması için6, C ve 1 saat süre ile 600 x g&#39;de döndürün. </li><li> Kültürü inkübatöründe organoid-virüs konacak ve transdüksiyonunu izin vermek için bir kültürü inkübatöründe 37 ° C&#39;de 1 saat süreyle inkübe edin. </li><li> İsteğe bağlı olarak: bundan başka, iletim etkinliğini geliştirmek kültür inkübatörü içinde 37 ° C&#39;de 3 saat daha organoids kuluçkalanması. </li><li> Organoids peletleştirmek için 850 x g&#39;de 5 dakika boyunca bir mikrosantrifüj içinde bir mikrosantrifüj tüpü ve santrifüj içine transfer organoid kültür ortamının 1 ml ilave edilir ve organoid-virüs karışımı tekrar süspansiyon. </li><li> Süpernatantı ve buz-soğuk matrigel 20 ul pelletini. Daha sıcak olduğunda malzeme katılaşır beri, birkaç kez soğuk PBS yukarı pipetleme ve buz aşağı soğutulmuş olan pipet uçları kullanın. </li><li> 48 kuyu plakasında iyi olarak ortasında damlacık koyun ve katılaşmaya 15 dakika için 37 ° C &#39;de kültür inkübatörü içinde inkübe edilir. </li><li> 15 dakika sonra, dikkatli bir şekilde ilave edilmiş organoid kültür ortamında 250 ul10 mM nikotinamid, 10 uM Chir99021 ve 10 uM Y27632. Küçük kesintiye organoid parçaları 24 saat içinde küçük kistik organoids içine oluşturacak. </li></ol> 5. Gün: <ol start="16"><li> Orta yenileme ve bir seçim antibiyotiği ile ek (puromisin için, 4 ug / ml kullanımı). </li></ol> Gün 7: <ol start="17"><li> Seçim antibiyotik ile desteklenmiş standart organoid kültür ortamı için orta değiştirin. Not: geliştirici Chir99021 çekilmesinden sonra 2-3 hafta tamamlanır. Seçimden sonra, organoids seçimi antibiyotiksiz yetiştirilebilir. </li></ol> Kantitatif RT-PCR veya Mikrodizi 4. organoid RNA Preparasyonu <ol><li> RNA preparasyonu için, üreticinin protokolüne göre, ticari bir kit kullanılır. Organoids orta çıkarın ve düz matrigel içeren organoids kubbesi üzerindeki β-mercaptoethanol ile takviye tampon RLT 350 ul ekleyin. Materyali yeniden süspanseP1000 mikropipet kullanarak pipetleme RLT al. </li><li> Üreticinin protokolüne göre başka bir RNA hazırlık yapın. </li><li> İsteğe bağlı olarak: üreticinin protokolüne uygun olarak 50 ug toplam RNA, ilk giriş gerektiren Ovation Pico WTA sistemi kullanılarak amplifikasyon RNA verimi artar. </li><li> İsteğe bağlı olarak: RNA, mikrodizi önce kalite kontrolü için, üreticinin protokolüne uygun olarak 8.5 ya da daha çok (Şekil 5) bir RNA bütünlüğü numarası (RIN) hedefleyen bir ökaryot toplam RNA, nano çipini kullanan bir 2100 Bioanalyzer çalışacak örnekleri. </li></ol> Parafin Yerleştirilmiş ve İmmünohistokimya için 5. İşleme Organoids <ol><li> Önceki gömme organoid başlamasından parafin sıvı tutmak için 70 ° C &#39;de delikli inkübatör bağlantı 12 mm çaplı cam tüpler bir alüminyum blok önceden ısıtın. </li><li> Bozulmamış gömülü organoids bırakarak, tam yetiştirilen organoids tek bir kuyu alın ve orta kaldırmak. </li><li> 1 ml ilave edilir% 4 paraformaldehid, PBS içinde düz iyi ve 1 saat her yerde, 4 ° C&#39;de gece boyunca sabitlemek. </li><li> Buz soğukluğunda PBS, 1 ml paraformaldehid yerine. İsteğe bağlı olarak: bu aşamadan sonra, 4 ° C&#39;de 1 hafta kadar PBS içinde sabit organoids tutun. </li><li> 1 ml PBS içinde yeniden süspanse edin ve sabit organoids bir cam şişe içine koyun. </li><li> , Organoids 1 dakika boyunca dibine çöker olsun PBS süzün ve eozin çözeltisinin damlacıklarının bir çift gömme işlemi boyunca organoids görselleştirme sağlamak için çözülmüş olan% 70 etanol ile değiştirin. </li><li> Oda sıcaklığında% 70 etanol içinde organoids bırakın. 30 dakika sonra, dikkatli bir şekilde, çünkü hafif pembe eozin renk gözle organoids görselleştirmek için güçlü olmak, boşaltılarak% 70 etanol çıkarın. % 96 etanol ile gömme çözüm değiştirin. </li><li> Adımı yineleyin 5.7, yanında bir gömme çözüm yerini her zaman. % 70 etanol,% 90 etanol,% 96 etanol,% 100 etano: daha sonra, aşağıdaki çözeltiler ile organoids geçirinmetanol,% 100 etanol, ksilen, ksilen. </li><li> Son ksilen yıkama Durusu ve tüpe parafin dökün. 30 dakika boyunca 70 ° C&#39;de önceden ısıtılmış alüminyum bloğunun Tüp hemen koyun ve yeni, temiz parafin ile parafin değiştirin. </li><li> Geniş açıklığı bulunan bir önceden ısıtılmış Pasteur pipet kullanarak parafin blok kalıp içine kapalı parafin ve pipet organoids dökün. Bunsen beki kullanarak Pasteur pipet sıcak tutun. Sıvı parafin, küçük bir tabaka halinde parafin blok kalıp tüm organoids yerleştirin. </li><li> Mümkün olduğunca, bir ısıtılmış diseksiyon iğne kullanılarak parafin blok kalıp merkezine doğru tüm organoids işlemek için deneyin. Bunsen beki kullanarak diseksiyon iğne sıcak tutun. </li><li> Kalıptaki organoids lokalizasyonu tatmin edici olduğunda, soğuk kalıp biraz parafin tabakası katılaşmaya. </li><li> Üst daha fazla parafin dökülerek blok bitirin ve bir standart histolojik gömme kaseti ekleyin. </li></ol>

<ol>
	<li>Sato, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche.">Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.</a> Nature. 459 (7244), 262-265 (2009).</li><li>Al-Nafussi, A. I., Wright, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+epidermal+growth+factor+(EGF)+on+cell+proliferation+of+the+gastrointestinal+mucosa+in+rodents.">The effect of epidermal growth factor (EGF) on cell proliferation of the gastrointestinal mucosa in rodents.</a> Virchows Archiv. B, Cell Pathology Including Molecular Pathology. 40 (1), 63-69 (1982).</li><li>Haramis, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+crypt+formation+and+juvenile+polyposis+on+BMP+inhibition+in+mouse+intestine.">De novo crypt formation and juvenile polyposis on BMP inhibition in mouse intestine.</a> Science. 303 (5664), 1684-1686 (2004).</li><li>Zhao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=R-spondin1,+a+novel+intestinotrophic+mitogen,+ameliorates+experimental+colitis+in+mice.">R-spondin1, a novel intestinotrophic mitogen, ameliorates experimental colitis in mice.</a> Gastroenterology. 132 (4), 1331-1343 (2007).</li><li>Schwank, G., Andersson-Rolf, A., Koo, B. K., Sasaki, N., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+BAC+transgenic+epithelial+organoids.">Generation of BAC transgenic epithelial organoids.</a> PLoS One. 8 (10), e76871 (2013).</li><li>Koo, B. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+gene+expression+in+primary+Lgr5+organoid+cultures.">Controlled gene expression in primary Lgr5 organoid cultures.</a> Nature Methods. 9 (1), 81-83 (2012).</li><li>Heijmans, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ER+stress+causes+rapid+loss+of+intestinal+epithelial+stemness+through+activation+of+the+unfolded+protein+response.">ER stress causes rapid loss of intestinal epithelial stemness through activation of the unfolded protein response.</a> Cell Rep. 3 (4), 1128-1139 (2013).</li><li>Roe, T., Reynolds, T. C., Yu, G., Brown, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+murine+leukemia+virus+DNA+depends+on+mitosis.">Integration of murine leukemia virus DNA depends on mitosis.</a> The EMBO Journal. 12 (5), 2099-2108 (1993).</li><li>Lau, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lgr5+homologues+associate+with+Wnt+receptors+and+mediate+R-spondin+signalling.">Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling.</a> Nature. 476 (7360), 293-297 (2011).</li><li>Bahnson, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrifugal+enhancement+of+retroviral+mediated+gene+transfer.">Centrifugal enhancement of retroviral mediated gene transfer.</a> Journal Of Virological Methods. 54 (2-3), 131-143 (1995).</li></ol>

The authors have nothing to disclose.

Mevcut video protokolü primer bağırsak epiteli ve kantitatif RNA teknikleri ve immünohistokimya kullanılarak, bu organoids aşağı doğru analizden organoids lentiviral iletimini tarif etmektedir. Lentiviral transdüksiyon genellikle kültür plakaları içinde yapışık veya yüzen hücrelerin içinde gerçekleştirilir. Organoids bölgesinin üç boyutlu yapısı, viral parçacıklar tarafından nüfuz onları zor hale yana etkinliğini artırmak için bir dizi yöntem kullanılır....

bağırsak epitel kanser ve kök hücreleri üzerinde araştırma geniş ilgi çekmek için neden olmuştur en hızlı çoğalan vücut dokuları biridir. 2009 yılında bir teknik 3 boyutlu bir yapı 1 korunması, matrigel küçük bağırsak kript uzun ömürlü kültürlerini oluşturmak için yayınlandı. Bu yapılar, bağırsak organoids orta BMP-sinyal yolu önleyicisi Noggin (Nog) de dahil olmak üzere tanımlanmış bir büyüme faktörü, bir dizi ile takviye çevreleyen, standart teknikler kullanılarak kültür edilebilir olarak adlandırılan 1 (Rspo1) rspondin Wnt-sinyal yolu güçlendirici ve epidermal büyüme faktörü (EGF) tüm bağırsak proliferasyonunu 2-4 arttırdığı bulunmuştur. Organoids, kök hücre hiyerarşi korumuştur, bunlar olmayan mutasyona olduğunu yönleriyle geleneksel kanser hücre hatları aşmak doğmakta olan küçük int bulunan tüm hücre soylarının içine sağlam hücresel kutuplaşma ve sergi farklılaşmasını görüntülerestinal epitel. Bunlar transgenlerin taşımak için aktarılacak olan veya RNA interferans 5 oluşturur olduğundan, eksiksiz ve hız yönü de, transjenik fareler kullanılarak deneyler outweighing, spesifik genetik elemanları incelemek için kullanılır. Organoids transjenik ifadesi sıçangil retroviral veya lentiviral vektörler 6,7 kullanarak gerçekleştirilebilir. Nedeniyle mitotik hücre özel olarak 8 transduse edebildiği sıçangil retrovirüslerin sınırlamaları, lentiviral transdüksiyon daha sık örneğin organoids olarak enfekte zor olan hücreler için kullanılır. Viral transdüksiyonlu ve stabil bir şekilde eksprese eden transgenik organoids analiz eder ve immünohistokimya kantitatif RNA dahil olmak üzere, akış aşağı analizler çok sayıda için kullanılabilir. Birlikte ele alındığında, primer intestinal epitel hücrelerinden organoids kültürü özel laboratuvar şartlarına olmadan uygulanması kolay bir rutin tekniği haline gelmiştir, ve ce roman standart haline gelmiştirbağırsak epiteli üzerinde araştırma ll kültürü. Viral iletimi ve organoids sonraki mansap analiz teknikleri gerçekleştirmek ve kültürlü organoids lentiviral transdüksiyon için yöntemler göstererek, bu video protokolü oluşturulan organoid deneyler yardım için sıkıcı bulunmaktadır. Biz ayrıca verimi artırmak ve dolayısıyla RNA teknikleri veya immunhistokimya kullanan alt analiz performansını artırabilirsiniz ne kadar doğru işlem organoids arasında gösteriyor. Tarif edilen teknikler de kolon organoids tatbik edilebilir, ancak protokol, ince bağırsak kript türetilen organoids sadece kullanılmıştır.

<table border="0" cellpadding="0" cellspacing="0" width="539">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">Reagent</td>
			<td style="width:91px;">Company&#160;</td>
			<td style="width:103px;">Cat. No.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">Polyethylene imine</td>
			<td style="width:91px;">polysciences</td>
			<td style="width:103px;">23966-2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">DMEM medium</td>
			<td style="width:91px;">Lonza</td>
			<td style="width:103px;">BE12-614F</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">Fetal calf serum</td>
			<td style="width:91px;">Lonza</td>
			<td style="width:103px;">DE14-801F</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">Penicillin-streptomycin</td>
			<td style="width:91px;">Invitrogen</td>
			<td style="width:103px;">15140-122</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">Glutamin</td>
			<td style="width:91px;">Invitrogen</td>
			<td style="width:103px;">25030-024</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">matrigel</td>
			<td style="width:91px;">BD</td>
			<td style="width:103px;">BD 356231</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">Advanced DMEM-F12</td>
			<td style="width:91px;">Gibco</td>
			<td style="width:103px;">12634-010</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">N2</td>
			<td style="width:91px;">Invitrogen</td>
			<td style="width:103px;">17502-048</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">B27</td>
			<td style="width:91px;">Invitrogen</td>
			<td style="width:103px;">17504-044</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">N-acetyl cysteine</td>
			<td>Sigma</td>
			<td>A9165-1G</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mouse Egf</td>
			<td>Invitrogen</td>
			<td>PMG8045</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hepes 1M</td>
			<td>Invitrogen</td>
			<td>15630-056</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">glutamax 100x</td>
			<td>Invitrogen</td>
			<td>35050-038</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chir 99021</td>
			<td>axon</td>
			<td>1386</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Y27632&#160;</td>
			<td>Sigma</td>
			<td>Y0503-5MG</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">polybrene</td>
			<td>Sigma</td>
			<td>107689</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">nicotinamide</td>
			<td>Sigma</td>
			<td>N0636</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin</td>
			<td>Lonza</td>
			<td>BE02-007E</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">puromycin</td>
			<td>sigma</td>
			<td>P 7255</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rneasy mini kit</td>
			<td>Qiagen</td>
			<td>74106</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">b-mercaptoethanol</td>
			<td>Merck</td>
			<td>8,057,400,250</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ovation Pico WTA system</td>
			<td>NuGen</td>
			<td>3300-12</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">paraformaldehyde</td>
			<td>Sigma</td>
			<td>252549-1L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">glass vial conical 12mm x 75mm 5ml</td>
			<td>VWR</td>
			<td>LSUKM12</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eosin Yellowish</td>
			<td>VWR</td>
			<td>1,159,350,025</td>
		</tr>
	</tbody>
</table>

a protocol for lentiviral transduction and downstream analysis of intestinal organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

Organoid lentiviral transdüksiyon Lentiviral parçacıklar kullanılarak organoid iletimi tekniği öncesi ve transdüksiyon sırasında organoids doğru kullanımı bağlıdır. Organoids (Şekil 3A) kültürlenmiştir ve bir kript (Şekil 3B) içine dağıtıldı. Daha önce belirtildiği gibi, GSK3 inhibitörü Chir99021 mevcudiyetinde kültürlenmiş, bu tek kript kistik crypts 9 (Şekil 3C) olmuştur. Daha sonra org...

Watch this Scientific Journal Video about A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids at JoVE.com

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

Lentiviral İletim ve Bağırsak Organoids Mansabında Analizi için Protokol

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

a-protocol-for-lentiviral-transduction-downstream-analysis-intestinal

Research

JoVE Journal

Biology

30.4K Görüntüleme.  Academical Medical Center. In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry. 

Video: A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Nörotoksisite Yüksek İçerik Analizi Nöronal ve Astrosit Hücre Co-Kültür Testi

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

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 ...

Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc, and Klf4 is capable of inducing pluripotency in mouse fibroblasts.1 Subsequent reports have demonstrated the utility of the doxycycline (DOX) inducible lentiviral delivery system for the generation of both primary and secondary iPS cells from a variety of other adult mouse somatic cell types.2,3
Induced pluripotent stem (iPS) cells are similar to embryonic stem (ES) cells in morphology, proliferation and ability to induce teratoma formation. Both types of cell can be used as the pluripotent starting material for the generation of differentiated cells or tissues in regenerative medicine.4-6 iPS cells also have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction.
Here we demonstrate the protocol for reprogramming mouse embryonic fibroblast (MEF) cells with the Stemgent DOX Inducible Mouse TF Lentivirus Set. We also demonstrate that the Stemgent DOX Inducible Mouse TF Lentivirus Set is capable of expressing each of the four transcription factors upon transduction into MEFs thereby inducing a pluripotent stem cell state that displays the pluripotency markers characteristic of ES cells.

The Stemgent Dox Inducible Mouse TF Lentivirus Set can reprogram mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. Here we demonstrate the protocol for DOX-inducible expression of mouse reprogramming transcription factors Oct4, Sox2, Klf4 and c-Myc to generate iPS colonies that express common mES pluripotency markers.

Dört Transkripsiyon Faktör Fare Embriyonik Fibroblastlar yeniden programlanması Bağlı Pluripotent Kök Hücre Üretimi, Doksisiklin lentiviral İletim Sistemi Đndüklenebilir

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and ...

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale quantitative analysis of formalin fixed and paraffin embedded (FFPE) tissue. The procedure comprises four distinct steps. The first one is the preparation of sections from the FFPE material and microdissection of cells of interest. In the second step the isolated cells are lysed and processed using 'filter aided sample preparation' (FASP) technique. In this step, proteins are depleted from reagents used for the sample lysis and are digested in two-steps using endoproteinase LysC and trypsin. 
After each digestion, the peptides are collected in separate fractions and their content is determined using a highly sensitive fluorescence measurement. Finally, the peptides are fractionated on 'pipette-tip' microcolumns. The LysC-peptides are separated into 4 fractions whereas the tryptic peptides are separated into 2 fractions. In this way prepared samples allow analysis of proteomes from minute amounts of material to a depth of 10,000 proteins. Thus, the described workflow is a powerful technique for studying diseases in a system-wide-fashion as well as for identification of potential biomarkers and drug targets.

Archival formalin fixed and paraffin embedded (FFPE) clinical samples are valuable material for investigation of diseases. Here we demonstrate a sample preparation workflow allowing in-depth proteomic analysis of microdissected FFPE tissue.

Formalin gelen Proteomic Numune Hazırlama parafin blok doku Sabit ve

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale ...

Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single procedure. Due to advances in microscope technology it is relatively easy to capture live cell images for the purpose of investigating cellular events in real time with minimal concern regarding phototoxicity to the cells. This protocol describes how to take live cell timelapse images of primary rat neonatal cardiomyocytes using a confocal spinning disk microscope following lentiviral and adenoviral transduction to modulate properties of the cell. The application of two different types of viruses makes it easier to achieve an appropriate transduction rate and expression levels for two different genes. Well focused live cell images can be obtained using the microscope&#8217;s autofocus system, which maintains stable focus for long time periods. Applying this method, the functions of exogenously engineered proteins expressed in cultured primary cells can be analyzed. Additionally, this system can be used to examine the functions of genes through the use of siRNAs as well as of chemical modulators.

This protocol describes a method of live cell imaging using primary rat neonatal cardiomyocytes following lentiviral and adenoviral transduction using confocal spinning disk microscopy. This enables detailed observations of cellular processes in living cardiomyocytes.

İlköğretim Rat Yenidoğan kardiyomiyositlerin Canlı Hücre Görüntüleme Adenoviral izlenerek ve Lentiviral İletimi Confocal Disk Mikroskopi Spinning kullanma

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single ...

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these &#39;mini-guts&#39;, also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.

This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.

İlköğretim Bağırsak organoid Kültür Retrovirütik Enfeksiyon Bir Video Protokolü

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...

Clock Scan Protocol for Image Analysis: ImageJ Plugins

The clock scan protocol for image analysis is an efficient tool to quantify the average pixel intensity within, at the border, and outside (background) a closed or segmented convex-shaped region of interest, leading to the generation of an averaged integral radial pixel-intensity profile. This protocol was originally developed in 2006, as a visual basic 6 script, but as such, it had limited distribution. To address this problem and to join similar recent efforts by others, we converted the original clock scan protocol code into two Java-based plugins compatible with NIH-sponsored and freely available image analysis programs like ImageJ or Fiji ImageJ. Furthermore, these plugins have several new functions, further expanding the range of capabilities of the original protocol, such as analysis of multiple regions of interest and image stacks. The latter feature of the program is especially useful in applications in which it is important to determine changes related to time and location. Thus, the clock scan analysis of stacks of biological images may potentially be applied to spreading of Na+ or Ca++ within a single cell, as well as to the analysis of spreading activity (e.g., Ca++ waves) in populations of synaptically-connected or gap junction-coupled cells. Here, we describe these new clock scan plugins and show some examples of their applications in image analysis.

This paper describes two novel ImageJ plugins for &#39;Clock Scan&#39; image analysis. These plugins expand the functionality of the original visual basic 6 program and, most importantly, make the program available to a large research community by bundling it with the ImageJ free image analysis software package.

Görüntü Analizi için Saat Tarama Protokolü: ImageJ Eklentileri

The clock scan protocol for image analysis is an efficient tool to quantify the average pixel intensity within, at the border, and outside (background) a ...

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

Su Macroinvertebrates kullanma gruba özel rDNA astar genetik Gut içerik analizleri için laboratuvar iletişim kuralı

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...

Laser-assisted Lentiviral Gene Delivery to Mouse Fertilized Eggs

Lentiviruses are efficient vectors for gene delivery to mammalian cells. Following transduction, the lentiviral genome is stably incorporated into the host chromosome and is passed on to progeny. Thus, they are ideal vectors for creation of stable cell lines, in vivo delivery of indicators, and transduction of single cell fertilized eggs to create transgenic animals. However, mouse fertilized eggs and early stage embryos are protected by the zona pellucida, a glycoprotein matrix that forms a barrier against lentiviral gene delivery. Lentiviruses are too large to penetrate the zona and are typically delivered by microinjection of viral particles into the perivitelline cavity, the space between the zona and the embryonic cells. The requirement for highly skilled technologists and specialized equipment has minimized the use of lentiviruses for gene delivery to mouse embryos. This article describes a protocol for permeabilizing the mouse fertilized eggs by perforating the zona with a laser. Laser-perforation does not result in any damage to embryos and allows lentiviruses to gain access to embryonic cells for gene delivery. Transduced embryos can develop into blastocyst in vitro, and if implanted in pseudopregnant mice, develop into transgenic pups. The laser used in this protocol is effective and easy to use. Genes delivered by lentiviruses stably incorporate into mouse embryonic cells and are germline transmittable. This is an alternative method for creation of transgenic mice that requires no micromanipulation and microinjection of fertilized eggs.

Mouse fertilized eggs and early stage embryos are protected by the zona pellucida, a glycoprotein matrix that forms a barrier against gene delivery. This article describes a protocol for perforating the zona with a laser to transduce embryonic cells with lentiviral vectors and to create transgenic mice.

Fare Lentiviral Gene lazer destekli teslim yumurta döllenmiş

Lentiviruses are efficient vectors for gene delivery to mammalian cells. Following transduction, the lentiviral genome is stably incorporated into the ...

3D Culturing of Organoids from the Intestinal Villi Epithelium Undergoing Dedifferentiation

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is compartmentalized into crypts and villi: the stem and proliferating cells are confined to the crypts, whereas the villi epithelium contains only differentiated cells. Hence, the normal intestinal crypts, but not the villi, can give rise to organoids in 3D cultures. The procedure described here is applicable only to villus epithelium undergoing dedifferentiation leading to stemness. The method described uses the Smad4-loss-of-function:&#946;-catenin gain-of-function (Smad4KO:&#946;-cateninGOF) conditional mutant mouse. The mutation causes the intestinal villi to dedifferentiate and generate stem cells in the villi. Intestinal villi undergoing dedifferentiation are scraped off the intestine using glass slides, placed in a 70 &#181;m strainer and washed several times to filter out any loose cells or crypts prior to plating in BME-R1 matrix to determine their organoid-forming potential. Two main criteria were used to ensure that the resulting organoids were developed from the dedifferentiating villus compartment and not from the crypts: 1) microscopically evaluating&#160;the isolated villi to ensure absence of any tethered crypts, both before and after plating in the 3D matrix, and 2) monitoring the time course of organoid development from the villi. Organoid initiation from the villi occurs only two to five days after plating and appears irregularly shaped, whereas the crypt-derived organoids from the same intestinal epithelium are apparent within sixteen hours of plating and appear spherical. The limitation of the method, however, is that the number of organoids formed, and the time required for organoid initiation from the villi vary depending on the degree of dedifferentiation. Hence, depending upon the specificity of the mutation or the insult causing the dedifferentiation, the optimal stage at which villi can be harvested to assay their organoid forming potential, must be determined empirically.

The procedure describes isolation of the villi from the mouse intestinal epithelium undergoing dedifferentiation to determine their organoid forming potential.

Organoidlerin Bağırsak Villi Epitelinden 3D Kültasyonu Adanmışlık Geçiriyor

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is ...