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).

1. Preparazione di polietilenimmina (PEI) come Transfection Reagent <ol><li> Disciogliere circa 150 mg di PEI in 100 ml di H 2 O. </li><li> Regolare soluzione a pH 7.4 con l&#39;aggiunta di HCl fino soluzione diventa chiara e mescolare fino a completa dissoluzione. Questo può richiedere tra 10 e 60 min e aggiungere acqua ad una concentrazione finale di 1 mg / ml. </li><li> Quando chiaro, filtrare la soluzione PEI attraverso sterile filtro da 0,22 micron e conservare in un -80 ° C freezer in aliquote di 5 ml. </li></ol> 2. Produzione di particelle lentivirali Giorno 1: <ol><li> HEK293T cellule Spalato al 60% -80% di confluenza in 162 cm 2 fiasco o grande Petri in linea cellulare terreno di coltura (DMEM supplementato con 10% FCS, 1% di penicillina / streptomicina e integratore di glutammina 2 mm). </li></ol> 2 ° giorno: <ol start="2"><li> Preparare la soluzione trasfezione di DNA contenente 45 mg di DNA totale plasmide sommandovettori lentivirali imballaggio (7 mg di pVSVg, 5 mcg di pRSV giri; 13 mcg di pMDL) e 20 microgrammi di plasmide lentivirali codificanti del gene di interesse o shRNA di interesse. Regolare ad un volume di 1 ml utilizzando DMEM. </li><li> Preparare la soluzione trasfezione PEI aggiungendo 90 ml di 1 mg / ml PEI a 930 ml di DMEM e incubare per 5 minuti a temperatura ambiente. </li><li> Aggiungere la soluzione trasfezione DNA soluzione PEI. Vortex o invertire un numero di volte e incubare per 5 min a temperatura ambiente per ottenere una soluzione trasfezione di DNA. </li><li> Drip 2 ml della soluzione di trasfezione di DNA sulle cellule HEK293T e incubare per 4 ore in un incubatore umidificato coltura cellulare da 37 ° C. </li><li> Dopo 4 ore, aggiornare il terreno di coltura per rimuovere PEI. Non è necessario lavare cellule prima di aggiungere nuovo mezzo. NOTA: PEI è citotossici e di incubazione più di 4 ore può causare danni alle cellule HEK293T. </li></ol> 4 ° giorno: <ol start="7"><li> Sostituire supernatant con il nuovo terreno di coltura. Tenere surnatante (contenente virus); questo sarà usato nella fase 2.10. </li><li> Mettere surnatante in 15 ml pallone. Per rimuovere le cellule morte, centrifugare per 5 min a 500 x g. </li><li> Spingere surnatante tramite 0,45 micron filtro con una grossa siringa da 60 ml. Conservare notte a 4 ° C. </li></ol> 5 ° giorno: <ol start="10"><li> Raccogliere il secondo lotto di surnatante; centrifuga e filtro come in passaggi 2,8-2,9. </li><li> Pool il surnatante da passi 2.9 e 2.10 in tubi ultracentrifuga e centrifugare a 50.000 xg in un&#39;ultracentrifuga per 90 min. </li><li> Estrarre capsule contenenti attentamente le provette ultracentrifuga e messo in una cappa a flusso laminare, ricordando l&#39;orientamento del tubo all&#39;interno della centrifuga. </li><li> Aprire capsula tenendo il tubo un&#39;ultracentrifuga e medie decantare attentamente in modo tale che il pellet è sul lato superiore del tubo. Dal pellet virale può essere difficile da visualizzare, ricordare su qualilato del tubo un pellet sarà formata. Prendere una micropipetta e rimuovere ultimo bit di media facendo attenzione a non agitare il pellet marrone opaco che è visibile sul lato del fondo del tubo ultracentrifuga. </li><li> Risospendere questo pellet in 500 ml di terreno di coltura organoide integrati con nicotinamide 10 mm, 10 micron Chir99021, 10 micron Y27632 e 8 mg / ml polibrene. Risospensione in questo mezzo è importante poiché virus titolo elevato viene utilizzato per transduct direttamente organoidi. </li><li> Facoltativamente: congelare il virus in questo mezzo frazionato in due lotti di 250 microlitri a -80 ° C. </li></ol> 3. lentivirali trasduzione di organoidi Giorno 0: <ol><li> Dividere una completa 0,95 centimetri 2 e di organoidi due giorni prima trasduzione in un nuovo pozzo, al fine di ottenere circa 50 piccoli organoidi. Split organoidi secondo il protocollo precedentemente pubblicato 1 (Figura 3A, B). </li><li> Supplemento organoide mezzo di coltura con 10 micron Chir99021 e nicotinamide 10 mm per ottenere iper cistica cripte proliferative (Figura 3C). NOTA: cripte cistica svilupperanno meglio quando organoidi appena divisi sono coltivati ​​in presenza di Chir99021. </li></ol> 2 ° giorno: <ol start="3"><li> Organoidi Harvest pipettando su e giù per il matrigel e media, interrompendo in tal modo il composto con una micropipetta p1000. Posizionare la miscela in una provetta da 15 ml. </li><li> Disrupt ulteriormente utilizzando Pasteur pipetta in cui l&#39;apertura distale è stato diminuito da fusione. Organoidi centrifuga a pellet per 5 min a 100 x g. NOTA: A seconda della dimensione della centrifuga, utilizza una velocità di centrifuga diversa. È necessario trovare l&#39;esatta velocità in cui interrotte organoidi sono separati dalla miscela. </li><li> Rimuovere il surnatante e aggiungere 500 ml di tripsina preriscaldata 1x (simile al 0,25% tripsina). Risospendere organoidi in tripsina e incubare3 min in un bagno di 37 ° C dell&#39;acqua. </li><li> Inattivare tripsina aggiungendo 3,5 ml di terreno di coltura linea cellulare (DMEM supplementato con 10% FCS, 1% di penicillina / streptomicina e glutammina). Centrifugare per 5 min a 500 x g. </li><li> Rimuovere il surnatante di lasciare pellet in circa 20 ml di media. Trasferire i organoidi in questa ultima piccola quantità di mezzo in un pozzo in un piatto di 48 pozzi. </li><li> Aggiungi alto lentivirus titolo nel medio trasduzione come descritto al punto 2.14, risospendere e continuare con il passo 3.10. Quando incontra bassa efficacia di trasduzione, punto 3.9 possono essere aggiunti. NOTA: Per la trasduzione efficiente, tipicamente aggiungere un&#39;aliquota di 250 microlitri di alta lentivirus titolo ad un pozzetto di organoidi. Questo rappresenta il 50% di tutti i lentivirus raccolti da una singola produzione come descritto al punto 2 di questo protocollo. </li><li> Facoltativamente: per migliorare l&#39;efficacia trasduzione, effettuare spinoculation mettendo 48 pozzetti contenente organoidi in un pre-riscaldato centrifuga da 326; C e ruotare a 600 xg per 1 ora. </li><li> Mettere miscela organoide-virus in cultura incubatore e incubare per 1 ora a 37 ° C in un incubatore culturale per consentire trasduzione. </li><li> Facoltativamente: per migliorare ulteriormente l&#39;efficacia di trasduzione, incubare organoidi Per ulteriori 3 ore a 37 ° C in un incubatore cultura. </li><li> Aggiungere 1 ml di terreno di coltura organoide e risospendere la miscela organoide-virus, il trasferimento in una provetta e centrifugare in una microcentrifuga per 5 minuti a 850 xg per far sedimentare organoidi. </li><li> Rimuovere il surnatante e risospendere il pellet in 20 ml di Matrigel ghiacciata. Poiché il materiale solidifica quando diventa caldo, utilizzare puntali che vengono refrigerati pipettando su e giù ghiacciata PBS per un numero di volte. </li><li> Mettere la goccia nel mezzo di un pozzetto in una piastra 48 pozzetti e incubare in coltura incubatore a 37 ° C per 15 minuti per solidificare. </li><li> Dopo 15 minuti, aggiungere con cautela 250 ml di terreno di coltura organoide integrato conNicotinamide 10 mM, 10 mM Chir99021 e 10 pM Y27632. Piccoli frammenti organoide perturbato formeranno in piccoli organoidi cistica entro 24 ore. </li></ol> 5 ° giorno: <ol start="16"><li> Aggiorna medio e integrare con un antibiotico selezione (per puromicina, utilizzare 4 mg / ml). </li></ol> 7 ° giorno: <ol start="17"><li> Sostituire mezzo per terreno di coltura organoide serie, integrato con la selezione antibiotica. NOTA: in erba sarà completa di 2-3 settimane dopo Chir99021 ritiro. Dopo la selezione, organoidi possono essere coltivate senza selezione antibiotica. </li></ol> 4. organoide RNA Preparazione quantitativa RT-PCR o Microarray <ol><li> Per la preparazione di RNA, utilizzare un kit commerciale secondo il protocollo del produttore. Rimuovere il supporto da organoidi e aggiungere 350 ml di tampone RLT, integrati con β-mercaptoetanolo dritto sulla cupola di Matrigel contenente organoidi. Risospendere le Material in RLT pipettando utilizzando p1000 micropipetta. </li><li> Eseguire inoltre RNA preparazione secondo il protocollo del produttore. </li><li> Facoltativamente: aumentare la resa di RNA mediante amplificazione utilizzando il sistema Ovation Pico WTA, richiede input iniziale di 50 mg di RNA totale secondo il protocollo del produttore. </li><li> Facoltativamente: per il controllo di qualità prima di RNA microarray, campioni eseguito su un bioanalizzatore 2100 con un totale di chip eucariote RNA nano, puntando per un numero di integrità dell&#39;RNA (RIN) di 8.5 o più (Figura 5) secondo il protocollo del produttore. </li></ol> 5. TRATTAMENTO organoidi per paraffina e immunoistochimica <ol><li> Prima di iniziare organoide incorporamento pre-riscaldare un blocco di alluminio con fori 12 millimetri tubi di vetro di diametro montaggio in incubatore a 70 ° C per mantenere la paraffina liquida. </li><li> Prendete un singolo bene con organoidi pienamente sviluppati e rimuovere il supporto, lasciando intatte le organoidi incorporati. </li><li> Aggiungere 1 mldel 4% paraformaldeide in PBS dritto al bene e fissare a 4 ° C ovunque da 1 ora a notte. </li><li> Sostituire paraformaldeide con 1 ml di ghiaccio freddo PBS. Facoltativamente: mantenere organoidi fissi in PBS fino a 1 settimana a 4 ° C dopo questo passaggio. </li><li> Risospendere organoidi fissi in 1 ml di PBS e riporre in flacone di vetro. </li><li> Let organoidi depositano inferiore per 1 min, decantare PBS e sostituire con il 70% di etanolo in cui una coppia di goccioline di soluzione eosina vengono sciolti per consentire la visualizzazione del organoidi tutto il processo di incorporazione. </li><li> Lasciare organoidi in etanolo al 70% a temperatura ambiente. Dopo 30 minuti, rimuovere il 70% di etanolo con attenzione decantazione, essendo in grado di visualizzare i organoidi occhio a causa del leggero colore rosato eosina. Sostituire soluzione incasso con 96% di etanolo. </li><li> Ripetere passaggio 5.7, ogni volta sostituendo la soluzione incorporamento con la successiva. Passare organoidi attraverso le seguenti soluzioni successivamente: il 70% etanolo, 90% etanolo, 96% etanolo, 100% ethanol, 100% etanolo, xilene, xilene. </li><li> Decantare l&#39;ultimo lavaggio xilene e versare la paraffina nel tubo. Inserire il tubo immediatamente nel blocco di alluminio pre riscaldato a 70 ° C per 30 min e sostituire paraffina con paraffina nuova pulita. </li><li> Versare la paraffina off e pipette organoidi nello stampo blocco di paraffina con una pipetta Pasteur preriscaldata con grande apertura. Mantenere Pasteur pipetta calda usando un becco Bunsen. Mettere tutti organoidi nello stampo blocco di paraffina in un piccolo strato di paraffina liquida. </li><li> Per quanto possibile, cercare di manipolare tutti organoidi verso il centro dello stampo blocco di paraffina utilizzando un ago dissezione riscaldato. Mantenere dissezione ago calda usando becco Bunsen. </li><li> Quando localizzazione organoidi nello stampo è soddisfacente, conchiglia leggermente a solidificare lo strato di paraffina. </li><li> Termina il blocco versando più di paraffina sulla parte superiore e aggiungere una cassetta embedding istologico standard. </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.

Il protocollo video corrente descrive trasduzione lentivirale di organoidi da epitelio intestinale primaria e l&#39;analisi a valle di queste organoidi utilizzando tecniche RNA quantitative e immunoistochimica. Trasduzione lentivirali è spesso eseguita in cellule aderenti o galleggianti in piastre di coltura. Poiché la struttura tridimensionale di organoidi li rende difficile da penetrare da particelle virali, sono utilizzati diversi metodi per aumentare l&#39;efficacia. Pretrattamento di ...

L&#39;epitelio intestinale è uno dei tessuti corporei più rapida proliferazione, che ha causato di attrarre grande interesse dalla ricerca sulle cellule tumorali e staminali. Nel 2009 una tecnica è stata pubblicata per generare culture durevoli di piccole cripte intestinali in matrigel, conservando una struttura dimensionale 3 1. Tali strutture, denominate organoidi intestinali, possono essere coltivate utilizzando tecniche standard, con circostante medio integrato con una serie di fattori di crescita definiti, compreso il BMP-segnalazione inibitore pathway zucca (Nog), il Wnt-via di segnalazione enhancer rspondin 1 (Rspo1) e fattore di crescita epidermico (EGF) tutto ha trovato per migliorare la proliferazione intestinale 2-4. Organoidi superano linee tradizionali di cellule di cancro negli aspetti che sono non-mutato, hanno mantenuto la gerarchia di cellule staminali, mostrano la polarizzazione cellulare intatta e la differenziazione mostra in tutte le linee cellulari presenti nel piccolo int nascenteepitelio estinal. Dal momento che possono essere trasdotte per trasportare transgeni o RNA interference costruisce 5, vengono utilizzati per studiare elementi genetici specifici, compensa esperimenti con topi transgenici in aspetti di costo e di velocità. Espressione transgenica in organoidi può essere eseguita utilizzando retrovirale murino o vettori lentivirali 6,7. A causa delle limitazioni di retrovirus murini, in grado di trasduzione cellule mitotiche esclusivamente 8, trasduzione lentivirali viene più frequentemente utilizzata per le celle che sono difficili da infettare, come organoidi. Viralmente trasdotte e esprimono stabilmente organoidi transgenici possono essere usati per una moltitudine di analisi a valle, tra cui RNA quantitativo analisi ed immunoistochimica. Nel loro insieme, la cultura di organoidi da cellule epiteliali intestinali primarie si è evoluto in una tecnica di routine che è facile da implementare, senza requisiti specifici di laboratorio, ed è diventato il romanzo standard cecultura ll nella ricerca sul epitelio intestinale. Tecniche di trasduzione virale e successiva analisi a valle in organoidi sono noiosi da eseguire e per aiutare gli esperimenti organoide abbiamo generato questo protocollo video, che mostra i metodi per lentivirali trasduzione del organoidi coltivate. Mostriamo inoltre come la corretta elaborazione del organoidi può aumentare la resa e quindi migliorare le prestazioni di analisi a valle con tecniche di RNA o immunoistochimica. Nel protocollo, organoidi che sono derivati ​​da piccole cripte intestinali sono stati usati esclusivamente, sebbene le tecniche descritte possono essere applicate a organoidi colon pure.

<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.

Organoide trasduzione lentivirali La tecnica di trasduzione organoide utilizzando particelle lentivirali dipende corretta gestione dei organoidi prima e durante la trasduzione. Organoidi (Figura 3A) sono state coltivate e furono interrotto in cripte singoli (Figura 3B). Come riportato in precedenza, queste singole cripte, quando coltivate in presenza dell&#39;inibitore GSK3 Chir99021 divennero cripte cistiche 9 (Figura 3C).</stron...

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.

Un protocollo per lentivirali trasduzione e analisi valle di intestinale organoidi

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

30.2K Views.  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.

Un neuronale e astrociti Co-Cultura Assay per l&#39;analisi dei contenuti elevati di neurotossicità

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

Ricerca

Biologia

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.

Rilevamento di proteine ​​Ubiquitinazione

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.

Generazione di cellule staminali pluripotenti indotte da Riprogrammazione fibroblasti embrionali di topo con un fattore quattro trascrizione, Doxiciclina inducibile lentivirali sistema di trasduzione

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

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.

Imaging cellulare dal vivo di primaria ratto neonato Cardiomyocytes seguito Adenoviral e lentivirali trasduzione Uso confocale Spinning Disk Microscopia

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.

Un protocollo video di infezione retrovirale in Primaria intestinale organoide Cultura

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

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.

Protocollo di laboratorio per analisi genetiche del contenuto dell'intestino del rDNA acquatica macroinvertebrati Using Group-specific Primers

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.

Laser-assistita lentivirali Gene consegna al Mouse fecondate le uova

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.

3D Coltivazione di Organoidi dall'Epitelio Villi Intestinale sottoposti a Dedifferenziazione

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

Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe

Reactive oxygen species (ROS) play essential roles in intestinal homeostasis. ROS are natural by-products of cell metabolism. They are produced in response to infection or injury at the mucosal level as they are involved in antimicrobial responses and wound healing. They are also critical secondary messengers, regulating several pathways, including cell growth and differentiation. On the other hand, excessive ROS levels lead to oxidative stress, which can be deleterious for cells and favor intestinal diseases like chronic inflammation or cancer. This work provides a straightforward method to detect ROS in the intestinal murine organoids by live imaging and flow cytometry, using a commercially available fluorogenic probe. Here the protocol describes assaying the effect of compounds that modulate the redox balance in intestinal organoids and detect ROS levels in specific intestinal cell types, exemplified here by the analysis of the intestinal stem cells genetically labeled with GFP. This protocol may be used with other fluorescent probes.

The present protocol describes a method to detect reactive oxygen species (ROS) in the intestinal murine organoids using qualitative imaging and quantitative cytometry assays. This work can be potentially extended to other fluorescent probes to test the effect of selected compounds on ROS.

Analisi dello stress ossidativo negli organoidi intestinali murini utilizzando la sonda fluorogenica sensibile alle specie reattive dell'ossigeno

Reactive oxygen species (ROS) play essential roles in intestinal homeostasis. ROS are natural by-products of cell metabolism. They are produced in ...

Coltura 3D di Organoidi da Cripte Intestinali Murine e Singola Cellula Staminale

The 3D Culturing of Organoids from Murine Intestinal Crypts and a Single Stem Cell for Organoid Research

At present, organoid culture represents an important tool for in vitro studies of different biological aspects and diseases in different organs. Murine small intestinal crypts can form organoids that mimic the intestinal epithelium when cultured in a 3D extracellular matrix. The organoids are composed of all cell types that fulfill various intestinal homeostatic functions. These include Paneth cells, enteroendocrine cells, enterocytes, goblet cells, and tuft cells. Well-characterized molecules are added into the culture medium to enrich the intestinal stem cells (ISCs) labeled with leucine-rich repeats containing G protein-coupled receptor 5 and are used to drive differentiation down specific lineages; these molecules include epidermal growth factor, Noggin (a bone morphogenetic protein), and R-spondin 1. Additionally, a protocol to generate organoids from a single erythropoietin-producing hepatocellular receptor B2 (EphB2)-positive ISC is also detailed. In this methods article, techniques to isolate small intestinal crypts and a single ISC from tissues and ensure the efficient establishment of organoids are described.

Autore in primo piano: La coltura 3D di organoidi da cripte intestinali murine e una singola cellula staminale per la ricerca sugli organoidi  

We describe a protocol to isolate murine small intestinal crypts and culture intestinal 3D organoids from the crypts. Additionally, we describe a method to generate organoids from a single intestinal stem cell in the absence of a sub-epithelial cellular niche.

La coltura 3D di organoidi da cripte intestinali murine e una singola cellula staminale per la ricerca sugli organoidi

At present, organoid culture represents an important tool for in vitro studies of different biological aspects and diseases in different organs. Murine ...