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. Préparation de Polyéthylènimine (PEI) comme réactif de transfection <ol><li> Dissoudre environ 150 mg de PEI dans 100 ml d&#39;H 2 O. </li><li> Réglez solution à pH 7,4 par addition de HCl jusqu&#39;au solution devient limpide et remuer jusqu&#39;à dissolution complète. Cela peut prendre entre 10 et 60 min et ajouter de l&#39;eau à une concentration de 1 mg / ml final. </li><li> Lorsque claire, filtrer la solution à travers le filtre PEI 0,22 um stérile et stocker dans un congélateur à -80 ° C en aliquots de 5 ml. </li></ol> 2. Production de Particules Lentivirales Jour 1: <ol><li> Des cellules HEK293T Split à 60% -80% de confluence dans 162 cm 2 fiole ou une grande boîte de Pétri dans un milieu de culture de lignée cellulaire (DMEM supplémenté avec 10% de FCS, 1% de pénicilline / streptomycine et supplément de glutamine 2 mM). </li></ol> Jour 2: <ol start="2"><li> Préparer la solution de transfection d&#39;ADN contenant 45 pg d&#39;ADN de plasmide total, additionnerles vecteurs lentiviraux d&#39;emballage (7 pg de pVSVg; 5 ug de pRSV rev; 13 pg de PMDL) et 20 ug de plasmide lentiviraux codant pour le gène d&#39;intérêt ou shRNA d&#39;intérêt. Ajuster à un volume de 1 ml en utilisant du DMEM. </li><li> Préparer la solution de transfection PEI en ajoutant 90 ul de 1 mg / ml de PEI à 930 pl de DMEM et incuber pendant 5 minutes à température ambiante. </li><li> Ajouter la solution de transfection de l&#39;ADN à la solution PEI. Vortex ou inverser un certain nombre de fois et incuber pendant 5 minutes à température ambiante pour obtenir une solution de transfection de l&#39;ADN. </li><li> Goutte à goutte 2 ml de la solution de transfection de l&#39;ADN sur les cellules HEK293T et incuber pendant 4 h dans un incubateur humidifié de culture de cellules à 37 ° C. </li><li> Après 4 h, rafraîchir le milieu de culture pour éliminer PEI. Il ne est pas nécessaire de laver les cellules avant d&#39;ajouter nouveau média. REMARQUE: PEI est cytotoxique et d&#39;incubation fois plus longtemps que 4 heures peuvent causer des dommages aux cellules HEK293T. </li></ol> Jour 4: <ol start="7"><li> Remplacer supernatant avec le nouveau milieu de culture. Gardez surnageant (contenant le virus); il sera utilisé dans l&#39;étape 2.10. </li><li> Mettez surnageant dans 15 ml flacon. Pour éliminer les cellules mortes, centrifugeuse pendant 5 min à 500 x g. </li><li> Poussez surnageant de 0,45 um filtre utilisant une grande seringue de 60 ml. Conservez une nuit à 4 ° C. </li></ol> Jour 5: <ol start="10"><li> Recueillir le deuxième lot de surnageant; centrifugeuse et le filtre comme dans les étapes 2.8 à 2.9. </li><li> Mutualiser les surnageants des étapes 2,9 et 2,10 dans des tubes d&#39;ultracentrifugation et centrifuger à 50 000 xg dans une ultracentrifugation pendant 90 min. </li><li> Sortez capsules contenant très soigneusement les tubes d&#39;ultracentrifugation et de mettre dans une hotte à flux laminaire, se souvenant de l&#39;orientation du tube à l&#39;intérieur de la centrifugeuse. </li><li> Ouvrir le tube de maintien de capsule soigneusement ultracentrifugeuse milieu décanter et de telle façon que la pastille est sur le côté supérieur du tube. Depuis pellets viraux peuvent être difficiles à visualiser, rappeler dans quellecôté du tube une pastille sera formée. Prenez une micropipette et retirez dernier bit de milieu tout en prenant soin de ne pas agiter le culot brun opaque qui est visible sur le côté de la partie inférieure du tube d&#39;ultracentrifugation. </li><li> Remettre en suspension cette pastille dans 500 ul de milieu de culture additionné de nicotinamide organoïde 10 mM, 10 pM Chir99021, 10 uM Y27632 et 8 ug / ml de polybrène. La remise en suspension dans ce milieu est important car le virus à titre élevé est utilisé pour transduct directement organites. </li><li> Optionnellement: geler le virus dans ce milieu aliquotés en deux lots de 250 pl dans -80 ° C. </li></ol> 3. Lentiviral transduction des organites Jour 0: <ol><li> Diviser une pleine 0,95 cm 2 et des organites deux jours avant de transduction dans un nouveau puits, visant à obtenir environ 50 petits organites. Diviser organoïdes selon le protocole précédemment publié une (figure 3A, B). </li><li> Supplément organoïde milieu de culture avec 10 uM Chir99021 et 10 mM de nicotinamide pour obtenir hyper prolifératives kystique cryptes (figure 3C). REMARQUE: cryptes kystique développeront mieux quand organoïdes fraîchement fendus sont cultivées en présence de Chir99021. </li></ol> Jour 2: <ol start="3"><li> Organoïdes récolte par aspiration et refoulement du matrigel et moyennes, perturbant ainsi le mélange avec une micropipette P1000. Placer le mélange dans un tube de 15 ml. </li><li> Perturber outre l&#39;utilisation d&#39;une pipette Pasteur dans lequel l&#39;ouverture distale a été diminué par fusion. organoïdes à centrifuger pour sédimenter pendant 5 min à 100 x g. NOTE: En fonction de la taille de la centrifugeuse, utiliser une vitesse de centrifugeuse différente. Il est nécessaire de trouver la vitesse exacte dont perturbés organites sont séparés du mélange. </li><li> Eliminer le surnageant et ajouter 500 ul de trypsine 1x préchauffé (similaire à la trypsine 0,25%). Remettre en suspension dans de la trypsine organites et incuber3 min dans un bain d&#39;eau à 37 C °. </li><li> Inactiver la trypsine par addition de 3,5 ml de milieu de culture de lignée cellulaire (DMEM supplémenté avec 10% de FCS, 1% de pénicilline / streptomycine et glutamine). Centrifuger pendant 5 minutes à 500 x g. </li><li> Eliminer le surnageant de quitter culot dans environ 20 pi de milieu. Transférez les organites dans cette dernière petite quantité de milieu dans un puits sur une plaque de 48 puits. </li><li> Ajouter haute lentivirus du titre en milieu de transduction comme décrit à l&#39;étape 2.14, remettre en suspension et passez à l&#39;étape 3.10. En rencontrant une faible efficacité de transduction, l&#39;étape 3.9 peuvent être ajoutés. NOTE: Pour la transduction efficace, ajouter généralement une aliquote de 250 pi de haute lentivirus de titre à un puits d&#39;organites. Cela représente 50% de tous les lentivirus récolté dans une seule production comme décrit dans l&#39;étape 2 de ce protocole. </li><li> En option: pour améliorer l&#39;efficacité de transduction, effectuez spinoculation en mettant les 48 et plaques contenant organites dans une centrifugeuse préchauffé sur 326; C et tourner à 600 g pendant 1 h. </li><li> Mettre mélange organoïde-virus en culture incubateur et incuber pendant 1 heure à 37 ° C dans un incubateur de culture pour permettre la transduction. </li><li> En option: pour améliorer encore l&#39;efficacité transduction, incuber organoïdes pour supplémentaire de 3 heures à 37 ° C dans un incubateur de culture. </li><li> Ajouter 1 ml de milieu de culture organoïde et remettre le mélange organoïde-virus, transférer dans un tube à centrifuger et centrifuger dans une microcentrifugeuse pendant 5 min à 850 xg pour sédimenter organites. </li><li> Retirer le surnageant et remettre le culot dans 20 ul d&#39;glacée matrigel. Comme le matériau se solidifie quand il devient plus chaud, utiliser des embouts de pipette qui sont refroidis par aspiration et refoulement PBS glacé pour un certain nombre de fois. </li><li> Mettez la goutte au milieu d&#39;un puits dans une plaque de 48 puits et incuber dans la culture incubateur à 37 ° C pendant 15 min pour solidifier. </li><li> Après 15 min, ajouter avec précaution 250 ul de milieu de culture supplémenté avec organoïdeNicotinamide 10 mM, 10 uM et 10 uM Chir99021 Y27632. Fragments organoïdes perturbé petits feront en petits organites kystiques dans les 24 heures. </li></ol> Jour 5: <ol start="16"><li> Actualiser moyen et compléter avec un antibiotique de sélection (pour puromycine, utiliser 4 pg / ml). </li></ol> Jour 7: <ol start="17"><li> Remplacer milieu pour milieu de culture organoïde standard, complétée par le choix des antibiotiques. REMARQUE: en herbe sera terminée 2-3 semaines après le retrait Chir99021. Après la sélection, organites peuvent être cultivées sans sélection antibiotique. </li></ol> 4. Organoid ARN Préparation de RT-PCR quantitative ou puces à ADN <ol><li> Pour la préparation d&#39;ARN, en utilisant un kit commercial selon le protocole du fabricant. Retirer le support de organites et ajouter 350 ul de tampon RLT, complétées par des β-mercaptoéthanol droite sur le dôme de matrigel contenant organites. Reprendre les material dans RLT par pipetage utilisant p1000 micropipette. </li><li> Effectuer d&#39;autres ARN préparation selon le protocole du fabricant. </li><li> Optionnellement: augmenter le rendement en ARN par amplification en utilisant le système Ovation Pico WTA, nécessitant l&#39;apport initial de 50 ug d&#39;ARN total selon le protocole du fabricant. </li><li> En option: contrôle de la qualité avant ARN microarray, échantillons se exécutent sur ​​un bioanalyseur 2100 en utilisant une puce ARN nano eucaryote totale, visant un certain nombre d&#39;intégrité de l&#39;ARN (RIN) de 8,5 ou plus (Figure 5) selon le protocole du fabricant. </li></ol> 5. Traitement organoïdes pour inclusion dans la paraffine et immunohistochimie <ol><li> Avant l&#39;instauration du organoïde intégration pré-chauffer un bloc d&#39;aluminium avec des trous de 12 mm tubes de verre de diamètre de montage en incubateur à 70 ° C pour empêcher le liquide de paraffine. </li><li> Prenez un seul bien avec organoïdes plein grandi et éliminer le milieu, laissant les organites embarqués intacte. </li><li> Ajouter 1 mlde 4% de paraformaldehyde dans PBS directement à bien et fixer à 4 ° C allant de 1 heure à une nuit. </li><li> Remplacer paraformaldéhyde avec 1 ml de PBS glacé. En option: garder organoïdes fixes dans du PBS jusqu&#39;à 1 semaine à 4 ° C après cette étape. </li><li> Remettre en suspension organites fixes dans 1 ml de PBS et les placer dans un flacon de verre. </li><li> Laissez organites se enfoncent vers le bas pendant 1 min, décanter et remplacer avec du PBS 70% d&#39;éthanol dans laquelle un couple de gouttelettes de solution d&#39;éosine sont dissous pour permettre la visualisation des organites au long du processus d&#39;enrobage. </li><li> Laisser organites dans 70% d&#39;éthanol à température ambiante. Après 30 min, retirez 70% d&#39;éthanol par décantation attentivement, être en mesure de visualiser les organites à l&#39;oeil à cause de légère couleur rose éosine. Remplacer solution intégrant avec 96% d&#39;éthanol. </li><li> Répétez l&#39;étape 5.7, à chaque fois que le remplacement de la solution enrobage avec la suivante. Passez organoïdes travers les solutions suivantes: par la suite 70% d&#39;éthanol, 90% d&#39;éthanol, 96% d&#39;éthanol, 100% ethanol, éthanol à 100%, le xylène, le xylène. </li><li> Décanter le dernier lavage de xylène et versez la paraffine dans le tube. Mettez immédiatement le tube dans le bloc d&#39;aluminium pré réchauffé à 70 ° C pendant 30 min et remplacer la paraffine avec le nouveau paraffine propre. </li><li> Verser de paraffine et organites pipette dans le moule du bloc de paraffine à l&#39;aide d&#39;une pipette Pasteur préchauffé à grande ouverture. Gardez Pasteur pipette tiède avec un bec Bunsen. Placez les organites dans le moule du bloc de paraffine dans une petite couche de paraffine liquide. </li><li> Autant que possible, essayer de manipuler tous les organites vers le centre du moule de bloc de paraffine en utilisant une aiguille de dissection réchauffé. Gardez dissection aiguille chaude en utilisant un brûleur Bunsen. </li><li> Lorsque localisation des organites dans le moule est satisfaisante, lingotière légèrement pour solidifier la couche de paraffine. </li><li> Terminez le bloc en versant plus de paraffine sur le dessus et ajouter une cassette d&#39;intégration histologique 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.

Le protocole vidéo actuel décrit transduction lentiviral des organites de l&#39;épithélium intestinal primaire et l&#39;analyse en aval de ces organites en utilisant des techniques d&#39;ARN quantitatifs et immunohistochimie. Transduction lentivirale est souvent réalisée dans des cellules adhérentes ou flottantes dans des plaques de culture. Étant donné que la structure tridimensionnelle des organites les rend difficiles à pénétrer par des particules virales, un certain nombre de...

L&#39;épithélium intestinal est l&#39;un des tissus les plus proliférant rapidement corporels, qui a causé d&#39;attirer un grand intérêt de la recherche sur les cellules cancéreuses et souches. En 2009, une technique a été publié pour générer cultures durables de petites cryptes intestinales dans matrigel, conserver une structure en 3 dimensions 1. Ces structures, appelées organites intestinaux, peuvent être cultivées en utilisant des techniques standard, avec entourant milieu supplémenté avec un certain nombre de facteurs de croissance définis, y compris la BMP-signalisation inhibiteur de la voie noggine (cheville en bois), la voie Wnt-signalisation activateur rspondin 1 (Rspo1) et le facteur de croissance épidermique (EGF) tout trouvé pour améliorer la prolifération intestinal 2-4. Organites dépassent lignées cellulaires de cancer dans les aspects traditionnels qu&#39;ils sont non mutée, ont maintenu hiérarchie de cellules souches, afficher polarisation cellulaire intacte et la différenciation d&#39;exposition dans toutes les lignées cellulaires trouvés dans la petite int naissantépithélium Estinal. Comme ils peuvent être transduites pour mener transgènes ou interférence ARN constructions 5, ils sont utilisés pour étudier des éléments génétiques spécifiques, l&#39;emportant sur ​​des expériences utilisant des souris transgéniques en facettes de coût et de vitesse. L&#39;expression transgénique dans organites peut être effectuée en utilisant soit retroviral murin ou vecteurs lentiviraux 6,7. En raison des limitations de retrovirus murins, capables de transduire des cellules mitotiques exclusivement 8, la transduction lentivirale est plus fréquemment utilisé pour des cellules qui sont difficiles à infecter, tels que les organites. Virally transduction et exprimant de façon stable organoïdes transgéniques peuvent être utilisés pour une multitude d&#39;analyses en aval, y compris ARN analyses quantitatives et immunohistochimie. Pris ensemble, la culture de organites de cellules épithéliales intestinales primaires a évolué pour devenir une technique de routine qui est facile à mettre en œuvre sans exigences spécifiques de laboratoire, et est devenu le roman standard CEll culture dans la recherche sur l&#39;épithélium intestinal. Techniques de transduction virale et l&#39;analyse aval ultérieure dans organites sont fastidieux d&#39;effectuer des expériences et d&#39;aider organoïdes nous avons généré ce protocole vidéo, montrant des méthodes pour lentiviral transduction des organites culture. Nous montrons en outre la façon dont le traitement correct des organites peuvent augmenter le rendement et donc d&#39;améliorer les performances de l&#39;analyse en aval en utilisant des techniques d&#39;ARN ou immunohistochimie. Dans le protocole, organites qui sont dérivés de petites cryptes intestinales ont été exclusivement utilisées, bien que les techniques décrites peuvent être appliquées aux organites côlon ainsi.

<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.

Transduction lentiviral Organoid La technique de transduction organoïde utilisant des particules lentiviraux dépend de manipulation correcte des organites avant et pendant la transduction. Organites (figure 3A) ont été cultivées et ils ont été perturbés dans cryptes simples (figure 3B). Comme indiqué précédemment, ces cryptes simples, lorsqu&#39;elles sont cultivées en présence de l&#39;inhibiteur de GSK3 Chir99021 cryptes sont devenues...

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 protocole pour Lentiviral transduction et analyse aval de intestinale organoïdes

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

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Research

JoVE Journal

Biology

30.2K vues.  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. 

Vidéo: 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 et astrocytes de co-culture de dosage pour l&#39;analyse du contenu haute de la neurotoxicité

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

Recherche

Biologie

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.

La détection des protéines ubiquitination

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.

Génération de cellules souches pluripotentes induites par la reprogrammation des fibroblastes embryonnaires de souris avec un facteur de transcription Quatre, doxycycline inductible système de transduction lentiviraux

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.

Imagerie de cellules vivantes primaires de rats néonatals cardiomyocytes Suite à adénovirus et Lentivirales transduction confocale à disque rotatif Microscopie

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 protocole de vidéo infections rétrovirales dans intestinale primaire 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 ...

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.

Protocole de laboratoire pour les Analyses de contenu Gut génétiques de macroinvertébrés aquatiques Using Group-specific ADNr amorces

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.

Oeufs fécondés de livraison assistée par laser des gènes gène aux souris

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.

Culture 3D d’organoïdes de l’épithélium intestinal des villosités en cours de dédifférenciation

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.

Analyse du stress oxydatif dans les organoïdes intestinaux murins à l’aide d’une sonde fluorogénique sensible aux espèces réactives de l’oxygène

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

Culture 3D d’organoïdes à partir de cryptes intestinales murines et de cellules souches uniques

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.

Pleins feux sur l’auteur : La culture 3D d’organoïdes à partir de cryptes intestinales murines et d’une seule cellule souche pour la recherche sur les organoïdes  

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 culture 3D d’organoïdes à partir de cryptes intestinales murines et d’une seule cellule souche pour la recherche organoïde

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