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. Herstellung von Polyethylenimin (PEI) als Transfektionsreagenz <ol><li> Man löst etwa 150 mg von PEI in 100 ml H 2 O. </li><li> Einstellen auf pH 7,4 durch Zugabe von HCl, bis die Lösung klar wird und rühre bis zur vollständigen Auflösung. Dieser kann zwischen 10 und 60 Minuten dauern und Wasser bis zu einer Endkonzentration von 1 mg / ml. </li><li> Wenn klar ist, filtern das PEI-Lösung durch ein steriles 0,22 um Filter und Speicher in einem -80 ° C Gefrierschrank in Aliquots von 5 ml. </li></ol> 2. Herstellung von Lentivirale Partikel Tag 1: <ol><li> Split HEK293T-Zellen zu 60% bis 80% Konfluenz in 162 cm 2 -Kolben oder große Petrischale in Zellinie Kulturmedium (DMEM mit 10% FCS, 1% Penicillin / Streptomycin und 2 mM Glutamin ergänzt Ergänzung). </li></ol> Tag 2: <ol start="2"><li> Bereiten DNA Transfektion Lösung mit 45 ug Gesamt-Plasmid-DNA durch Additionlentiviralen Verpackungsvektoren (7 ug pVSVg; 5 ug pRSV rev; 13 ug pMDL) und 20 ug lentiviralen Plasmid, der das Gen von Interesse oder shRNA von Interesse kodiert. Einzustellen, um ein Volumen von 1 ml mit DMEM. </li><li> Bereiten PEI Transfektion Lösung durch Zugabe von 90 ul 1 mg / ml PEI 930 ul DMEM und Inkubation für 5 min bei Raumtemperatur. </li><li> In DNA-Transfektion Lösung PEI-Lösung. Vortex oder Invertzucker mehrfach und Inkubation für 5 Minuten bei Raumtemperatur, um DNA-Transfektion Lösung zu erhalten. </li><li> Tropft 2 ml des DNA-Transfektions-Lösung auf die HEK293T Zellen und Inkubation für 4 Stunden in einem befeuchteten Zellkulturbrutschrank bei 37 ° C. </li><li> Nach 4 Stunden, aktualisieren Sie die Kulturmediums auf PEI zu entfernen. Es ist nicht notwendig, Zellen zu waschen, bevor neues Medium. HINWEIS: PEI ist zytotoxischen und Inkubationszeiten länger als 4 Stunden kann zu Schädigungen der HEK293T-Zellen führen. </li></ol> Tag 4: <ol start="7"><li> Supe ersetzenrnatant mit neuen Kulturmedium. Halten Stand (Virus enthalten); Dies wird in Schritt 2.10 verwendet werden. </li><li> Legen Überstand in 15 ml-Kolben. Um tote Zellen, Zentrifuge für 5 Minuten bei 500 x g zu entfernen. </li><li> Schieben Stand durch ein 0,45 um-Filter unter Verwendung einer großen Spritze 60 ml. Speichern Nacht bei 4 ° C. </li></ol> Tag 5: <ol start="10"><li> Sammeln Sie die zweite Charge von Überstand; Zentrifuge und Filter, wie in den Schritten 2,8-2,9. </li><li> Pool der Überstände aus den Schritten 2.9 und 2.10 in Ultrazentrifugenröhrchen und Zentrifuge bei 50.000 xg in einer Ultrazentrifuge für 90 min. </li><li> Nehmen Sie die Kapseln Ultrazentrifugenröhrchen, die sehr sorgfältig und in eine Sterilbank, die Erinnerung an die Ausrichtung des Rohres in der Zentrifuge. </li><li> Offene Kapselhalteultrazentrifugenröhrchen dekantiert Medium sorgfältig in einer solchen Weise, daß das Pellet an der Oberseite des Rohrs. Da virale Pellets kann schwierig sein, zu visualisieren, zu erinnern, wasSeite des Rohrs ein Pellet gebildet haben. Werfen Sie einen Mikropipette und entfernen letzte bisschen Medium während kümmert sich nicht um die undurchsichtigen braunen Pellets, die auf der Seite der Unterseite des Ultrazentrifugenröhrchen sichtbar zu agitieren. </li><li> Resuspendieren dieses Pellet in 500 ul organoiden Kultur-Medium mit 10 mM Nicotinamid, 10 uM Chir99021, 10 uM Y27632 und 8 ug / ml Polybren ergänzt. Resuspension in diesem Medium ist wichtig, da mit hohem Titer-Virus wird verwendet, um Organoide direkt transduzieren. </li><li> Optional: einfrieren, das Virus in diesem Medium in zwei Chargen von 250 ul in -80 ° C aliquotiert. </li></ol> 3. Lentivirale Transduktion Organoide Tag 0: <ol><li> Split eine vollständige 0,95 cm 2 und der Organoide zwei Tage vor dem in eine neue und Weiterleitung mit dem Ziel, rund 50 kleine Organoide erhalten. Aufgeteilt Organoiden nach früher veröffentlichten Protokoll 1 (3A, B). </li><li> Ergänzen organoide Kulturmedium mit 10 &amp; mgr; Chir99021 und 10 mM Nicotinamid zystische Hyperproliferations Krypten (3C) zu erhalten. HINWEIS: Cystic Krypten am besten entwickeln, wenn frisch gespalten Organoide werden in Gegenwart von Chir99021 gewachsen. </li></ol> Tag 2: <ol start="3"><li> Ernte Organoide durch Auf- und Abpipettieren der Matrigel und mittlere, wodurch das Gemisch mit einer Mikropipette p1000 zu stören. Legen Sie die Mischung in einem 15-ml-Tube. </li><li> Weiter stören mit Pasteur-Pipette, in dem sich die distale Öffnung hat, durch Schmelzen verringert. Zentrifuge Organoiden für 5 min bei 100 x g pelletieren. HINWEIS: Abhängig von der Größe der Zentrifuge, verwenden Sie ein anderes Zentrifugendrehzahl. Es ist notwendig, die genaue Geschwindigkeit, mit der gestörten Organoide werden aus dem Gemisch abgetrennt finden. </li><li> Entfernen Sie den Überstand und fügen Sie 500 ul vorgewärmtes 1x Trypsin (ähnlich wie 0,25% Trypsin). Resuspendieren Organoide in Trypsin und Inkubation3 min in einem 37 ° C Wasserbad. </li><li> Inaktivierung von Trypsin durch Zugabe von 3,5 ml Zelllinie Kulturmedium (DMEM, ergänzt mit 10% FCS, 1% Penicillin / Streptomycin und Glutamin). Zentrifuge 5 min bei 500 x g. </li><li> Entfernen Sie den Überstand in Pellet in etwa 20 ul Medium zu verlassen. Übertragen Sie die Organoide in diesem letzten kleinen Menge des Mediums in einen Brunnen auf einem 48-Mulden Platte. </li><li> In hohem Titer Lentivirus in Übertragungsmedium, wie in Schritt 2.14, resuspendieren beschrieben und fahren Sie mit Schritt 3.10. In der Begegnung mit niedrigen Übertragungswirksamkeit, Schritt 3.9 hinzugefügt werden. HINWEIS: Für eine effiziente Weiterleitung, in der Regel fügen Sie ein Aliquot von 250 ul mit hohem Titer Lentivirus um eine Vertiefung Organoide. Dies entspricht 50% der gesamten Ernte Lentivirus aus einer Produktion wie in Schritt 2 dieses Protokolls beschrieben. </li><li> Optional: zu Übertragungswirksamkeit zu verbessern, führen spinoculation, indem Sie die 48 Well-Platte mit Organoide in einem vorgewärmten Zentrifuge auf 326; C und rotieren bei 600 × g für 1 Std. </li><li> Setzen organoide-Virus-Mischung in Kulturbrutschrank und Inkubation für 1 h bei 37 ° C in einem Brutschrank zum Übertragung ermöglichen. </li><li> Optional: zur weiteren Verbesserung der Wirksamkeit der Transduktion inkubieren Organoiden weitere 3 h bei 37 ° C in einem Brutschrank. </li><li> 1 ml der organoiden Kulturmedium und resuspendieren organoide-Virus-Mischung, Transfer in ein Mikrozentrifugenröhrchen und Zentrifuge in einer Mikrozentrifuge für 5 Minuten bei 850 xg pelletiert Organoide. </li><li> Überstand verwerfen und das Pellet in 20 ul eiskaltem Matrigel. Da das Material verfestigt, wenn es wärmer wird, verwenden Sie die Pipettenspitzen, die durch Auf- und Abpipettieren eiskaltem PBS für eine Reihe von mal gekühlt werden. </li><li> Setzen des Tröpfchens in der Mitte einer Vertiefung in einer 48 Vertiefungen Inkubieren in Kulturbrutschrank bei 37 ° C für 15 min zu verfestigen. </li><li> Nach 15 Minuten vorsichtig mit 250 ul organoide Kulturmedium, ergänzt10 mM Nicotinamid, 10 uM und 10 uM Chir99021 Y27632. Kleine gestört organoide Fragmente werden in kleine zystische Organoide innerhalb von 24 Stunden zu bilden. </li></ol> Tag 5: <ol start="16"><li> Aktualisieren Medium und zu ergänzen, mit einer Auswahl Antibiotikum (für Puromycin, verwenden Sie 4 ug / ml). </li></ol> 7. Tag: <ol start="17"><li> Ersetzen Medium für Standard organoide Kulturmedium, mit Auswahl Antibiotikum ergänzt. HINWEIS: Angehende wird komplett 2-3 Wochen nach Chir99021 Rückzug sein. Nach der Auswahl können Organoide ohne Selektionsantibiotikum angebaut werden. </li></ol> 4. Organoide RNA Vorbereitung Quantitative RT-PCR oder Microarray- <ol><li> Zur RNA-Präparation, mit einem kommerziellen Kit nach Herstellerprotokoll. Entfernen Sie das Medium aus Organoide und fügen Sie 350 ul Puffer RLT, mit β-Mercaptoethanol ergänzt gerade auf der Kuppel des Matrigel enthält Organoide. Resuspendieren der Material in RLT durch Pipettieren mit p1000 Mikropipette. </li><li> Der weitere RNA prep nach dem Protokoll des Herstellers. </li><li> Optional: RNA-Ausbeute zu erhöhen durch Amplifikation mit Ovation Pico WTA Systems und schreibt erste Eingabe von 50 ug Gesamt-RNA nach Herstellerprotokoll. </li><li> Optional: für die Qualitätskontrolle vor der RNA-Microarray, laufen Proben auf einem 2100 Bioanalyzer mit einem Eukaryoten Gesamt-RNA Nano-Chip, mit dem Ziel für eine RNA Integrity Number (RIN) von 8,5 oder mehr (5) nach dem Protokoll des Herstellers. </li></ol> 5. Die Verarbeitung Organoide für Paraffinausgießstation und Immunhistochemie <ol><li> Vor Beginn der organoiden Einbindung vorwärmen ein Aluminiumblock mit Bohrungen passend Durchmesser 12 mm Glasröhrchen im Brutschrank bei 70 ° C bis Paraffin flüssig zu halten. </li><li> Werfen Sie einen einzigen gut mit ausgewachsenen Organoide und entfernen Sie das Medium, so dass die eingebetteten Organoide intakt. </li><li> 1 mlvon 4% Paraformaldehyd in PBS direkt zu gut und zu beheben bei 4 ° C überall von 1 Stunde über Nacht. </li><li> Para Ersetzen mit 1 ml eiskaltem PBS. Optional: halten fest Organoide in PBS bis zu 1 Woche bei 4 ° C nach diesem Schritt. </li><li> Resuspendieren Fest Organoide in 1 ml PBS aufnehmen und in Glasfläschchen. </li><li> Lassen Organoiden sinken nach unten für 1 min, zu dekantieren und zu ersetzen PBS mit 70% Ethanol in dem ein paar Tropfen von Eosin-Lösung gelöst, um die Visualisierung von Organoiden gesamten Einbettungsprozesses zu ermöglichen. </li><li> Verlassen Organoiden in 70% Ethanol bei Raumtemperatur. Nach 30 Minuten, entfernen Sie 70% Ethanol durch vorsichtiges Dekantieren, die Möglichkeit, die Organoide von Auge, weil der leichte rosa Farbe Eosin sichtbar zu machen. Ersetzen Sie die Einbettung Lösung mit 96% Ethanol. </li><li> Wiederholen Sie Schritt 5.7 wird jedes Mal, Austausch der Einbettung Lösung mit dem nächsten. Pass Organoide durch die folgenden Lösungen anschließend: 70% Ethanol, 90% Ethanol, 96% Ethanol, 100% ethanol, 100% Ethanol, Xylol, Xylol. </li><li> Dekantiert man die letzten Xylol Wasch und gießen Paraffin in die Röhre. Das Glas sofort in der Pre erwärmt Aluminiumblock bei 70 ° C für 30 min und ersetzen Paraffin mit neuen sauberen Paraffin. </li><li> Gießen Paraffin aus und Pipetten Organoide in den Paraffinblock Form unter Verwendung einer vorgewärmten Pasteur-Pipette mit großer Öffnung. Halten Pasteur-Pipette warmen mit einem Bunsenbrenner. Legen Sie alle Organoide im Paraffinblockform in einer kleinen Schicht von flüssigem Paraffin. </li><li> So viel wie möglich, versuchen Sie, alle Organoide in Richtung der Mitte des Paraffinblocks Form unter Verwendung einer erwärmten Dissektion Nadel manipulieren. Halten Sie Dissektion Nadel warmen mit Bunsenbrenner. </li><li> Wenn Lokalisierung Organoide in der Form ist zufriedenstellend, Kokille leicht auf die Paraffinschicht verfestigt. </li><li> Beenden Sie den Block durch Gießen mehr Paraffin auf und fügen Sie ein Standard-histologische Einbettung Kassette. </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.

Das aktuelle Video-Protokoll beschreibt lentivirale Transduktion Organoide von der Grund Darmepithel und nachgelagerten Analyse dieser Organoide mittels quantitativer RNA-Techniken und Immunhistochemie. Lentivirale Transduktion wird oft in anhaftenden oder schwimmende Zellen in Kulturplatten durchgeführt. Da die dreidimensionale Struktur von Organoiden macht sie schwierig durch virale Partikel eindringen, sind eine Reihe von Verfahren, um die Wirksamkeit zu erhöhen. Eine Vorbehandlung der ...

Das Darmepithel ist eine der am schnellsten vermehren Körpergewebe, die verursacht hat es großes Interesse aus der Forschung über Krebs und Stammzellen zu gewinnen. Im Jahr 2009 wurde eine Technik veröffentlicht, um lang anhaltende Kulturen von Klein Darmkrypten in Matrigel erzeugen, Erhaltung eine 3-dimensionale Struktur 1. Diese Strukturen, sogenannte Darm Organoiden, kann unter Verwendung von Standardtechniken gezüchtet werden, mit Umgebungsmedium mit einer Anzahl von definierten Wachstumsfaktoren, einschließlich BMP-Signalweg-Inhibitors Noggin (nOgh) ergänzt, die Wnt-Signalwegs Enhancer rspondin 1 (Rspo1) und epidermalen Wachstumsfaktor (EGF) alle eine Darm Proliferation 2-4 verbessern. Organoide übertreffen traditionelle Krebszelllinien in den Aspekten, dass sie nicht-mutierten sind, haben Stammzellen Hierarchie beibehalten, zeigen intakten Zellpolarisation und Ausstellungs Differenzierung in allen Zelllinien in der im Entstehen begriffenen kleine int gefundenestinal Epithel. Da sie transduziert Transgene tragen oder RNA-Interferenz-Konstrukte 5, werden sie verwendet, um spezifische genetische Elemente zu untersuchen, überwiegt Experimenten mit transgenen Mäusen in Facetten Kosten und Geschwindigkeit. Transgene Expression in Organoiden kann unter Verwendung von entweder murine retroviralen oder lentiviralen Vektoren 6,7 werden. Aufgrund der Einschränkungen der murine Retroviren, in der Lage zu transduzieren mitotischen Zellen ausschließlich 8 wird lentiviralen Transduktion häufiger für Zellen, die nur schwer zu infizieren, wie Organoiden verwendet. Viral transduziert und stabil exprimierenden transgenen Organoiden kann für eine Vielzahl von Downstream-Analysen verwendet werden, einschließlich quantitative RNA-Analysen und Immunhistochemie. Zusammengenommen hat Kultur Organoide von primären intestinalen Epithelzellen in eine Routine-Technik, die einfach und ohne spezielle Laboranforderungen umzusetzen ist entwickelt, und hat sich die neue Norm in cell Kultur in Forschung auf das Darmepithel. Techniken der virale Transduktion und nachfolgende Downstream-Analyse in Organoide sind mühsam durchzuführen und um organoide Experimente helfen wir generiert dieses Video-Protokoll und zeigt Methoden zur lentivirale Transduktion der kultivierten Organoide. Wir zeigen außerdem, wie die korrekte Verarbeitung Organoide kann die Ausbeute zu erhöhen und damit die Leistung zu verbessern nachgeschalteter Analyse mittels RNA-Techniken oder die Immunhistochemie. In dem Protokoll wurden Organoiden, die von kleinen Darmkrypten abgeleitet werden ausschließlich verwendet, obwohl die beschriebenen Techniken können zum Kolon Organoide als auch angewendet werden.

<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 lentivirale Transduktion Die Technik der organoiden Transduktion unter Verwendung lentivirale Partikel hängt von richtigen Umgang mit Organoide vor und während der Weiterleitung. Organoide (3A) wurden kultiviert und sie in einzelne Krypten (3B) gestört wurden. Wie bereits berichtet, diese einzelnen Krypten, wenn sie in Gegenwart der GSK3-Inhibitor Chir99021 kultiviert wurde zystische Krypten 9 (3C). Anschlie?...

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.

Ein Protokoll für Lentivirale Transduktion und Downstream-Analyse der Darm Organoide

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

30.3K Aufrufe.  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. 

Serie: 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.

Eine Neuronale und Astrocyte Co-Kultur-Assay für High Content Analyse der Neurotoxizität

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

Forschung

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.

Detektion von Protein 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.

Erzeugung von induzierten pluripotenten Stammzellen durch Reprogrammierung embryonalen Maus-Fibroblasten mit einem Vier Transcription Factor, induzierbare Doxycycline Lentivirale Transduktionssystems

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.

Live Cell Imaging von primären Ratten-Kardiomyozyten Neonatal Nach Adenovirale und Lentivirale Transduktion mittels konfokaler Mikroskopie Spinning-Disk-

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.

Ein Video-Protokoll von retrovirale Infektion in der Grunddarm Organoide Kultur

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.

Laborprotokoll für genetische Darm Inhaltsanalysen der aquatischen Makroinvertebraten Using gruppenspezifische rDNA Primer

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.

Lasergestützte Lentivirale gen Lieferung an Maus befruchtete Eier

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-Kultivierung von Organoiden aus dem intestinalen Zottenepithel, das einer Dedifferenzierung unterzogen wird

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 von oxidativem Stress in murinen Darmorganoiden mit reaktiver Sauerstoffspezies-sensitiver fluorogener Sonde

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

3D-Kultur von Organoiden aus murinen Darmkrypten und einzelnen Stammzellen

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.

Autor im Rampenlicht: Die 3D-Kultivierung von Organoiden aus murinen Darmkrypten und einer einzelnen Stammzelle für die Organoidforschung  

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.

Die 3D-Kultivierung von Organoiden aus murinen Darmkrypten und einer einzelnen Stammzelle für die Organoidforschung

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