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. Preparación de polietilenimina (PEI) como reactivo de transfección <ol><li> Disolver aproximadamente 150 mg de PEI en 100 ml de H 2 O. </li><li> Ajuste solución a pH 7,4 mediante la adición de HCl hasta que la solución se vuelva clara y revuelva hasta que se disuelva por completo. Esto puede tomar entre 10 y 60 min y añadir agua a una concentración final de 1 mg / ml. </li><li> Cuando clara, filtrar la solución PEI a través del filtro de 0,22 micras estéril y guárdelo en un congelador a -80ºC en alícuotas de 5 ml. </li></ol> 2. Producción de partículas de Lentiviral Día 1: <ol><li> Células HEK293T de Split a 60% -80% de confluencia en 162 cm 2 matraz grande o placa de Petri en medio de cultivo línea celular (DMEM suplementado con 10% de FCS, 1% de penicilina / estreptomicina y suplemento de glutamina 2 mM). </li></ol> Día 2: <ol start="2"><li> Preparar la solución de transfección de ADN que contiene 45 g de ADN plasmídico total sumandovectores lentivirales de embalaje (7 g de pVSVg; 5 g de pRSV rev; 13 g de pMDL) y 20 g de plásmido lentiviral que codifican el gen de interés o shRNA de interés. Ajustar a un volumen de 1 ml usando DMEM. </li><li> Preparar la solución de transfección PEI mediante la adición de 90 l de 1 mg / ml de PEI a 930 l de DMEM y se incuba durante 5 min a temperatura ambiente. </li><li> Añadir solución de transfección de ADN a la solución de PEI. Vortex o invertir un número de veces e incubar durante 5 min a temperatura ambiente para obtener solución de transfección de ADN. </li><li> Gotear 2 ml de la solución de transfección de ADN en las células HEK293T y se incuba durante 4 h en una incubadora de cultivo celular humidificado en 37 ° C. </li><li> Después de 4 horas, refrescar el medio de cultivo para eliminar PEI. No es necesario lavar las células antes de añadir nuevo medio. NOTA: PEI es citotóxicos e incubación veces más que 4 horas pueden causar daño a las células HEK293T. </li></ol> Día 4: <ol start="7"><li> Reemplace supernatant con el nuevo medio de cultivo. Mantenga sobrenadante (que contiene virus); esto será usado en la etapa 2.10. </li><li> Ponga sobrenadante en un matraz de 15 ml. Para eliminar las células muertas, centrifugar durante 5 min a 500 x g. </li><li> Empuje sobrenadante a través de 0,45 micras filtro utilizando una jeringa grande 60 ml. Guarde la noche a 4 ° C. </li></ol> Día 5: <ol start="10"><li> Recoger el segundo lote de sobrenadante; centrífuga y filtro como en los pasos 2.8 a 2.9. </li><li> Reúnase el sobrenadantes de pasos 2.9 y 2.10 en tubos de ultracentrífuga y centrifugar a 50.000 xg en una ultracentrífuga durante 90 min. </li><li> Sacar cápsulas que contienen los tubos de ultracentrífuga muy cuidadosamente y poner en una campana de flujo laminar, recordando la orientación del tubo dentro de la centrífuga. </li><li> Abrir cápsula sosteniendo el tubo de ultracentrífuga y medio decantar cuidadosamente de tal manera que la pastilla está en el lado superior del tubo. Desde bolitas virales pueden ser difíciles de visualizar, recordar en quélado del tubo de un pellet se habrá formado. Tome una micropipeta y quitar el último bit de medio, mientras que teniendo cuidado de no agitar el sedimento marrón opaco que es visible en el lado de la parte inferior del tubo de ultracentrífuga. </li><li> Resuspender este precipitado en 500 l de medio de cultivo suplementado con organoide nicotinamida 10 mM, 10 mM Chir99021, 10 mM Y27632 y 8 mg / ml de polibreno. Resuspensión en este medio es importante, ya que el virus de título elevado se usa para transduct directamente organoides. </li><li> Opcionalmente: congelar el virus en este medio en alícuotas en dos lotes de 250 l en -80 ° C. </li></ol> 3. de Lentiviral Transducción de organoides Día 0: <ol><li> Dividir un total de 0,95 cm 2 y de organoides dos días antes de transducción en un nuevo pozo, con el objetivo de obtener unos 50 pequeños organoides. Dividir organoides acuerdo con el protocolo previamente publicado 1 (Figura 3A, B). </li><li> Suplemento organoid medio de cultivo con 10 mM Chir99021 y nicotinamida 10 mM para obtener criptas proliferativas hiper quística (Figura 3C). NOTA: criptas quísticas desarrollarán mejor cuando organoides recién divididas se cultivan en presencia de Chir99021. </li></ol> Día 2: <ol start="3"><li> Organoides Cosecha de la pipeta hacia arriba y abajo de la matrigel y medio, alterando de esta manera la mezcla con una micropipeta p1000. Colocar la mezcla en un tubo de 15 ml. </li><li> Interrumpir adicionalmente usando una pipeta Pasteur en el que la abertura distal se ha reducido por fusión. Organoides centrífuga para sedimentar durante 5 min a 100 x g. NOTA: Dependiendo del tamaño de la centrífuga, utilice una velocidad centrífuga diferente. Es necesario encontrar la velocidad exacta en la que interrumpen organoides se separan de la mezcla. </li><li> Eliminar el sobrenadante y añadir 500 l de tripsina 1x precalentado (similar al 0,25% de tripsina). Resuspender organoides en tripsina y se incuba3 min en un baño a 37 ° C. </li><li> Inactivar la tripsina mediante la adición de 3,5 ml de medio de cultivo línea celular (DMEM suplementado con 10% de FCS, 1% de penicilina / estreptomicina y glutamina). Centrifugar durante 5 min a 500 x g. </li><li> Eliminar el sobrenadante dejar pellet en aproximadamente 20 l de medio. La transferencia de los organoides en esta última pequeña cantidad de medio a un pozo en una placa de 48 pozos. </li><li> Añadir alta lentivirus título en medio de transducción tal como se describe en el paso 2.14, volver a suspender y continuar con el paso 3.10. Al encontrarse con baja eficacia de transducción, paso 3.9 se puede añadir. NOTA: Para la transducción eficiente, por lo general añadir una alícuota de 250 l de alta lentivirus título a un pocillo de organoides. Esto representa el 50% de todo el lentivirus cosechado de una sola producción, como se describe en el paso 2 de este protocolo. </li><li> Opcionalmente: para mejorar la eficacia de transducción, realice espinoculación poniendo los 48 y la placa que contiene organoides en un pre-calentado centrífuga de 326; C y giran a 600 xg durante 1 hr. </li><li> Ponga la mezcla organoide-virus en cultivo incubadora y se incuba durante 1 hora a 37 ° C en una incubadora de cultivo para permitir la transducción. </li><li> Opcionalmente: para mejorar aún más la eficacia de transducción, incubar organoides de 3 horas adicionales a 37 ° C en una incubadora de cultivo. </li><li> Añadir 1 ml de medio de cultivo organoide y resuspender la mezcla organoide-virus, transferir a un tubo de microcentrífuga y se centrifuga en una microcentrífuga durante 5 min a 850 xg para sedimentar organoides. </li><li> Eliminar el sobrenadante y resuspender el precipitado en 20 l de Matrigel enfriado con hielo. Dado que el material se solidifica cuando se vuelve más caliente, utilizar puntas de pipeta que se enfrían por pipeteando arriba y abajo PBS enfriado con hielo para un número de veces. </li><li> Ponga la gotita en el medio de un pozo en una placa de 48 pocillos e incubar en la cultura incubadora a 37 ° C durante 15 min para solidificar. </li><li> Después de 15 min, añadir cuidadosamente 250 l de medio de cultivo suplementado con organoideNicotinamida 10 mM, 10 mM y 10 mM Chir99021 Y27632. Fragmentos de organoides alteradas pequeñas formarán en pequeños organoides quísticas dentro de las 24 horas. </li></ol> Día 5: <ol start="16"><li> Actualizar medio y complementar con un antibiótico de selección (por puromicina, utilice 4 g / ml). </li></ol> Día 7: <ol start="17"><li> Reemplace medio de medio de cultivo organoid norma, complementado con la selección de antibióticos. NOTA: en ciernes será completa 2-3 semanas después de la retirada Chir99021. Después de la selección, organoides pueden ser cultivadas sin selección de antibióticos. </li></ol> 4. organoide Preparación de ARN para RT-PCR cuantitativa o Microarray <ol><li> Para la preparación de ARN, utilizar un kit comercial de acuerdo con el protocolo del fabricante. Retire el medio de organoides y añadir 350 l de tampón RLT, complementados con β-mercaptoetanol recta en la cúpula de matrigel contiene organoides. Resuspender las material en RLT pipeteando usando micropipeta p1000. </li><li> Realizar más de preparación de ARN de acuerdo con el protocolo del fabricante. </li><li> Opcionalmente: aumentar el rendimiento de ARN mediante amplificación usando el sistema Ovation Pico WTA, requiriendo de entrada inicial de 50 g de ARN total de acuerdo con el protocolo del fabricante. </li><li> Opcionalmente: para el control de calidad antes de RNA microarrays, las muestras se ejecutan en un Bioanalyzer 2100 utilizando un chip de ARN total de nano eucariota, con el objetivo de un número de integridad del ARN (RIN) de 8,5 o más (Figura 5) de acuerdo con el protocolo del fabricante. </li></ol> 5. organoides de transformación de parafina y inmunohistoquímica <ol><li> Antes de iniciar el organoid incrustar pre-calentar un bloque de aluminio con agujeros de montaje de vidrio tubos de diámetro 12 mm de incubadora a 70 ° C para mantener el líquido de parafina. </li><li> Tome un solo pozo con organoides completamente desarrollados y eliminar el medio, dejando los organoides incrustados intacta. </li><li> Añadir 1 mlde paraformaldehído al 4% en PBS directamente al pozo y fijar a 4 ° C en cualquier lugar de 1 hora a la noche. </li><li> Reemplazar paraformaldehído con 1 ml de PBS enfriado con hielo. Opcionalmente: mantener organoides fijos en PBS hasta 1 semana a 4 ° C después de este paso. </li><li> Resuspender organoides fijos en 1 ml de PBS y colocar en vial de vidrio. </li><li> Deje organoides se hunden hacia abajo durante 1 min, se decanta PBS y se reemplazan con etanol al 70% en el que un par de gotas de solución de eosina se disuelven para permitir la visualización de organoides todo el proceso de incrustación. </li><li> Deja organoides en etanol al 70% a temperatura ambiente. Después de 30 min, eliminar el etanol 70% en cuidadosamente decantación, ser capaz de visualizar los organoides por el ojo a causa de leve del color rosáceo eosina. Reemplace solución incrustar con etanol al 96%. </li><li> Repita el paso 5.7, cada vez que la sustitución de la solución de la incrustación con la siguiente. Pase organoides a través de las siguientes soluciones a continuación: etanol al 70%, etanol al 90%, etanol al 96%, 100% ethanol, etanol 100%, xileno, xileno. </li><li> Decantar el último lavado xileno y verter la parafina en el tubo. Poner el tubo inmediatamente en el bloque de aluminio pre calentado a 70 ° C durante 30 min y reemplazar parafina con parafina nuevo limpio. </li><li> Vierta de parafina apagado y pipeta organoides en el molde de bloque de parafina utilizando una pipeta precalentado Pasteur con gran abertura. Mantenga pipeta Pasteur de tibia con un mechero Bunsen. Coloque todos los organoides en el molde bloque de parafina en una pequeña capa de parafina líquida. </li><li> En lo posible, trate de manipular todos los organoides hacia el centro del molde bloque de parafina utilizando una aguja de disección calentado. Mantenga disección aguja caliente usando un mechero Bunsen. </li><li> Cuando localización de organoides en el molde es satisfactorio, coquilla ligeramente para solidificar la capa de parafina. </li><li> Termina el bloque mediante el vertido de más de parafina en la parte superior y añadir una cinta de incrustación histológico estándar. </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.

El protocolo de vídeo actual describe la transducción lentiviral de organoides de epitelio intestinal primaria y análisis de aguas abajo de estos organoides utilizando técnicas de ARN cuantitativos e inmunohistoquímica. Transducción lentiviral se realiza a menudo en las células adherentes o flotantes en placas de cultivo. Dado que la estructura tridimensional de organoides los hace difíciles de penetrar por las partículas virales, se utiliza un número de métodos para aumentar la e...

El epitelio intestinal es uno de los tejidos corporales que proliferan más rápidamente, lo que ha causado a atraer amplio interés de la investigación sobre células cancerosas y madre. En 2009 se publicó una técnica para generar culturas de larga duración de las pequeñas criptas intestinales en matrigel, conservando una estructura dimensional 3 1. Estas estructuras, denominadas organoides intestinales, pueden ser cultivadas utilizando técnicas estándar, y la zona medio suplementado con un número de factores de crecimiento definidos, incluyendo la BMP-noggin inhibidor de la vía de señalización (Nog), el potenciador de la vía Wnt de señalización rspondin 1 (Rspo1) y factor de crecimiento epidérmico (EGF) encuentran todos para mejorar la proliferación intestinal 2-4. Organoides superan las líneas tradicionales de células de cáncer en los aspectos que son no mutado, han mantenido jerarquía de células madre, muestran la polarización celular intacta y la diferenciación de exposiciones en todos los linajes de células que se encuentran en la naciente pequeña intepitelio estinal. Puesto que pueden ser transducidas para llevar a transgenes o la interferencia de ARN construye 5, que se utilizan para estudiar elementos genéticos específicos, que prevalezca experimentos usando ratones transgénicos en facetas de costo y la velocidad. La expresión transgénica en organoides se puede realizar utilizando cualquiera retroviral murino o vectores lentivirales 6,7. Debido a las limitaciones de los retrovirus murinos, capaces de transducir células mitóticas exclusivamente 8, la transducción lentiviral se utiliza con más frecuencia para las células que son difíciles de infectar, tales como organoides. Virally transducidas y que expresan establemente organoides transgénicos pueden ser utilizados para una multitud de análisis aguas abajo, incluyendo ARN análisis cuantitativos e inmunohistoquímica. En conjunto, la cultura de organoides de células epiteliales intestinales primarias se ha convertido en una técnica rutinaria que es fácil de implementar, sin requisitos específicos de laboratorio, y se ha convertido en la novela de serie en cecultura ll en la investigación sobre el epitelio intestinal. Técnicas de transducción viral y análisis de aguas abajo posterior en organoides son tediosos para realizar y ayudar experimentos organoides generamos este protocolo de vídeo, que muestra los métodos para la transducción lentiviral de organoides cultivadas. Tenemos, además, muestra cómo el procesamiento correcto de organoides puede aumentar el rendimiento y, por tanto, mejorar el rendimiento de análisis de aguas abajo usando técnicas de ARN o inmunohistoquímica. En el protocolo, se utilizaron exclusivamente organoides que se derivan de pequeñas criptas intestinales, aunque las técnicas descritas pueden aplicarse a organoides colon también.

<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.

Transducción lentiviral organoid La técnica de la transducción organoid utilizando lentiviral partículas depende de la correcta manipulación de los organoides antes y durante la transducción. Organoides (Figura 3A) se cultivaron y se fueron interrumpidas en criptas individuales (Figura 3B). Como se informó anteriormente, estas criptas individuales, cuando se cultivan en presencia del inhibidor de GSK3 Chir99021 se convirtieron en criptas quí...

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 protocolo para la de Lentiviral Transducción y Análisis de Aguas abajo de Intestinal organoides

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

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

Research

JoVE Journal

Biology

30.2K vistas.  Academical Medical Center. In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry. 

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

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

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

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

A neuronales y astrocitos Co-cultivo de ensayo para el análisis de alto contenido de neurotoxicidad

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

Investigación

Biología

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 detección de ubiquitinación de proteínas

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.

Generación de células madre pluripotentes inducidas por reprogramación de fibroblastos embrionarios de ratón con un factor de transcripción Cuatro, doxiciclina inducible sistema de transducción de lentivirales

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.

Imágenes de células vivas de primarias de rata neonatal cardiomiocitos Siguiendo Adenoviral y lentiviral transducción Usando Confocal Spinning disco Microscopía

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 Protocolo de vídeo de la infección retroviral en Primaria Intestinal organoide Cultura

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

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

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

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

Protocolo de laboratorio para análisis genéticos de contenido intestinal de macroinvertebrados acuáticos con grupo-específica rDNA cartillas

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.

Entrega de Gene lentivirales asistida por láser ratón huevos fertilizados

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.

Cultivo 3D de organoides del epitelio de las vellosidades intestinales sometidos a desdiferenciación

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.

Análisis del estrés oxidativo en organoides intestinales murinos utilizando una sonda fluorogénica sensible a las especies reactivas de oxígeno

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

Cultivo 3D de organoides a partir de criptas intestinales murinos y células madre individuales

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 destacado: El cultivo en 3D de organoides de criptas intestinales murinas y una sola célula madre para la investigación de organoides  

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.

El cultivo 3D de organoides a partir de criptas intestinales murinas y una sola célula madre para la investigación de organoides

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