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. Preparação de polietilenimina (PEI) como Reagente de Transfecção <ol><li> Dissolve-se cerca de 150 mg de PEI em 100 ml de H 2 O. </li><li> Ajuste a solução para pH 7,4 pela adição de HCl até que a solução torna-se clara e mexa até dissolver completamente. Isto pode levar entre 10 e 60 min e adicionar água a uma concentração final de 1 mg / ml. </li><li> Quando clara, filtrar a solução PEI através estéril filtro de 0,22 um e armazenar em um freezer -80 ° C em alíquotas de 5 ml. </li></ol> 2. Produção de Lentivirus Particles Dia 1: <ol><li> Células HEK293T Dividir a 60% -80% de confluência em 162 cm2 de frasco ou placa de petri grande em linha de células de meio de cultura (DMEM suplementado com 10% de FCS, 1% de penicilina / estreptomicina e glutamina 2 mM). </li></ol> Dia 2: <ol start="2"><li> Preparar a solução de transfecção de DNA contendo 45? G de DNA total do plasmídeo adicionando juntosvectores lentivirais de embalagem (7 ug de pVSVg; 5 ug de pRSV rev; 13 ug de pMDL) e 20 ug de plasmídeo de lentivírus que codificam o gene de interesse ou shRNA de interesse. Ajustar para um volume de 1 ml, utilizando meio DMEM. </li><li> Preparar a solução de transfecção PEI por adição de 90 ul de 1 mg / ml de PEI a 930 ul de DMEM e incubar durante 5 min à temperatura ambiente. </li><li> Adicionar solução de transfecção de ADN a uma solução de PEI. Vortex ou inverter um número de vezes e incubar durante 5 min à temperatura ambiente para se obter a solução de transfecção de ADN. </li><li> Gotejamento 2 ml da solução de transfecção de ADN para as células HEK293T e incubar durante 4 horas numa incubadora de cultura de células em humidificada a 37 ° C. </li><li> Após 4 horas, atualize o meio de cultura para remover PEI. Não é necessário lavar as células antes da adição de meio novo. NOTA: PEI é citotóxico e incubação vezes mais do que 4 horas pode causar danos às células HEK293T. </li></ol> Dia 4: <ol start="7"><li> Substitua supernatant com o novo meio de cultura. Manter sobrenadante (contendo vírus); este vai ser utilizado no passo 2.10. </li><li> Coloque sobrenadante em 15 ml frasco. Para remover as células mortas, centrifugar durante 5 minutos a 500 x g. </li><li> Empurrar sobrenadante através de 0,45 um filtro usando um grande seringa de 60 mL. Armazenar durante a noite a 4 ° C. </li></ol> Dia 5: <ol start="10"><li> Recolhe-se o segundo lote do sobrenadante; centrifugar e filtrar tal como nos passos 2,8-2,9. </li><li> Reúnem-se os sobrenadantes dos passos 2.9 e 2.10 em tubos de ultracentrífuga e centrifugar a 50000 xg numa ultracentrífuga durante 90 min. </li><li> Retire cápsulas contendo os tubos de ultracentrífuga muito cuidadosamente e colocados numa câmara de fluxo laminar, lembrando-se a orientação do tubo para dentro da centrífuga. </li><li> Abrir de retenção da cápsula e tubo de ultracentrífuga meio decantar cuidadosamente, de tal forma que o pelete é no lado superior do tubo. Desde pelotas virais podem ser difíceis de visualizar, para o que se lembrarlado do tubo, um sedimento terá formado. Tomar uma micropipeta e remover último bit de meio, tendo cuidado para não agitar o sedimento castanha opaca que é visível do lado da parte inferior do tubo de ultracentrífuga. </li><li> Ressuspender este sedimento em 500 ul de meio de cultura organï¿½de suplementadas com 10 mM de nicotinamida, 10 uM Chir99021, 10 uM Y27632 e 8 ug / mL de polibreno. A ressuspensão neste meio é importante já que o vírus de título elevado é usado para transduct Organóides directamente. </li><li> Opcionalmente: congelar o vírus neste meio aliquotadas em dois lotes de 250 ul em -80 ° C. </li></ol> 3. Transdução Lentivirus de Organóides Dia 0: <ol><li> Dividir uma completa 0,95 centímetros 2 poço de Organóides dois dias antes transdução em um novo poço, com o objetivo de obter cerca de 50 pequenas Organóides. Dividir Organóides acordo com o protocolo previamente publicado 1 (Figura 3A, B). </li><li> Suplemento organï¿½de meio de cultura com 10 uM Chir99021 e 10 mM de nicotinamida para obter hiper cística criptas proliferativas (Figura 3C). NOTA: criptas cística irá desenvolver melhor quando Organóides recém divididas são cultivadas na presença de Chir99021. </li></ol> Dia 2: <ol start="3"><li> Organóides colheita por pipetagem cima e para baixo do matrigel e médio, interrompendo assim a mistura com uma micropipeta P1000. Coloque a mistura em um tubo de 15 ml. </li><li> Perturbar ainda mais usando pipeta Pasteur em que a abertura distal foi diminuído por fusão. Organóides Centrifugar para sedimentar durante 5 min a 100 x g. NOTA: Dependendo do tamanho da centrífuga, centrífuga de usar uma velocidade diferente. É necessário encontrar a velocidade exacta em que interrompeu Organóides são separadas da mistura. </li><li> Remover o sobrenadante e adicionar 500 ul de tripsina (semelhante à tripsina 0,25%) pré-aquecida 1x. Ressuspender Organóides em tripsina e incubar3 min num banho de água a 37 ° C. </li><li> Inactivar a tripsina por adição de 3,5 ml de meio de cultura de linha celular (DMEM suplementado com 10% de FCS, 1% de penicilina / estreptomicina e glutamina). Centrifugar durante 5 minutos a 500 x g. </li><li> Remover o sobrenadante para deixar o granulado em cerca de 20 ul de meio. Transferir os Organóides neste último pequena quantidade de meio para um poço numa placa de 48-poços. </li><li> Adicionar alta lentivírus título na forma de transdução, tal como descrito no passo 2.14, ressuspender e continuar para o passo 3.10. Ao encontrar baixa eficácia de transdução, passo 3.9 podem ser adicionados. NOTA: Para a transdução eficiente, tipicamente adicionar uma alíquota de 250 ul de elevado título de lentivírus para um poço de Organóides. Isso representa 50% de todo o lentivírus colhidas a partir de uma única unidade de produção, tal como descrito no passo 2 deste protocolo. </li><li> Opcionalmente: para aumentar a eficácia de transdução, execute spinoculation colocando os 48 poços contendo Organóides em uma pré-aquecido centrífuga em 326; C e gire a 600 g durante 1 hora. </li><li> Coloque a mistura organï¿½de-vírus em cultura incubadora e incubar durante 1 hora a 37 ° C numa incubadora de cultura para permitir a transdução. </li><li> Opcionalmente, para melhorar ainda mais a eficácia de transdução, Organóides incubar durante 3 horas adicionais a 37 ° C numa incubadora de cultura. </li><li> Adicionar 1 ml de meio de cultura organï¿½de e ressuspender a mistura organï¿½de-vírus, transferir para um tubo de microcentrifugação e centrifuga-se numa microcentrífuga durante 5 min a 850 xg para sedimentar Organóides. </li><li> Remover o sobrenadante e ressuspender o sedimento em 20 mL de matrigel arrefecido com gelo. Uma vez que o material solidifica quando se torna mais quente, usar as pontas de pipeta que são refrigerados por pipetagem para cima e para baixo PBS gelado para um número de vezes. </li><li> Coloque a gota no meio de um poço numa placa de 48 poços e incuba-se a cultura numa estufa a 37 ° C durante 15 minutos para solidificar. </li><li> Após 15 min, adicionar cuidadosamente 250 ul de meio de cultura suplementado com organï¿½de10 mM de nicotinamida, 10 uM e 10 uM Chir99021 Y27632. Pequenos fragmentos organï¿½de rompidas vai formar em pequenas Organóides císticas dentro de 24 horas. </li></ol> Dia 5: <ol start="16"><li> Actualizar médio e completar com um antibiótico de selecção (por puromicina, utilizar 4 ug / ml). </li></ol> Dia 7: <ol start="17"><li> Substituir médio por meio de cultura organóide standard, complementada com a seleção do antibiótico. NOTA: brotamento será completa de 2-3 semanas após a suspensão Chir99021. Após selecção, Organóides podem ser cultivadas sem selecção de antibiótico. </li></ol> 4. organï¿½de Preparação do RNA para RT-PCR quantitativo ou Microarray <ol><li> Para a preparação de ARN, utilizar um kit comercial de acordo com o protocolo do fabricante. Retirar o meio de Organóides e adicionar 350 mL de tampão RLT, suplementadas com β-mercaptoetanol em frente a cúpula da matrigel contendo Organóides. Volte a suspender as materiai em RLT pipetando usando p1000 micropipeta. </li><li> Realize mais prep RNA de acordo com o protocolo do fabricante. </li><li> Opcionalmente: aumentar a produção de RNA por amplificação utilizando o sistema Ovation Pico WTA, exigindo a entrada inicial de 50 mg de RNA total de acordo com o protocolo do fabricante. </li><li> Opcionalmente: para controle de qualidade antes de RNA microarray, as amostras são executados em um Bioanalyzer 2100 usando um chip nano RNA eukaryote totais, apontando para um número de integridade do RNA (RIN) de 8,5 ou mais (Figura 5), de acordo com o protocolo do fabricante. </li></ol> 5. O tratamento Organóides para inclusão em parafina e imuno-histoquímica <ol><li> Antes do início da organï¿½de incorporação pré-aquecer um bloco de alumínio com furos de ajuste 12 milímetros tubos de vidro de diâmetro em estufa a 70 ° C para manter a parafina líquida. </li><li> Tome um único bem com Organóides adultas e remover o meio, deixando as Organóides embutidos intacta. </li><li> Adicionar 1 mlde 4% de paraformaldeído em PBS e linear para fixar bem a 4 ° C em qualquer lugar a partir de 1 hora até durante a noite. </li><li> Substituir paraformaldeído com 1 ml de PBS gelado. Opcionalmente: Organóides manter fixos em PBS até 1 semana a 4 ° C, após este passo. </li><li> Ressuspender Organóides fixos em 1 ml de PBS e colocar em frascos de vidro. </li><li> Deixe Organóides afundar para baixo durante 1 min, decanta-se PBS e substituir com etanol a 70%, em que um par de gotas de solução de eosina são dissolvidos para permitir a visualização de Organóides todo o processo de incorporação. </li><li> Deixar Organóides em etanol a 70% à temperatura ambiente. Após 30 min, remover o etanol 70% por decantação com cuidado, ser capaz de visualizar os Organóides por olho por causa de cor ligeiramente rosada eosina. Substituir a incorporação de solução com 96% de etanol. </li><li> Repetir o passo 5.7, substituindo cada vez que a incorporação da solução com o seguinte. Organóides passar através das seguintes soluções: posteriormente, etanol a 70%, etanol 90%, etanol 96%, 100% ethanol, etanol a 100%, xileno, xileno. </li><li> Decantar a última lavagem xileno e despeje parafina para dentro do tubo. Colocar imediatamente o tubo no bloco de alumínio pré aquecido a 70 ° C durante 30 min e substituir com parafina nova limpeza da parafina. </li><li> Despeje parafina off e pipeta Organóides no molde bloco de parafina usando um pré-aquecido pipeta Pasteur com grande abertura. Mantenha Pasteur pipeta morna usando um bico de Bunsen. Coloque todos os Organóides no molde de bloco de parafina em uma pequena camada de parafina líquida. </li><li> Tanto quanto possível, tentar manipular todas Organóides para o centro do molde de bloco de parafina, utilizando uma agulha de dissecção aquecido. Mantenha dissecção agulha quente usando bico de Bunsen. </li><li> Quando localização de Organóides no molde é satisfatória, molde a chill ligeiramente para solidificar a camada de parafina. </li><li> Termine o bloco derramando mais de parafina na parte superior e adicione uma cassete incorporação histológica convencional. </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.

O protocolo de vídeo actual descreve transdução lentiviral de Organóides de epitélio intestinal primária e análise a jusante destas Organóides RNA utilizando técnicas quantitativas e imuno-histoquímica. Transdução de lentivírus é freqüentemente realizada em células aderentes ou flutuantes em placas de cultura. Uma vez que a estrutura tridimensional de Organóides torna difícil de penetrar por partículas virais, um número de métodos para aumentar a eficácia são utilizado...

O epitélio intestinal é um dos tecidos corporais mais proliferam rapidamente, o que fez com que ele atrair grande interesse da pesquisa sobre as células cancerosas e tronco. Em 2009, uma técnica foi publicado para gerar culturas de longa duração de pequenas criptas intestinais em matrigel, conservando uma estrutura dimensional 3 1. Estas estruturas, denominado Organóides intestinal, pode ser cultivado usando técnicas convencionais, com áreas meio suplementado com um número de factores de crescimento definidas, incluindo a BMP-inibidor da via de sinalização noggin (nog), o intensificador de via de sinalização Wnt-1 rspondin (RSPO1) e factor de crescimento epidérmico (EGF) todos encontrados para aumentar a proliferação intestinal 2-4. Organóides superar tradicionais linhas celulares de cancro nos aspectos que são não mutado, mantiveram hierarquia de células-tronco, exibir polarização celular intacto e diferenciação exposição em todas as linhagens de células encontradas na nascente pequeno intepitélio estinal. Uma vez que podem ser transduzidas para transportar transgenes ou interferência de ARN constrói 5, eles são utilizados para estudar os elementos genéticos específicos, compensando experiências utilizando ratinhos transgénicos em facetas de custo e de velocidade. A expressão transgénica em Organóides pode ser realizada utilizando retrovírus murino ou vectores lentivirais 6,7. Devido às limitações de retrovírus murino, capazes de transdução de células mitóticas exclusivamente 8, transdução lentiviral é mais frequentemente usado para células que são difíceis de infectar, como Organóides. Viralmente e transduzidas que expressam estavelmente Organóides transgénicas podem ser usadas para uma multiplicidade de análises a jusante, incluindo ARN análises quantitativa e imuno-histoquímica. Tomados em conjunto, a cultura de Organóides de células epiteliais do intestino primárias evoluiu para uma técnica de rotina que é fácil de implementar, sem requisitos laboratoriais específicos, e tornou-se o novo padrão em cecultura ll em pesquisas sobre o epitélio intestinal. Técnicas de transdução viral e análise jusante subsequente em Organóides são entediantes para executar e para ajudar experimentos organóide geramos este protocolo de vídeo, mostrando métodos para a transdução de lentivírus de Organóides cultivadas. Nós, adicionalmente, mostrar como correto processamento de Organóides pode aumentar o rendimento e, portanto, melhorar o desempenho da análise a jusante, utilizando técnicas de RNA ou imuno-histoquímica. No protocolo, Organóides que são derivados a partir de pequenas criptas intestinais foram utilizados exclusivamente, embora as técnicas descritas podem ser aplicadas a Organóides do cólon, bem.

<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.

Transdução lentiviral organóide A técnica de transdução organóide utilizando partículas de lentivírus depende manipulação correta de Organóides antes e durante a transdução. Organóides (Figura 3A) foram cultivadas e foram interrompidas em criptas individuais (Figura 3B). Como relatado anteriormente, estes criptas individuais, quando cultivadas na presença do inibidor de GSK3 Chir99021 tornou criptas císticas 9 (Fi...

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.

Um Protocolo de Transdução Lentivirus e Análise de Downstream da Intestinal Organóides

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

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JoVE Journal

Biology

30.2K visualizações.  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. 

Vídeo: 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 Neuronal e astrocitária Ensaio Co-Cultura de Análise de Conteúdo de Alta de neurotoxicidade

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

Biologia

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

Detecção de Ubiquitinação Protein

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.

Geração de células-tronco pluripotentes induzidas pela reprogramação fibroblastos de rato embrionárias com um fator de transcrição Quatro, Doxiciclina induzível Sistema de Transdução Lentivirus

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.

Imagens de células vivas de primários de ratos neonatos Cardiomyocytes Após Adenoviral e Transdução Lentivirus Usando confocal Spinning Disk Microscopia

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

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

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

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

Um Protocolo de vídeo de infecção retroviral na Primária Intestinal organóide 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 do laboratório para análises genéticas de conteúdo intestino de rDNA usando de macroinvertebrados aquáticos grupo-específicos Primers

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

Laser-assisted Lentiviral Gene Delivery to Mouse Fertilized Eggs

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

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

Entrega de gene lentiviral assistida por laser em óvulos fertilizados de camundongos

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.

Culturação 3D de Organoides do Epitélio Intestinal Villi passando por Dediferente

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.

Analisando o estresse oxidativo em organoides intestinais murinos usando sonda fluorogênica sensível à espécie de oxigênio reativa

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

Cultura 3D de Organoides de Criptas Intestinais Murinas e Célula-Tronco Única

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 em destaque: A cultura 3D de organoides de criptas intestinais murinas e uma única célula-tronco para pesquisa 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.

A Cultura 3D de Organoides de Criptas Intestinais Murinas e uma Única Célula-Tronco para Pesquisa de Organoides

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