Reconhecemos o apoio financeiro do CPRIT (RP100119) e da Fundação Welch (I-1665).

Protocolo inteiro leva cerca de 4 dias. 1. Preparação de células para siRNA e Reporter transfecção Vamos introduzir aqui um protocolo para interrogar bibliotecas de siRNA usando um pequeno conjunto de siRNAs (384 matriz diferente piscinas siRNA em formato de 96 poços com cada conjunto constituído por siRNAs 4x destinada a um único gene) a partir de uma biblioteca de siRNA de todo o genoma para fins ilustrativos. Para este estudo particular, vamos explorar HCT116 células que apresentam sinalização Wnt e Kras desviantes e atividade p53. A linha celular adequada em estudos destinados a interrogar outros processos celulares de interesse terão de ser identificado e avaliado de uma maneira semelhante. <ol><li> Lavar 5 x 10 cm 2 placas de 80-90% confluentes, as células HCT-116 com PBS, em seguida as células por tripsinização de colheita utilizando 1 ml de solução de tripsinização / placa, seguido de neutralização com 10 mL de Meio Eagle Modificado de Dulbecco (DMEM) contendo 2% feTal soro de bovino (FBS) e 1% de penicilina / estreptomicina / placa. </li><li> Transferência de células em suspensão para um tubo cónico de 50 ml e centrifugar a ~ 130 x g durante 5 minutos, remover o sobrenadante, e re-suspender o sedimento celular em 10 ml de DMEM / FBS a 2% / 1% de penicilina / estreptomicina. </li><li> Conte o número de células com um contador de células e em seguida, fazer uma solução de células com 10 5 células / ml, manter 150 ml de suspensão celular para o próximo passo. Placa 10 4 células (100 ul) em cada poço de uma placa de cultura de células de 96 poços utilizando um dispensador de liquido Multidrop automatizado (doravante Multidrop). A suspensão de células deve ser suficiente, neste caso, para chapeamento de 12 x placas de 96 poços. </li><li> Incubar as placas com as células a 37 ° C numa incubadora com 5% de CO 2 durante a preparação da mistura de transfecção. </li></ol> 2. Preparando-se construir o Reporter Banco Solution <ol><li> Isolar repórter de alta qualidade construir DNA usando o padrão midiprep kits (NucleoBond Xtra Midi produzido porClontech) e diluir o ADN para uma concentração final de 1 mg / ml para cada repórter. </li><li> Prepare a 100 ul de solução de reserva de construção repórter combinando 30 ul de p53-FL, 30 ul de 8X TCF-RL, 30 ul de Elk1-Gal4, 6 ul de UAS-CL e 4 mL de H 2 O para obter uma mistura de ADN com uma proporção final 1:1:1:0.2 dos repórteres. Concentração de ADN final desta solução estoque mistura de ADN é de 0,3 mg / ml. </li></ol> 3. Preparando o siRNA / DNA Mix transfecção Nesta parte do protocolo, vamos preparar siRNA / DNA misturas de transfecção, que será suficiente para a transfecção de 12 x placas de 96 poços. Para este ensaio de biblioteca de 4 x placa de 96 poços de siRNAs, vamos transfectar cada pool de siRNA em triplicado. O reagente de transfecção usaremos é chamado Effectene (Qiagen). Nas nossas mãos este é o único reagente, que funciona para a entrega simultânea de siRNA e DNA em células em cultura. <ol><li> Diluir 80 ml de repórter construção estoque soluição em 8 ml de tampão de CE (Effectene Kit) para obter uma solução de DNA com uma concentração final de 3 ng / ml. Em geral, preparar suficiente mistura solução de ADN para uso imediato. </li><li> Placa de 20 ul desta solução de ADN para cada poço de um de 96 poços / placa. O número de placas de 96 poços vai depender de quantas piscinas siRNA será testado. Por exemplo, neste caso, vamos precisar de 4 x placas de 96 poços para avaliar 384 diferentes conjuntos de siRNA. </li><li> Os siRNAs a serem testadas devem estar a uma concentração de estoque de 5 mM. Caso contrário, eles podem ser semeadas réplicas e diluída para esta concentração, utilizando o tampão de diluição recomendada associado com cada biblioteca. </li><li> Transferir 2 ul de cada unidade siRNA 5 uM para o correspondente poço da placa de PCR contendo o estoque de ADN diluída com uma Biomek Automated Liquid Handler. Dependendo da escala do estudo, uma pipeta multi-canal pode ser suficiente. </li><li> Agora, será adicionado 1 ul de solução de realçador (Effectene kit) para cada poço de DNA / siRNA plate. Isto também pode ser conseguido ou com um manipulador de líquidos ou automatizado pipetador multicanal. </li><li> Permitir que a reacção Enhancer a ocorrer à temperatura ambiente durante 5 min, em seguida, adicionar 3 ml de reagente de transfeco Effectene a cada poço e incubar à temperatura ambiente durante um adicional de 10 min. </li><li> Para parar a formação de complexos de transfecção, adicionam-se 10 ul de DMEM / FBS a 2% / 1% de penicilina / estreptomicina a cada poço da placa de PCR. </li><li> Transferem-se 10 ul da mistura de transfecção a cada poço da placa de 96 poços contendo as células (recuperados a partir da célula incubadora). À medida que cada transfecção serão realizados em triplicado, a mesma mistura será aplicada a duas placas de 96 poços contendo células adicionais. </li><li> Incubar as células com misturas de transfecção adicionados a 37 ° C num ambiente humidificado com 5% de CO 2 durante 36 horas. </li></ol> 4. Determinar as respectivas actividades de luciferase a partir de amostras de células Após 36 horas, as atividades de luciferase estão prontos para a mediçãoem células transfectadas. O desfecho deve ser determinada em uma base per-experimento. Em nosso estudo, um tempo de incubação de 36 horas já havia rendido um sinal suficientemente robusta para significando determinação resultado. Como este estudo incorpora múltiplos repórteres (um segregada para o meio de cultura, e os outros dois expresso no citoplasma da célula), vamos obter a partir de medições tanto meio de cultura, assim como um lisado celular. <ol><li> Detecção de actividade de CL no meio de cultura: <ol><li> 20 mL de meio de cultura é réplica banhado em uma placa de 96 poços opaco branco (usando o líquido Handler Biomek ou pipeta multicanal). </li><li> Adicionar 20 ul de tampão de ensaio CL (Targeting Systems) a cada poço. </li><li> Adicionar 10 ul de substrato luciferina Cypridina (Targeting Systems) a cada poço e detectar a actividade CL utilizando um luminómetro (o leitor de placas Pherastar produzido por BMG, por exemplo). </li></ol></li><li> Detecção de actividade de FL e RL: <ol><li> Lyse do cells primeiro por remoção do meio de cultura. Isto pode ser realizado rapidamente rodando placa invertida e jogando médio em uma pia. Meio restante pode ser removido batendo suavemente a placa de cabeça para baixo sobre toalhas de papel. Adicionar 30 ul de 1x tampão de lise passiva (Promega) a cada poço de células usando o Multidrop e coloque numa plataforma oscilante (Bélico é uma empresa que fabrica estes) definir uma velocidade média, durante 5 min à TA. </li><li> Adicionar 20 ul de Reagente de Ensaio da Luciferase II (parte do kit Promega Luciferase Duplo) utilizando a actividade FL Multidrop e medir imediatamente usando o luminómetro. Em seguida, adicione Stop &amp; Glo Reagente (Promega), que irá saciar a atividade FL e permitir a medição da atividade RL. Nota: a menos que você estiver usando misturas de substratos que permitem duração do sinal estendido (como Kits de luciferase Dual-Glo da Promega), o uso do flash kits exige uma rápida medição com a adição de substrato (dentro de 5 min). </li></ol></li></ol>

<ol>
	<li>Bronstein, I., Fortin, J., Stanley, P. E., Stewart, G. S., Kricka, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemiluminescent+and+bioluminescent+reporter+gene+assays.">Chemiluminescent and bioluminescent reporter gene assays.</a> Analytical Biochemistry. 219, 169-181 (1994).</li><li>Miraglia, L. J., King, F. J., Damoiseaux, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+the+light:+luminescent+reporter+gene+assays.">Seeing the light: luminescent reporter gene assays.</a> Combinatorial Chemistry &amp; High Throughput Screening. 14, 648-657 (2011).</li><li>McGuire, S. E., Roman, G., Davis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+systems+in+Drosophila:+a+synthesis+of+time+and+space.">Gene expression systems in Drosophila: a synthesis of time and space.</a> Trends in Genetics: TIG. 20, 384-391 (2004).</li><li>Smirnova, N. A., 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=Development+of+Neh2-luciferase+reporter+and+its+application+for+high+throughput+screening+and+real-time+monitoring+of+Nrf2+activators.">Development of Neh2-luciferase reporter and its application for high throughput screening and real-time monitoring of Nrf2 activators.</a> Chem. Biol. 18, 752-765 (2011).</li><li>Chen, 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=Small+molecule-mediated+disruption+of+Wnt-dependent+signaling+in+tissue+regeneration+and+cancer.">Small molecule-mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer.</a> Nat. Chem. Biol. 5, 100-107 (2009).</li><li>Tang, 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=A+genome-wide+RNAi+screen+for+Wnt/beta-catenin+pathway+components+identifies+unexpected+roles+for+TCF+transcription+factors+in+cancer.">A genome-wide RNAi screen for Wnt/beta-catenin pathway components identifies unexpected roles for TCF transcription factors in cancer.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 9697-9702 (2008).</li><li>Lum, L. D., Kulak, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+Luciferase+Reporter+Assay+Systems.">Multiplexed Luciferase Reporter Assay Systems.</a> United States patent. , (2012).</li><li>Funk, W. D., Pak, D. T., Karas, R. H., Wright, W. E., Shay, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transcriptionally+active+DNA-binding+site+for+human+p53+protein+complexes.">A transcriptionally active DNA-binding site for human p53 protein complexes.</a> Mol. Cell Biol. 12, 2866-2871 (1992).</li><li>DasGupta, R., Kaykas, A., Moon, R. T., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genomic+analysis+of+the+Wnt-wingless+signaling+pathway.">Functional genomic analysis of the Wnt-wingless signaling pathway.</a> Science. 308, 826-833 (2005).</li><li>Korinek, V., 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=Depletion+of+epithelial+stem-cell+compartments+in+the+small+intestine+of+mice+lacking+Tcf-4.">Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4.</a> Nat. Genet. 19, 379-383 (1998).</li><li>Korinek, V., 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=Two+members+of+the+Tcf+family+implicated+in+Wnt/beta-catenin+signaling+during+embryogenesis+in+the+mouse.">Two members of the Tcf family implicated in Wnt/beta-catenin signaling during embryogenesis in the mouse.</a> Mol. Cell Biol. 18, 1248-1256 (1998).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+molecular+characterization+of+human+colon+and+rectal+cancer.">Comprehensive molecular characterization of human colon and rectal cancer.</a> Nature. 487, 330-337 (2012).</li><li>Koboldt, D. C., 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=Comprehensive+molecular+portraits+of+human+breast+tumours.">Comprehensive molecular portraits of human breast tumours.</a> Nature. , (2012).</li><li>Wood, L. D., 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=The+genomic+landscapes+of+human+breast+and+colorectal+cancers.">The genomic landscapes of human breast and colorectal cancers.</a> Science. 318, 1108-1113 (2007).</li><li>Berndt, J. D., Biechele, T. L., Moon, R. T., Major, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrative+analysis+of+genome-wide+RNA+interference+screens.">Integrative analysis of genome-wide RNA interference screens.</a> Science Signaling. 2, pt4 (2009).</li><li>Falschlehner, C., Steinbrink, S., Erdmann, G., Boutros, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+RNAi+screening+to+dissect+cellular+pathways:+a+how-to+guide.">High-throughput RNAi screening to dissect cellular pathways: a how-to guide.</a> Biotechnology Journal. 5, 368-376 (2010).</li><li>Boutros, M., Bras, L. P., Huber, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+cell-based+RNAi+screens.">Analysis of cell-based RNAi screens.</a> Genome Biology. 7, R66 (2006).</li><li>Gilbert, D. F., 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=A+novel+multiplex+cell+viability+assay+for+high-throughput+RNAi+screening.">A novel multiplex cell viability assay for high-throughput RNAi screening.</a> PLoS ONE. 6, e28338 (2011).</li></ol>

OK e LL são autores de uma patente pendente relativa ao multiplexados tecnologia luciferase.

Existem vários fornecedores de sistemas repórter baseados em luciferase (tais como Promega, Targeting Systems, New England Biolabs, e Thermo Fisher) que fornecem úteis recursos on-line para aqueles entreter uma tela de alto rendimento para a primeira vez. Além disso, uma série de excelentes comentários sobre a otimização do uso de telas de alto rendimento para a descoberta dos genes e como baseada em RNAi resultados de rastreio pode ser integrado com conjuntos de dados-omics baseados outro deve ser útil 15...

Uma variedade de repórteres de luciferase de base para monitorizar uma matriz diversificada de processos biológicos celulares estão comercialmente disponíveis. A maioria destas construções de ADN que codificam proteínas de luciferase são induzidas para expressar por estímulos específicos de células ou perturbações. Os sólidos repórteres maioria baseados na transcrição colocar a expressão de uma enzima da luciferase sob o controlo de elementos potenciadores sintéticos ou regiões promotoras de genes bem estabelecidos para a sua fiabilidade em um relato de eventos de transcrição 1,2 fisiologicamente relevante. Há também sistemas de trans-repórter de luciferase que utilizam GAL4-UAS para dirigir a expressão da proteína luciferase. Gal4 é um activador da transcrição de levedura e as UAS é um potenciador para o qual se liga especificamente Gal4 para activar a transcrição de sequências de genes colocados a jusante do mesmo 3. Estes tipos de sistemas de luciferase são tipicamente suportado por dois plasmídeos de ADN - uma codifica a proteína GAL4 fundido com o regulatory porção de uma proteína de interesse e o segundo codifica uma proteína da luciferase colocado sob o controlo de uma ou mais sequências UAS. Assim, o sinal de luciferase celular reflecte o nível da actividade da proteína de interesse. Outra utilização comum de repórteres de luciferase de base é para monitorizar a estabilidade da proteína em que uma proteína de interesse é fundida a uma enzima luciferase 4. Independentemente do tipo de repórter, um caveat.emptor é notado aqui como não se pode assumir a especificidade de qualquer repórter ter sido bem interrogados. Assim, due diligence é necessária a incorporação de novas construções repórter em qualquer estratégia de pesquisa. A selecção da enzima luciferase a leitura pode ser tão importante como a fiabilidade dos elementos de DNA que controlam a sua expressão. Considerando que a luciferase do pirilampo (FL) é a enzima mais utilizada em construções comercialmente disponíveis, o advento de novos sistemas de repórter da luciferase que não necessitam de ATP (como no casode reações baseadas FL) ou que incorporem enzimas mais estáveis ​​que emitem sinais mais fortes prometem melhorar a eficiência ea confiabilidade desta plataforma de pesquisa. Uma nota importante sobre a selecção de repórteres de luciferase em estudos químicos é a susceptibilidade de FL para inibição química que poderia dar origem a relatórios falsos. Na nossa experiência, outras enzimas, tais como a luciferase de Renilla, ou Gaussia (RL e GL, respectivamente), que utilizam como substrato coelenterazina são menos facilmente inibidas por moléculas pequenas 5. O formato mais comum para multiplexação de luciferase leia-outs implica a utilização de FL e RL, em grande parte devido à disponibilidade de fácil de usar kits com a capacidade de medir actividades enzimáticas sequencialmente a partir de uma única amostra. Na maioria dos jogos, as amostras são expostos primeiro a luciferina para revelar os níveis de actividade FL seguido por um reagente de extinção que é depositado simultaneamente com coelenterazina para originar um em segundasinal RL dary. Com a adição de outras luciferases de que podem ser segregadas, tais como GL ou que o uso de um outro substrato, tal como a luciferase Cypridina (CL), um maior número de possibilidades para a geração de dados de conteúdos elevados utilizando luciferases emergiram. Um exemplo de uma tela de RNAi do genoma usando esta técnica pode ser encontrada aqui 6. Estes avanços tecnológicos têm por sua vez, estimulou o desenvolvimento de mecanismos específicos para extinguir cada tipo de enzima luciferase a fim de limitar a conversa cruzada 7. Em nossas mãos, notamos uma maior freqüência de &quot;efeitos de borda&quot; (tendências inexplicáveis ​​na atividade celular associada à beira de placas de cultura de alta titulação), com luciferases secretadas como GL ou CL. Por outro lado, tais luciferases secretadas são úteis para ensaios cinéticos, pois permitem a amostragem do sinal sem adulterar a viabilidade celular. Para o nosso estudo apresentado aqui, vamos monitorar simultaneamente a atividade de três câncer relevanteprocessos celulares: o p53, Kras, e vias de sinalização Wnt. Os repórteres incorporados no nosso estudo são a FL repórter PP53-TA-Luc de Clontech (doravante referido como o repórter p53-FL) 8, um sistema repórter Elk1-GAL4/UAS-CL (doravante, Elk-1 repórter; Agilent), eo 8X TCF RL repórter que incorpora vários elementos potenciador sintéticos reconhecidos pela via Wnt reguladores de transcrição, conhecidos como fatores de células T (ou TCFs) 9-11.

<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>PathDetect Elk1 Trans-Reporting System</td>
			<td>Agilent Technologies Inc.</td>
			<td>219005</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pathway Profiling Luciferase System 4 pp53-TA-Luc Vector</td>
			<td>Clontech Lab Inc.</td>
			<td>631914</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Effectene Transfection Reagent</td>
			<td>Qiagen Inc.</td>
			<td>301427</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dual-Luciferase Reporter Assay System</td>
			<td>Promega Corp.</td>
			<td>E1960</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cypridina Luciferase Assay reagent</td>
			<td>Targeting Systems</td>
			<td>VLAR-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HCT116 cells</td>
			<td>ATCC</td>
			<td>CCL-247</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CytoTox-Fluo Cytotoxicity Assay</td>
			<td>Promega Corp.</td>
			<td>G9260</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>MultiFlo Microplate Dispenser</td>
			<td>Labsystems Inc.</td>
			<td>Model 832</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Biomek FXP Laboratory Automation Workstation</td>
			<td>Beckman Coulter Inc.</td>
			<td>A31842</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PHERAstar FS-multi-mode HTS microplate reader</td>
			<td>BMG Labtech Inc.</td>
			<td>0471-101A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bright-Line Reichert Hemacytometers</td>
			<td>Hausser Scientific Company</td>
			<td>1490</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

a multiplexed luciferase-based screening platform for interrogating cancer-associated signal transduction in cultured cells

Interrogatório da função do gene usando RNA de interferência (RNAi) genoma escala detém uma grande promessa para a rápida identificação de vulnerabilidades de células de câncer quimicamente tratáveis. Limitar o potencial desta tecnologia é a incapacidade de delinear rapidamente a base mecanicista de resultados fenotípicos e, assim, informar o desenvolvimento de estratégias terapêuticas molecularmente alvo. Nós delineamos aqui métodos para desconstruir fenótipos celulares induzidas pelo gene RNAi mediado alvo usando sistemas repórter multiplexados que permitem a monitorização do câncer de processos-chave associados a células. Esta metodologia de rastreio de alto conteúdo é versátil e pode ser facilmente adaptado para o rastreio de outros tipos de grandes bibliotecas moleculares.

Apesar dos avanços no mapeamento da paisagem mutacional de vários tipos de câncer usando enormes esforços de seqüenciamento do genoma 12-14, ainda não tem em lugar de uma abordagem sistemática para traduzir estas observações em estratégias de intervenção. No câncer colorretal (CRC), as mutações que estão previstos para afetar os componentes de três processos celulares - Wnt / β-catenina, p53, e Kras sinalização - são encontrados em 99% de todos os tumores, sugerindo que eles são fatores-...

Watch this Scientific Journal Video about A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells at JoVE.com

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Alcançar um nível de compreensão dos processos celulares de sistemas é uma meta da biologia celular moderna. Descrevemos aqui as estratégias para jornalistas luciferase multiplexação de várias funções celulares de pontos finais para interrogar a função do gene usando genoma escala bibliotecas RNAi.

A plataforma de triagem baseada luciferase diversificadas para interrogar associados ao câncer transdução de sinal em células cultivadas

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

a-multiplexed-luciferase-based-screening-platform-for-interrogating

Research

JoVE Journal

Medicine

11.0K visualizações.  UT Southwestern Medical Center. Alcançar um nível de compreensão dos processos celulares de sistemas é uma meta da biologia celular moderna. Descrevemos aqui as estratégias para jornalistas luciferase multiplexação de várias funções celulares de pontos finais para interrogar a função do gene usando genoma escala bibliotecas RNAi.

Vídeo: A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

How to Measure Cortical Folding from MR Images: a Step-by-Step Tutorial to Compute Local Gyrification Index

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will be identifiable at any age. As recently reviewed by Mangin and colleagues2, several methods exist to quantify different characteristics of gyrification. For instance, sulcal morphometry can be used to measure shape descriptors such as the depth, length or indices of inter-hemispheric asymmetry3. These geometrical properties have the advantage of being easy to interpret. However, sulcal morphometry tightly relies on the accurate identification of a given set of sulci and hence provides a fragmented description of gyrification. A more fine-grained quantification of gyrification can be achieved with curvature-based measurements, where smoothed absolute mean curvature is typically computed at thousands of points over the cortical surface4. The curvature is however not straightforward to comprehend, as it remains unclear if there is any direct relationship between the curvedness and a biologically meaningful correlate such as cortical volume or surface. To address the diverse issues raised by the measurement of cortical folding, we previously developed an algorithm to quantify local gyrification with an exquisite spatial resolution and of simple interpretation. Our method is inspired of the Gyrification Index5, a method originally used in comparative neuroanatomy to evaluate the cortical folding differences across species. In our implementation, which we name local Gyrification Index (lGI1), we measure the amount of cortex buried within the sulcal folds as compared with the amount of visible cortex in circular regions of interest. Given that the cortex grows primarily through radial expansion6, our method was specifically designed to identify early defects of cortical development.
In this article, we detail the computation of local Gyrification Index, which is now freely distributed as a part of the FreeSurfer Software (<a href="http://surfer.nmr.mgh.harvard.edu/">http://surfer.nmr.mgh.harvard.edu/</a>, Martinos Center for Biomedical Imaging, Massachusetts General Hospital). FreeSurfer provides a set of automated reconstruction tools of the brain's cortical surface from structural MRI data. The cortical surface extracted in the native space of the images with sub-millimeter accuracy is then further used for the creation of an outer surface, which will serve as a basis for the lGI calculation. A circular region of interest is then delineated on the outer surface, and its corresponding region of interest on the cortical surface is identified using a matching algorithm as described in our validation study1. This process is repeatedly iterated with largely overlapping regions of interest, resulting in cortical maps of gyrification for subsequent statistical comparisons (Fig. 1). Of note, another measurement of local gyrification with a similar inspiration was proposed by Toro and colleagues7, where the folding index at each point is computed as the ratio of the cortical area contained in a sphere divided by the area of a disc with the same radius. The two implementations differ in that the one by Toro et al. is based on Euclidian distances and thus considers discontinuous patches of cortical area, whereas ours uses a strict geodesic algorithm and include only the continuous patch of cortical area opening at the brain surface in a circular region of interest.

Measuring gyrification (cortical folding) at any age represents a window into early brain development. Hence, we previously developed an algorithm to measure local gyrification at thousands of points over the hemisphere1. In this paper, we detail the computation of this local gyrification index.

Como medir o Folding Cortical partir de imagens de MR: um tutorial passo-a-Passo para Calcular Índice Gyrification local

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will ...

Medicina

High Content Screening in Neurodegenerative Diseases

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be addressed as a matter of great urgency1-4. Multiple complementary 'omics' approaches have provided clues as to the genetic risk factors and pathogenic mechanisms underlying numerous neurodegenerative diseases, but most findings still require functional validation5. For example, a recent genome wide association study for Parkinson's Disease (PD), identified many new loci as risk factors for the disease, but the underlying causative variant(s) or pathogenic mechanism is not known6, 7. As each associated region can contain several genes, the functional evaluation of each of the genes on phenotypes associated with the disease, using traditional cell biology techniques would take too long. 
	There is also a need to understand the molecular networks that link genetic mutations to the phenotypes they cause. It is expected that disease phenotypes are the result of multiple interactions that have been disrupted. Reconstruction of these networks using traditional molecular methods would be time consuming. Moreover, network predictions from independent studies of individual components, the reductionism approach, will probably underestimate the network complexity8. This underestimation could, in part, explain the low success rate of drug approval due to undesirable or toxic side effects. Gaining a network perspective of disease related pathways using HT/HC cellular screening approaches, and identifying key nodes within these pathways, could lead to the identification of targets that are more suited for therapeutic intervention.
	High-throughput screening (HTS) is an ideal methodology to address these issues9-12. but traditional methods were one dimensional whole-well cell assays, that used simplistic readouts for complex biological processes. They were unable to simultaneously quantify the many phenotypes observed in neurodegenerative diseases such as axonal transport deficits or alterations in morphology properties13, 14. This approach could not be used to investigate the dynamic nature of cellular processes or pathogenic events that occur in a subset of cells. To quantify such features one has to move to multi-dimensional phenotypes termed high-content screening (HCS)4, 15-17. HCS is the cell-based quantification of several processes simultaneously, which provides a more detailed representation of the cellular response to various perturbations compared to HTS. 
	HCS has many advantages over HTS18, 19, but conducting a high-throughput (HT)-high-content (HC) screen in neuronal models is problematic due to high cost, environmental variation and human error. In order to detect cellular responses on a 'phenomics' scale using HC imaging one has to reduce variation and error, while increasing sensitivity and reproducibility. 
	Herein we describe a method to accurately and reliably conduct shRNA screens using automated cell culturing20 and HC imaging in neuronal cellular models. We describe how we have used this methodology to identify modulators for one particular protein, DJ1, which when mutated causes autosomal recessive parkinsonism21. 
	Combining the versatility of HC imaging with HT methods, it is possible to accurately quantify a plethora of phenotypes. This could subsequently be utilized to advance our understanding of the genome, the pathways involved in disease pathogenesis as well as identify potential therapeutic targets.

We describe a methodology combining automated cell culturing with high-content imaging to visualize and quantify multiple cellular processes and structures, in a high-throughput manner. Such methods can aid in the further functional annotation of genomes as well as identify disease gene networks and potential drug targets.

Screening alto teor em Doenças Neurodegenerativas

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be ...

The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

The lower urinary tract (LUT) functions as a dynamic reservoir that is able to store urine and to efficiently expel it at a convenient time. While storing urine, however, the bladder is exposed for prolonged periods to waste products. By acting as a tight barrier, the epithelial lining of the LUT, the urothelium, avoids re-absorption of harmful substances. Moreover, noxious chemicals stimulate the bladder's nociceptive innervation and initiate voiding contractions that expel the bladder's contents. Interestingly, the bladder's sensitivity to noxious chemicals has been used successfully in clinical practice, by intravesically infusing the TRPV1 agonist capsaicin to treat neurogenic bladder overactivity1. This underscores the advantage of viewing the bladder as a chemosensory organ and prompts for further clinical research. However, ethical issues severely limit the possibilities to perform, in human subjects, the invasive measurements that are necessary to unravel the molecular bases of LUT clinical pharmacology. A way to overcome this limitation is the use of several animal models2. Here we describe the implementation of cystometry in mice and rats, a technique that allows measuring the intravesical pressure in conditions of controlled bladder perfusion.
	After laparotomy, a catheter is implanted in the bladder dome and tunneled subcutaneously to the interscapular region. Then the bladder can be filled at a controlled rate, while the urethra is left free for micturition. During the repetitive cycles of filling and voiding, intravesical pressure can be measured via the implanted catheter. As such, the pressure changes can be quantified and analyzed. Moreover, simultaneous measurement of the voided volume allows distinguishing voiding contractions from non-voiding contractions3.
	Importantly, due to the differences in micturition control between rodents and humans, cystometric measurements in these animals have only limited translational value4. Nevertheless, they are quite instrumental in the study of bladder pathophysiology and pharmacology in experimental pre-clinical settings. Recent research using this technique has revealed the key role of novel molecular players in the mechano- and chemo-sensory properties of the bladder.

Cystometry is an efficient technique to measure bladder function of small animals in vivo. The bladder is continuously infused at rates controlled through an intravesical catheter, whereas the urethra is left free for micturition. This allows for repetitive filling and emptying of the bladder, while intravesical pressure and voided volume are recorded.

O Uso de cistometria em pequenos roedores: Um Estudo de Chemosensation Bexiga

	The lower urinary tract (LUT) functions as a  dynamic reservoir that is able to store urine and to efficiently expel it at a  convenient time. While ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

Triagem para Modificadores Melanoma usando um modelo de tumor Zebrafish autóctone

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Estudar o cancro do pâncreas Stem características celulares para desenvolver estratégias Novo tratamento

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

A High-content In Vitro Pancreatic Islet &amp;#946;-cell Replication Discovery Platform

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

A-alta conteúdo<em&gt; In Vitro</em&gt; Replicação plataforma de descoberta de células-β das ilhotas pancreáticas

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have been limited compounds available so far to attenuate TLR signaling and there have been no clinically approved TLR inhibitors (except the anti-malarial drug hydroxychloroquine) in clinical use. In light of rapid advances in nanotechnology, manipulation of immune responsiveness using nano-devices may provide a new strategy to treat these diseases. Herein, we present a high throughput screening method for quickly identifying novel bioactive nanoparticles that inhibit TLR signaling in phagocytic immune cells. This screening platform is built on THP-1 cell-based reporter cells with colorimetric and luciferase assays. The reporter cells are engineered from the human THP-1 monocytic cell line by stable integration of two inducible reporter constructs. One expresses a secreted embryonic alkaline phosphatase (SEAP) gene under the control of a promoter inducible by the transcription factors NF-&#954;B and AP-1, and the other expresses a secreted luciferase reporter gene under the control of promoters inducible by interferon regulatory factors (IRFs).Upon TLR stimulation, the reporter cells activate transcription factors and subsequently produce SEAP and/or luciferase, which can be detected using their corresponding substrate reagents. Using a library of peptide-gold nanoparticle (GNP) hybrids established in our previous studies as an example, we identified one peptide-GNP hybrid that could effectively inhibit the two arms of TLR4 signaling cascade triggered by its prototypical ligand, lipopolysaccharide (LPS). The findings were validated by standard biochemical techniques including immunoblotting. Further analysis established that this lead hybrid had a broad inhibitory spectrum, acting on multiple TLR pathways, including TLR2, 3, 4, and 5. This experimental approach allows a rapid assessment of whether a nanoparticle (or other therapeutic compounds) can modulate specific TLR signaling in phagocytic immune cells.

Toll-like receptor (TLR) signaling plays an important role in the pathophysiology of many human inflammatory diseases, and regulating TLR responses by bioactive nanoparticles is anticipated to be beneficial in many inflammatory conditions. THP-1 cell-based reporter cells provide a versatile and robust screening platform for identifying novel inhibitors of TLR signaling.

Triagem de Nanopartículas Bioativas em Células Imunes Fagocíticas para Inibidores da Sinalização de Receptores Toll-like

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have ...

Routine Screening Method for Microparticles in Platelet Transfusions

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood banks. Cells fragment off microparticles (MP) when they are stressed. Blood and blood components may contain cellular fragments from a variety of cells, most notably from activated platelets. When performing their roles as innate immune cells and major players in coagulation and hemostasis, platelets change shape and generate microparticles. With dynamic light scattering (DLS)-based microparticle detection, it is possible to differentiate activated (high microparticle) from non-activated (low microparticle) platelets in transfusions, and optimize the use of this scarce blood product. Previous research suggests that providing non-activated platelets for prophylactic use in hematology-oncology patients could reduce their risk of becoming refractory and improve patient care. The goal of this screening method is to routinely differentiate activated from non-activated platelets. The method described here outlines the steps to be performed for routine platelet inventory management in a hospital blood bank: obtaining a sample from a platelet transfusion, loading the sample into the capillary for DLS measurement, performing the DLS test to identify microparticles, and using the reported microparticle content to identify activated platelets.

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative in hospital blood banks. The goal is to differentiate activated from non-activated platelets to optimize the use of platelets. Providing non-activated platelets to hematology-oncology patients might reduce their high risk to become refractory.

Método de triagem para micropartículas em transfusões de plaquetas de rotina

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood ...

Hydra, a Computer-Based Platform for Aiding Clinicians in Cardiovascular Analysis and Diagnosis

Cardiovascular diseases (CVDs) are the leading cause of death throughout the world. The total risk of developing CVD is determined by the combined effect of different cardiovascular risk factors (e.g., diabetes, raised blood pressure, unhealthy diet, tobacco use, stress, etc.) that commonly coexist and act multiplicatively. Most CVDs can be prevented by an early identification of the highest risk factors and an appropriate treatment. The stratification of cardiovascular risk factors involves a wide range of parameters and tests that specialists use in their clinical practice. In addition to cardiovascular (CV) risk stratification, ambulatory blood pressure monitoring (ABPM) also provides relevant information for diagnostic and treatment purposes. This work presents a list of protocols based on the Hydra platform, a web-based system for clinical decision support which incorporates a set of functionalities and services that are required for complete cardiovascular analysis, risk assessment, early diagnosis, treatment and monitoring of patients over time. The program includes tools for inputting and managing comprehensive patient data, organized into different checkups to track the evolution over time. It also has a risk stratification tool to compute a CV risk factor based upon several risk stratification tables of reference. Additionally, the program includes a tool that incorporates ABPM analysis and allows the extraction of valuable information by monitoring blood pressure over a specific period of time. Finally, the reporting service summarizes the most relevant information in a set of reports that aid clinicians in their clinical decision-making process.

This article presents a protocol based on Hydra &#8212; a web-based system for clinical decision support that integrates a full and detailed set of functionalities and services required by physicians for complete cardiovascular analysis, risk assessment, early diagnosis, treatment, and monitoring over time.

Hydra, uma plataforma baseada em computador para auxiliar os médicos no diagnóstico e análise Cardiovascular

Cardiovascular diseases (CVDs) are the leading cause of death throughout the world. The total risk of developing CVD is determined by the combined effect ...

In Vitro Method to Control Concentrations of Halogenated Gases in Cultured Alveolar Epithelial Cells

Acute respiratory distress syndrome (ARDS) is a syndrome of diffuse alveolar injury with impaired alveolar fluid clearance and severe inflammation. The use of halogenated agents, such as sevoflurane or isoflurane, for the sedation of intensive care unit (ICU) patients can improve gas exchange, reduce alveolar edema, and attenuate inflammation during ARDS. However, data on the use of inhaled agents for continuous sedation in the ICU to treat or prevent lung damage is lacking. To study the effects of halogenated agents on alveolar epithelial cells under "physiologic" conditions, we describe an easy system to culture cells at the air-liquid interface and expose them to halogenated agents to provide precise controlled "air" fractions and "medium" concentrations for these agents. We developed a sealed air-tight chamber in which plates with human alveolar epithelial immortalized cells could be exposed to a precise, controlled fraction of sevoflurane or isoflurane using a continuous gas flow provided by an anesthetic machine circuit. Cells were exposed to 4% of sevoflurane and 1% of isoflurane for 24 hours. Gas mass spectrometry was performed to determine the concentration of halogenated agents dissolved in the medium. After the first hour, the concentrations of sevoflurane and isoflurane in the medium were 251 mg/L and 25 mg/L, respectively. The curves representing the concentrations of both sevoflurane and isoflurane dissolved in the medium showed similar courses over time, with a plateau reached at one hour after exposure. 
This protocol was specifically designed to reach precise and controlled concentrations of sevoflurane or isoflurane in vitro to improve our understanding of mechanisms involved in epithelial lung injury during ARDS and to test novel therapies for the syndrome.

In Vitro Method to Control Concentrations of Halogenated Gases in Cultured Alveolar Epithelial Cells

We describe an easy protocol specifically designed to reach precise and controlled concentrations of sevoflurane or isoflurane in vitro in order to improve our understanding of mechanisms involved in the epithelial lung injury and to test novel therapies for acute respiratory distress syndrome.

In Vitro Método de controle de concentrações de Gases halogenados em culturas de células epiteliais alveolares

Acute respiratory distress syndrome (ARDS) is a syndrome of diffuse alveolar injury with impaired alveolar fluid clearance and severe inflammation. The ...