Estes esforços de pesquisa foram apoiadas pela Divisão de Ciência da Fundação Nacional de Química, Bioengenharia, ambiental e sistemas de transporte (CBET) sob os números prêmio Instituto Nacional de Ciências de Saúde Ambiental (NIEHS) CBET e CBET-0853780-1159344 e do National Institutes of Health, sob o número prêmio 1R43ES022567-01, e do Instituto Nacional do Câncer, do Programa de Imagem Câncer sob o número prêmio CA127745-01. O IVIS Lumina instrumento utilizado neste trabalho foi obtido a partir do Programa de Instrumentação Exército dos EUA Defense University Research.

1. Preparação celular <ol><li> Recuperar um frasco de bioluminescentes HEK293 de azoto estoque congelada líquido e fazê-los crescer em meio de Eagle Modificado por Dulbecco (DMEM) suplementado com 10% de soro fetal bovino, 0,01 mM de aminoácidos não essenciais, 1 x antibiótico-antimicótico e 0,01 mM de piruvato de sódio num t 75 frascos de cultura de tecidos a 37 ° C e 5% de CO 2. Nota: Os meios de comunicação e componentes complementares varia de acordo com linhas de células e, portanto, deve ser escolhido de acordo. </li><li> Atualizar média a cada 2-3 dias até atingir ~ 80% de confluência. Nota: A quantidade de células necessárias irá basear-se no desenho experimental. Se necessário, mais as células podem ser obtidas por sub-culturas. </li><li> Células colheita para testes. <ol><li> Remover o meio e lava-se as células com solução salina tamponada com fosfato (PBS), agitando suavemente o balão e, em seguida, descartando o gasto de PBS. </li><li> Adicionar tripsina e incubar a 37 ° C durante 2 min,ou até que as células foram separadas do balão. </li><li> Recolha das células isoladas com PBS e transferi-las para um tubo de centrífuga limpo. Nota: Se as linhas de células não aderentes são usados, celas separadas por centrifugação e lave uma vez com PBS. </li></ol></li><li> Determinar a contagem total de células utilizando um hemacitómetro, ou outro sistema de contagem de células. </li><li> Centrifugar a suspensão de células a 300 xg durante 5 min. Descartar o sobrenadante e ressuspender as células em fresco, médio pré-aquecido. </li><li> Sementes de igual número de células em poços individuais de uma placa multi-bem opaco. Neste exemplo, a placa 5 x 10 5 células em um mL de volume em cada poço de uma placa de 24 cavidades preta 1. Placa de um volume igual de meio em poços em triplicado para actuar como controlo negativo. Nota: o número de células colocadas em cada poço, é flexível e pode ser optimizado para as experiências individuais. Para cada linha celular bioluminescente, determinar experimentalmente a relação entre o número de células e biolumiNESCent saída, bem como a célula mínima detectável contar antes de quaisquer testes de toxicidade. Para fazer isso, a bioluminescência medida através de uma vasta gama de tamanhos da população (por exemplo, 1 x outubro 3-01 x 10 6 culas) em condições padronizadas de imagem. </li><li> Tratar as células com o composto de teste (s), como descrito abaixo. </li></ol> 2. Preparação química <ol><li> Preparar o produto químico a ser testado como uma solução de reserva concentrada. Neste exemplo, o uso de 100 mg / ml de solução de um antibiótico da família da bleomicina. Nota: Para minimizar os potenciais efeitos tóxicos à base de veículo, especialmente se um solvente orgânico é utilizado como um veículo para a adição de produtos químicos, recomenda-se que a concentração de Estoque ser de pelo menos 1000 vezes a concentração pós-aplicação desejada. </li><li> Adicionar o material químico preparado directamente para as células. Neste exemplo, a dose do antibiótico de interesse em concentrações finais de 100, 200, 300 e 400 ug / ml em triplicate poços. Adicione três poços de células não tratadas como controlos. Nota: A concentração final do produto químico alvo devem ser selecionados com base em objetivos experimentais específicos. Uma vasta gama de concentrações é normalmente testados para obter um espectro completo dos efeitos tóxicos. </li></ol> 3. Imagem e Análise de Dados <ol><li> Imediatamente após a adição de produtos químicos, coloque a placa na câmara de imagem do instrumento adequado para aquisição de imagem e medição da bioluminescência. </li><li> Bioluminescência de medida a cada 15 minutos ao longo de um período de 24 horas utilizando um tempo de integração de 10 minutos para cada leitura. Nota: O tempo de integração usado neste exemplo é em uma base por placa e pode ser ajustado para a medição sobre uma base por poço usando outros luminómetros de escolha. O tempo de integração também podem ser ajustados com base na linha celular escolhida bioluminescente. Porque bioluminescência é produzida de forma autônoma sem destruição celular ou qualquer estímulo externo, lermentos podem ser tomadas em qualquer ponto de tempo desejado para cumprir objetivos experimentais específicos. </li><li> Quantificar a intensidade bioluminescente de cada poço através da identificação de uma região de interesse usando software compatível. Mostrar a saída de luz, quer como o fluxo total (fotões / segundo) ou a radiância média (fotões / segundo / cm steridian 2 /) de cada amostra. </li></ol>

<ol>
	<li>U.S. Food and Drug Administration. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+it+really+FDA+approved?.">Is it really FDA approved?.</a> FDA Consumer Health Information. 2, (2009).</li><li>Hartung, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicology+for+the+twenty-first+century.">Toxicology for the twenty-first century.</a> Nature. 460, 208-212 (2009).</li><li>Olson, H., 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=Concordance+of+the+toxicity+of+pharmaceuticals+in+humans+and+in+animals.">Concordance of the toxicity of pharmaceuticals in humans and in animals.</a> Regul. Toxicol. Pharmacol. 32, 56-67 (2000).</li><li>Stacey, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+developments+in+cell+culture+technology.">Current developments in cell culture technology.</a> Adv. Exp. Med. Biol. 745, 1-13 (2012).</li><li>Li, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+human+ADME/Tox+drug+properties+in+drug+discovery.">Screening for human ADME/Tox drug properties in drug discovery.</a> Drug Discov. Today. 6, 357-366 (2001).</li><li>Li, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+approaches+to+evaluate+ADMET+drug+properties.">In vitro approaches to evaluate ADMET drug properties.</a> Curr. Top. Med. Chem. 4, 701-706 (2004).</li><li>Tannous, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gaussia+luciferase+reporter+assay+for+monitoring+biological+processes+in+culture+and+in+vivo.">Gaussia luciferase reporter assay for monitoring biological processes in culture and in vivo.</a> Nat. Protoc. 4, 582-591 (2009).</li><li>Thompson, E. M., Nagata, S., Tsuji, F. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+expression+of+cDNA+for+the+luciferase+from+the+marine+ostracod+Vargula+hilgendorfii.">Cloning and expression of cDNA for the luciferase from the marine ostracod Vargula hilgendorfii.</a> Proc. Natl. Acad. Sci. U.S.A. 86, 6567-6571 (1989).</li><li>Lupold, S. E., Johnson, T., Chowdhury, W. H., Rodriguez, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+real+time+Metridia+luciferase+based+non-invasive+reporter+assay+of+mammalian+cell+viability+and+cytotoxicity+via+the+beta-actin+promoter+and+enhancer.">A real time Metridia luciferase based non-invasive reporter assay of mammalian cell viability and cytotoxicity via the beta-actin promoter and enhancer.</a> PLoS ONE. 7, (2012).</li><li>Meighen, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+biology+of+bacterial+bioluminescence.">Molecular biology of bacterial bioluminescence.</a> Microbiol. Rev. 55, 123-142 (1991).</li><li>Close, D. M., 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=Autonomous+bioluminescent+expression+of+the+bacterial+luciferase+gene+cassette+(lux)+in+a+mammalian+cell+line.">Autonomous bioluminescent expression of the bacterial luciferase gene cassette (lux) in a mammalian cell line.</a> PLoS ONE. 5, e12441 (2010).</li><li>Close, D. M., Hahn, R. E., Ripp, S. A., Sayler, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+toxicant+bioavailability+using+a+constitutively+bioluminescent+human+cell+line.">Determining toxicant bioavailability using a constitutively bioluminescent human cell line.</a> , 1-4 (2011).</li><li>Oliva-Trastoy, M., Defais, M., Larminat, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+to+the+antibiotic+Zeocin+by+stable+expression+of+the+Sh+ble+gene+does+not+fully+suppress+Zeocin-induced+DNA+cleavage+in+human+cells.">Resistance to the antibiotic Zeocin by stable expression of the Sh ble gene does not fully suppress Zeocin-induced DNA cleavage in human cells.</a> Mutagenesis. 20, 111-114 (2005).</li><li>Close, D. M., Webb, J. D., Ripp, S. A., Patterson, S. S., Sayler, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+detection+of+human+toxicants+in+real+time+using+a+human+optimized+bioluminescent+bacterial+luciferase+gene+cassette+bioreporter.">Remote detection of human toxicants in real time using a human optimized bioluminescent bacterial luciferase gene cassette bioreporter.</a> Proc. SPIE 8371, Sensing Technologies for Global Health, Military Medicine, Disaster Response, and Environmental Monitoring II; and Biometric Technology for Human Identification IX. 837117, (2012).</li><li>Close, D. M., Hahn, R. E., Patterson, S. S., Ripp, S. A., Sayler, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+human+optimized+bacterial+luciferase,+firefly+luciferase,+and+green+fluorescent+protein+for+continuous+imaging+of+cell+culture+and+animal+models.">Comparison of human optimized bacterial luciferase, firefly luciferase, and green fluorescent protein for continuous imaging of cell culture and animal models.</a> J. Biomed. Opt. 16, e12441 (2011).</li></ol>

DM Close, SA Ripp, e GS Sayler são fundadores e proprietários de 490 BioTech, Inc.

Este método demonstra o uso de células de mamíferos bioluminescentes autonomamente como um ensaio de rastreio de citotoxicidade in vitro que permite que as células vivas de ser monitorada continuamente durante a sua vida. Este protocolo é flexível e pode ser modificado para acomodar condições experimentais específicas, conforme necessário. Por exemplo: a experiência aqui apresentada é apropriada para rastrear os efeitos tóxicos agudos, mas pode ser adaptado para avaliar efeitos de actuaç?...

Em os EUA, farmacêuticos e outros produtos destinados ao consumo humano requer ampla avaliação antes de serem aprovadas para uso do consumidor pela Food and Drug Administration. Os encargos financeiros para executar este teste é colocada no revelador 1, o que aumenta substancialmente os custos de desenvolvimento de novos compostos e, portanto, se traduz num aumento de custos para os consumidores. Enquanto, tradicionalmente, grande parte desta seleção utilizou assuntos animais para atuar como proxies para hospedeiros humanos, este provou ser um grande ônus financeiro, com um valor estimado de 2,8 bilhões dólares gastos anualmente em ADME / Tox (absorção, distribuição, metabolismo, excreção e toxicidade ) triagem sozinho 2 e evidências sugerindo que os modelos animais não é possível prever com segurança respostas toxicológicos humanos 3. Por conseguinte, os testes in vitro de células humanas à base de cultura ganhou popularidade nos últimos dois décadas devido à sua relativamente baixade custos, maior produtividade e melhor representação de biodisponibilidade humana e toxicologia 4. Baseados em métodos de rastreio de cultura celular correntes toxicidade empregar vários parâmetros, tais como a medição de níveis de ATP, o rastreio da actividade de enzimas citoplasmáticos endogenamente disponíveis, sondando a integridade da membrana celular, ou o seguimento do nível de actividade mitocondrial, para avaliar a viabilidade celular 5 , 6. No entanto, independentemente da extremidade escolhido, estes métodos requerem a destruição da amostra antes da medição pode ser feita, portanto, apenas a produção de dados num único ponto de tempo. Como resultado, um grande número de amostras têm de ser preparados e tratados em paralelo para estudos toxicológicos básicos cinética, mais uma vez aumentando o custo e o trabalho necessários para o desenvolvimento de novos compostos. Em alternativa, os ensaios de luciferase utilizando secretada como Gaussia luciferase 7, Vargula luciferase 8 e 9 Metridia luciferasetêm sido desenvolvidos, que eliminam a necessidade de lise celular e necessitam de uma fracção dos meios para a medição de ponto de extremidade, no entanto, estes ainda estão limitados a amostragem em pontos de tempo predeterminados e também requerer a adição de substratos exógenos de luz de activação. Para evitar o detrimento da destruição da amostra requerida, bem como para eliminar o custo de substratos, uma linha celular humana foi modificado que expressa a bioluminescência completo (lux), cassete do gene (luxCDABEfrp) para permitir a monitorização contínua de células vivas que é semelhante para imagens de células vivas com base fluorescente-dye, mas sem os procedimentos adicionais de investigação photon-ativadores e microscópica. Esta linha celular é capaz de constitutivamente produzindo um sinal óptico para a detecção contínua, directa, sem a necessidade de estimulação externa, evitando assim a destruição da amostra. Mecanisticamente, o sinal bioluminescente gerado a partir destas células resultars quando a enzima luciferase luxAB formada catalisa a oxidação de um aldeído gordos de cadeia longa (sintetizado e regenerado pelos produtos do gene luxCDE utilizando os substratos endógenos), na presença de riboflavina fosfato reduzido (FMNH 2, que é reciclada a partir de FMN pelo gene frp produto) e oxigénio molecular 10. A expressão da cassete de lux na célula hospedeira, por conseguinte, permite que a luz seja produzida e detectada sem destruição celular ou adição de substrato exógeno. Da mesma forma, a interação entre os genes lux ea FMN disponível endogenamente, e O dois co-substratos, bem como a exigência de manutenção de um ambiente capaz de suportar a conversão de FMN para FMNH 2, garante que o sinal bioluminescente resultante só pode ser detectada de viver as células activas metabolicamente. Estes requisitos foram anteriormente explorados para demonstrar que lux-bassaída bioluminescente ed correlaciona fortemente com o tamanho da população celular 11 e que a exposição composto tóxico prejudica a produção autobioluminescent de uma forma dose-resposta 12. Aqui usamos um autobioluminescent rim embrionário humano previamente caracterizadas (HEK293) 11 linha de células para demonstrar a triagem automatizada toxicológico de um antibiótico da família da bleomicina com ADN conhecida actividade prejudicial como um exemplo representativo, para validar a aplicação de células de mamífero autobioluminescent para os ensaios de toxicidade.

<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comment</td>
		</tr>
		<tr>
			<td>IVIS Lumina</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>Other IVIS models and PMT-based plate readers can also be used</td>
		</tr>
		<tr>
			<td>Living Imaging 2.0</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>Newer updates of this software is available</td>
		</tr>
		<tr>
			<td>75 cm2 cell culture treated flasks</td>
			<td>Corning</td>
			<td>430641</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture-treated plates</td>
			<td>Costar</td>
			<td>07-200-83</td>
			<td></td>
		</tr>
		<tr>
			<td>Black 24-well plate</td>
			<td>Greiner Bio-One</td>
			<td>662174</td>
			<td>96- or 384-well plates can be used for higher throughput applications</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Hyclone</td>
			<td>SH30910</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM), phenol red-free</td>
			<td>Hyclone</td>
			<td>SH30284</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Hyclone</td>
			<td>SH3091003</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonessential amino acids, 100x</td>
			<td>Life Technologies</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-antimycotic, 100x</td>
			<td>Life Technologies</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate, 100mM</td>
			<td>Life Technologies</td>
			<td>11360070</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeocin, 100 mg/ml</td>
			<td>Life Technologies</td>
			<td>R25001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tripsin, 0.05%</td>
			<td>Life Technologies</td>
			<td>25300062</td>
			<td></td>
		</tr>
	</tbody>
</table>

autonomously bioluminescent mammalian cells for continuous and real-time monitoring of cytotoxicity

Células de mamíferos com base em ensaios in vitro têm sido amplamente empregados como alternativas aos testes em animais para estudos toxicológicos, mas foram limitados devido aos altos custos monetários e de tempo de preparação da amostra paralela que sejam necessárias devido à natureza destrutiva do vaga-lume métodos de rastreio baseados luciferase . Este vídeo descreve a utilização de células de mamífero de forma autónoma bioluminescentes, que não requerem a adição de um substrato destrutiva luciferina, tal como um método barato e fácil para a monitorização dos efeitos citotóxicos de um composto de interesse. As células de mamíferos que expressem estavelmente a bioluminescência completo (luxCDABEfrp) cassete do gene autonomamente produzirem um sinal óptico que um pico a 490 nm, sem a adição de um substrato luciferina caro e, possivelmente interferindo, a excitação por uma fonte de energia exterior ou a destruição da amostra que é tradicionalmente realizado durante o procedimento de imagiologia ópticas. Esta independência de estimulação externa coloca o fardo para manter a reacção bioluminescente unicamente sobre a célula, o que significa que o sinal resultante é detectado apenas activo durante o metabolismo. Esta característica faz com que a linha de células de lux que expressam um excelente candidato para o uso como um biosentinel contra os efeitos citotóxicos por causa mudanças na produção bioluminescente são indicativos de efeitos negativos sobre o crescimento e metabolismo celular. Do mesmo modo, a natureza autónoma e falta de destruição da amostra requerida permite imagiologia repetida da mesma amostra em tempo real ao longo do período de exposição tóxico e pode ser realizada através de várias amostras utilizando o equipamento existente de imagem de uma forma automatizada.

Neste estudo, a dinâmica de autobioluminescent as células HEK293 foram continuamente monitorizados ao longo de um período de 24 horas em resposta à exposição a antibióticos (Figura 1). Os efeitos tóxicos deste antibiótico, que é um membro da família de bleomicina conhecido por matar células vivas por ligação e clivagem de ADN 13, foram demonstradas por meio de um decréscimo na produção de bioluminescente em comparação com células não tratadas, o que pode ser visualizado d...

Watch this Scientific Journal Video about Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity at JoVE.com

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

As células de mamífero que expressam a cassete de gene de bioluminescência (<em&gt; Lux</em&gt;) Produzem luz de forma autônoma. A dinâmica bioluminescentes resultantes após a exposição química têm sido demonstrados para reflectir os efeitos do tratamento sobre o crescimento e metabolismo celular, tornando essas células um, contínuo e em tempo real ferramenta de rastreio de toxicidade baixo custo que pode ser facilmente adaptado para a automatização de alto rendimento.

Células de mamíferos autonomamente bioluminescentes para monitoramento contínuo e em tempo real de citotoxicidade

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

autonomously-bioluminescent-mammalian-cells-for-continuous-real-time

Research

JoVE Journal

Biology

10.0K visualizações.  Oak Ridge National Laboratory. As células de mamífero que expressam a cassete de gene de bioluminescência ( Lux) Produzem luz de forma autônoma. A dinâmica bioluminescentes resultantes após a exposição química têm sido demonstrados para reflectir os efeitos do tratamento sobre o crescimento e metabolismo celular, tornando essas células um, contínuo e em tempo real ferramenta de rastreio de toxicidade baixo custo que pode ser facilmente adaptado para a automatização de alto ...

Vídeo: Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

Trypsinizing e Subcultura células de mamíferos

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

Biologia

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

EsiRNA MISSÃO para a seleção RNAi em células de mamíferos

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

Mutagênese e Análise Funcional de canais iônicos Heterologously Expresso em células de mamíferos

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR (qPCR) Arrays

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower detection limit compared to other methods of gene expression profiling while using smaller amounts of input for each assay.  Automated qPCR setup has improved this field by allowing for greater reproducibility.  Its convenient and rapid setup allows for high-throughput experiments, enabling the profiling of many different genes simultaneously in each experiment.  This method along with internal plate controls also reduces experimental variables common to other techniques.  
We recently developed a qPCR assay for profiling of pre-microRNAs  (pre-miRNAs) using a set of 186 primer pairs.  MicroRNAs have emerged as a novel class of small, non-coding RNAs with the ability to regulate many mRNA targets at the post-transcriptional level.  These small RNAs are first transcribed by RNA polymerase II as a primary miRNA (pri-miRNA) transcript, which is then cleaved into the precursor miRNA (pre-miRNA).  Pre-miRNAs are exported to the cytoplasm where Dicer cleaves the hairpin loop to yield mature miRNAs.  Increases in miRNA levels can be observed at both the precursor and mature miRNA levels and profiling of both of these forms can be useful.  There are several commercially available assays for mature miRNAs; however, their high cost may deter researchers from this profiling technique.  Here, we discuss a cost-effective, reliable, SYBR-based qPCR method of profiling pre-miRNAs.  Changes in pre-miRNA levels often reflect mature miRNA changes and can be a useful indicator of mature miRNA expression.  However, simultaneous profiling of both pre-miRNAs and mature miRNAs may be optimal as they can contribute nonredundant information and provide insight into microRNA processing.  Furthermore, the technique described here can be expanded to encompass the profiling of other library sets for specific pathways or pathogens.

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR qPCR Arrays

We will demonstrate the setup and analysis of pre-microRNA 96-well arrays for QPCR using a robot as well as by hand with a Thermo Scientific Matrix multichannel pipette.

Perfil dos pré-micro RNAs e microRNAs usando Quantitative PCR em tempo real (qPCR) Arrays

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower ...

Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer

FRET is a process whereby energy is non-radiatively transferred from an excited donor molecule to a ground-state acceptor molecule through long-range dipole-dipole interactions1. In the present sensing assay, we utilize an interesting property of PDA: blue-shift in the UV-Vis electronic absorption spectrum of PDA (Figure 1) after an analyte interacts with receptors attached to PDA2,3,4,7. This shift in the PDA absorption spectrum provides changes in the spectral overlap (J) between PDA (acceptor) and rhodamine (donor) that leads to changes in the FRET efficiency. Thus, the interactions between analyte (ligand) and receptors are detected through FRET between donor fluorophores and PDA. In particular, we show the sensing of a model protein molecule streptavidin. We also demonstrate the covalent-binding of bovine serum albumin (BSA) to the liposome surface with FRET mechanism. These interactions between the bilayer liposomes and protein molecules can be sensed in real-time. The proposed method is a general method for sensing small chemical and large biochemical molecules. Since fluorescence is intrinsically more sensitive than colorimetry, the detection limit of the assay can be in sub-nanomolar range or lower8. Further, PDA can act as a universal acceptor in FRET, which means that multiple sensors can be developed with PDA (acceptor) functionalized with donors and different receptors attached on the surface of PDA liposomes.

We demonstrate FRET between conjugated polymer polydiacetylene (PDA) and fluorophore attached to the surface of PDA liposomes for the sensing of biomolecules. PDA liposomes also contained receptor molecules on their surfaces for biomolecules to be used as probes. Ligand-receptor interactions lead to changes in the FRET efficiency between the fluorophore and PDA which is the basis of the sensing mechanism.

Monitoramento em tempo real de Ligante-receptor Interações com transferência de energia de ressonância de fluorescência

FRET is a process  whereby energy is non-radiatively transferred from an excited donor molecule to  a ground-state acceptor molecule through long-range ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

FRET Microscopia para Monitoramento em Tempo Real de eventos de sinalização em células vivas utilizando biossensores unimolecular

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

Few naturally-occurring plasmids are maintained in mammalian cells. Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV), which cause multiple human malignancies 1-3. These two genomes are replicated in a licensed manner, each using a single viral protein and cellular replication machinery, and are passed to daughter cells during cell division despite their lacking traditional centromeres 4-8.
Much work has been done to characterize the replications of these plasmid genomes using methods such as Southern blotting and fluorescence in situ hybridization (FISH). These methods are limited, though. Quantitative PCR and Southern blots provide information about the average number of plasmids per cell in a population of cells. FISH is a single-cell assay that reveals both the average number and the distribution of plasmids per cell in the population of cells but is static, allowing no information about the parent or progeny of the examined cell.
Here, we describe a method for visualizing plasmids in live cells. This method is based on the binding of a fluorescently tagged lactose repressor protein to multiple sites in the plasmid of interest 9. The DNA of interest is engineered to include approximately 250 tandem repeats of the lactose operator (LacO) sequence. LacO is specifically bound by the lactose repressor protein (LacI), which can be fused to a fluorescent protein. The fusion protein can either be expressed from the engineered plasmid or introduced by a retroviral vector. In this way, the DNA molecules are fluorescently tagged and therefore become visible via fluorescence microscopy. The fusion protein is blocked from binding the plasmid DNA by culturing cells in the presence of IPTG until the plasmids are ready to be viewed.
This system allows the plasmids to be monitored in living cells through several generations, revealing properties of their synthesis and partitioning to daughter cells. Ideal cells are adherent, easily transfected, and have large nuclei. This technique has been used to determine that 84% of EBV-derived plasmids are synthesized each generation and 88% of the newly synthesized plasmids partition faithfully to daughter cells in HeLa cells. Pairs of these EBV plasmids were seen to be tethered to or associated with sister chromatids after their synthesis in S-phase until they were seen to separate as the sister chromatids separated in Anaphase10. The method is currently being used to study replication of KSHV genomes in HeLa cells and SLK cells. HeLa cells are immortalized human epithelial cells, and SLK cells are immortalized human endothelial cells. Though SLK cells were originally derived from a KSHV lesion, neither the HeLa nor SLK cell line naturally harbors KSHV genomes11. In addition to studying viral replication, this visualization technique can be used to investigate the effects of the addition, removal, or mutation of various DNA sequence elements on synthesis, localization, and partitioning of other recombinant plasmid DNAs.

A method of observing individual DNA molecules in live cells is described. The technique is based on the binding of a fluorescently tagged lac repressor protein to binding sites engineered into the DNA of interest. This method can be adapted to follow many recombinant DNAs in live cells over time.

Monitorando a replicação plasmídeo em células vivas de mamíferos longo de múltiplas gerações por microscopia de fluorescência

Few naturally-occurring plasmids are maintained in mammalian cells.  Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus  (EBV) ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Medição dos Níveis de heme em células de mamíferos Síntese

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

Baseado-Probe-PCR em tempo real Abordagens para a medição quantitativa de microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

Monitoração in situ de montagens moleculars formadas Transientemente do chaperone nas bactérias, no fermento, e nas pilhas humanas

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...