Estos esfuerzos de investigación fueron apoyados por la División Nacional Science Foundation de la Química, Bioingeniería, Medio Ambiente y Sistemas de Transporte (CBET) bajo el número de premios CBET-0853780 y CBET-1159344 y los Institutos Nacionales de Salud, Instituto Nacional de Ciencias de Salud Ambiental (NIEHS) bajo el número de concesión 1R43ES022567-01, y el Instituto Nacional del Cáncer, Programa de Imagen del Cáncer con el número premio CA127745-01. El instrumento IVIS Lumina utilizado en este trabajo se obtuvo del Programa de Instrumentación EE.UU. Ejército Defense University Research.

1. Preparación de células <ol><li> Recuperar un vial de células bioluminiscentes HEK293 de nitrógeno líquido stock congelado y crecer en medio de Eagle modificado por Dulbecco (DMEM) suplementado con 10% de suero fetal bovino, mM de aminoácidos no esenciales 0,01, 1x antibiótico-antimicótico, y 0,01 mM de piruvato de sodio en un T 75 frasco de cultivo de tejidos a 37 º C y 5% de CO 2. Nota: Los medios de comunicación y componentes complementarios pueden variar dependiendo de las líneas de células y por lo tanto deben ser elegidos en consecuencia. </li><li> Actualizar medio cada 2-3 días hasta alcanzar ~ 80% de confluencia. Nota: La cantidad de células necesarias se basa en el diseño experimental. Si es necesario, más células pueden obtenerse a través de subcultivo. </li><li> Células de la cosecha para la prueba. <ol><li> Retirar el medio y lavar las células con solución salina tamponada con fosfato (PBS) girando suavemente el matraz y luego desechar el estado de PBS. </li><li> Añadir tripsina e incubar a 37 ° C durante 2 min,o hasta que las células se separaron del matraz. </li><li> Recoger las células desprendidas en PBS y transferirlos a un tubo de centrífuga limpio. Nota: Si las líneas celulares adherentes se utilizan, las células separadas por centrifugación y se lavan una vez con PBS. </li></ol></li><li> Determinar el recuento total de células utilizando un hemocitómetro o de otro sistema de recuento de células. </li><li> Centrifugar la suspensión de células a 300 xg durante 5 min. Desechar el sobrenadante y resuspender las células en medio fresco, precalentado. </li><li> Semillas igual número de células en pocillos individuales de una placa de múltiples pocillos opaca. En este ejemplo, la placa de 5 x 10 5 células en un volumen de 1 ml en cada pocillo de una placa de 24 pocillos negro. Placa de un volumen igual de medio en pocillos por triplicado para actuar como el control negativo. Nota: El número de células sembradas en cada pocillo es flexible y puede ser optimizado para los experimentos individuales. Para cada línea celular bioluminiscente, determinar experimentalmente la relación entre el número de células y biolumiNESCent de salida, así como la célula mínima detectable cuentan antes de ningún ensayo de toxicidad. Para ello, la medida de bioluminiscencia en una amplia gama de tamaños de población (por ejemplo, 1 x 10 03 al 1 x 10 6 células) en condiciones de formación de imágenes estandarizadas. </li><li> Tratar las células con el compuesto de ensayo (s) según se describe a continuación. </li></ol> 2. Preparación química <ol><li> Preparar la sustancia estudiada como una solución madre concentrada. En este ejemplo, utilizar una 100 mg / ml de solución madre de un antibiótico de la familia de la bleomicina. Nota: Para reducir al mínimo los posibles efectos tóxicos vehículo a base de, especialmente si un disolvente orgánico se utiliza como un vehículo para la adición de productos químicos, se recomienda que la concentración de solución madre sea de al menos 1000 veces la concentración después de la aplicación deseada. </li><li> Añadir el caldo de química preparada directamente a las células. En este ejemplo, la dosis del antibiótico de interés a concentraciones finales de 100, 200, 300, y 400 mg / ml en triplicar pozos. Agregar tres pocillos de células no tratadas como controles. Nota: La concentración final de la sustancia química objetivo se debe seleccionar sobre la base de los objetivos experimentales específicas. Una amplia gama de concentraciones se prueba normalmente para obtener un espectro completo de los efectos tóxicos. </li></ol> 3. Imágenes y Análisis de Datos <ol><li> Inmediatamente después de la adición de productos químicos, coloque la placa en la cámara de imágenes de instrumento adecuado para la adquisición de imágenes y la medición de bioluminiscencia. </li><li> Medida bioluminiscencia cada 15 minutos durante un período de 24 horas con un tiempo de 10 min de integración para cada lectura. Nota: El tiempo de integración utilizado en este ejemplo es en una base por placa y se puede ajustar para la medición en una base por pocillos usando otros luminómetros de elección. El tiempo de integración también se puede ajustar sobre la base de la línea de células bioluminiscente elegido. Debido a que la bioluminiscencia se produce autónomamente sin la destrucción de células o ninguna estimulación externa, leernes se pueden tomar en cualquier punto en el tiempo que desee para cumplir con los objetivos experimentales específicas. </li><li> Cuantificar la intensidad bioluminiscente de cada pocillo mediante la identificación de una región de interés utilizando software compatible. Muestra la salida de luz, ya sea como el flujo total (fotones / segundo) o la luminosidad media (fotones / segundo / cm steridian 2 /) de cada muestra. </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 y GS Sayler son fundadores y propietarios de 490 BioTech, Inc.

Este método demuestra el uso de células de mamíferos bioluminiscentes forma autónoma como un ensayo de cribado de citotoxicidad in vitro que permite a las células en vivo para ser controlados continuamente durante toda su vida en. Este protocolo es flexible y puede ser modificado para adaptarse a las condiciones experimentales específicas según se requiera. Por ejemplo, el experimento que aquí se presenta es adecuado para el seguimiento de los efectos tóxicos agudos, pero puede ser adaptado pa...

En los EE.UU., los productos farmacéuticos y otros productos destinados al consumo humano requieren una evaluación extensiva antes de que sean aprobados para su uso por el consumidor por la Administración de Alimentos y Drogas. La carga financiera para la realización de esta prueba se coloca en el desarrollador de 1, lo que aumenta sustancialmente el coste de desarrollo compuesto nuevo y por lo tanto, se traduce en un aumento de coste para los consumidores. Si bien tradicionalmente la mayor parte de este examen ha utilizado materias animales para actuar como representantes de los huéspedes humanos, esto ha demostrado ser una gran carga financiera, con un estimado de 2,8 mil millones dólares gastados anualmente en ADME / Tox (absorción, distribución, metabolismo, excreción y toxicidad ) seleccionar solo 2 y la creciente evidencia que sugiere que los modelos animales no pueden predecir con certeza las respuestas toxicológicas humanos 3. Por lo tanto, las pruebas basadas en el cultivo de células humanas in vitro se ha ganado popularidad en las últimas dos décadas debido a su relativamente bajacoste, mayor rendimiento y una mejor representación de la biodisponibilidad humana y toxicología 4. Los métodos de cribado de toxicidad basada en el cultivo de células actuales emplean diversos criterios de valoración, tales como la medición de los niveles de ATP, la detección de la actividad de las enzimas citoplasmáticas endógenamente disponibles, el sondeo de la integridad de la membrana celular, o el seguimiento del nivel de la actividad mitocondrial, para evaluar la viabilidad celular 5 , 6. Sin embargo, independientemente del punto final elegido, todos estos métodos requieren la destrucción de la muestra antes de las mediciones se pueden tomar, por tanto, sólo la producción de datos en un solo punto de tiempo. Como resultado de ello, un gran número de muestras tienen que ser preparados y tratados en paralelo para estudios básicos cinética toxicológicos, de nuevo añadiendo al coste y mano de obra requerida para el nuevo desarrollo compuesto. Alternativamente, los ensayos de luciferasa utilizando secretadas tales como luciferasa Gaussia 7, luciferasa de Vargula 8, 9 y Metridia luciferasase han desarrollado que elimina la necesidad de la lisis celular y requiere una fracción de los medios de comunicación para la medición de la variable, sin embargo, éstos están todavía se limita a muestreo en puntos de tiempo predeterminados y también requiere la adición de sustratos exógenos luz de activación. Para evitar el detrimento de la destrucción de la muestra requerida, así como para eliminar el costo de sustratos, una línea celular humana ha sido diseñado que expresa la bioluminiscencia bacteriana completo (lux) casete del gen (luxCDABEfrp) para permitir la monitorización continua de células vivas que es similar de imágenes de células vivas fluorescente basado-dye, pero sin los procedimientos de investigación de fotones que activan y microscópicas adicionales. Esta línea celular es capaz de producir constitutivamente una señal óptica para la detección continua, directa y sin la necesidad de la estimulación externa, evitando así la destrucción de la muestra. Mecánicamente, la señal bioluminiscente generada a partir de estas células como resultados cuando la enzima luciferasa luxAB formado cataliza la oxidación de un aldehído graso de cadena larga (sintetizado y regenerado por los productos de los genes luxCDE usando sustratos endógenos) en presencia de la reducción de fosfato de riboflavina (FMNH 2, que se recicla desde FMN por el gen FRP producto) y oxígeno molecular 10. Por lo tanto, la expresión del casete de lux en la célula huésped permite que la luz a ser producido y detectado sin la destrucción celular o adición de sustrato exógeno. Del mismo modo, la interacción entre los genes lux y el FMN disponibles endógenamente, y O 2 co-sustratos, y los requisitos de mantenimiento de un entorno que puede apoyar la conversión de FMN a FMNH 2, asegura que la señal bioluminiscente resultante sólo puede ser detectado de vivir , las células metabólicamente activas. Estos requisitos previamente han sido explotados para demostrar que lux-bassalida ed bioluminiscente se correlaciona fuertemente con tamaño de la población celular 11 y que la exposición compuesto tóxico perjudica la producción autobioluminescent de una manera dosis-respuesta 12. Aquí se utiliza un autobioluminescent de riñón embrionario humano previamente caracterizado (HEK293) línea celular 11 para demostrar la detección toxicológica automatizado de un antibiótico de la familia de la bleomicina con conocida actividad que daña el ADN como un ejemplo representativo para validar la solicitud de células de mamífero autobioluminescent para las pruebas de toxicidad.

<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 basado en ensayos in vitro han sido ampliamente empleada como alternativas a los ensayos con animales para los estudios toxicológicos, pero se han limitado debido a los altos costos monetarios y el tiempo de preparación de la muestra paralela que se hicieron necesaria debido a la naturaleza destructiva de los métodos de detección basados ​​en luciferasa de luciérnaga . Este video describe la utilización de células de mamíferos bioluminiscentes forma autónoma, que no requiere la adición destructiva de un sustrato de luciferina, como un método económico y fácil para el seguimiento de los efectos citotóxicos de un compuesto de interés. Las células de mamífero que expresan establemente la bioluminiscencia bacteriana completo (luxCDABEfrp) casete del gen autónoma producen una señal óptica que picos a 490 nm sin la adición de un sustrato de luciferina caro y posiblemente interferir, de excitación por una fuente de energía externa, o la destrucción de la muestra que es tradicionalmente realizado durante el procedimiento de formación de imágenes ópticass. Esta independencia de la estimulación externa coloca la carga para el mantenimiento de la reacción bioluminiscente únicamente en la célula, lo que significa que la señal resultante sólo se detecta durante el metabolismo activo. Esta característica hace que la línea de células de lux que expresan un excelente candidato para el uso como un biosentinel contra los efectos citotóxicos debido a cambios en la producción bioluminiscente son indicativos de efectos adversos sobre el crecimiento celular y el metabolismo. Del mismo modo, el carácter autónomo y la falta de la destrucción de la muestra requerida permite formación de imágenes repetidas de la misma muestra, en tiempo real durante todo el período de exposición tóxica y se pueden realizar a través de múltiples muestras de imagen utilizando el equipo existente de forma automatizada.

En este estudio, la dinámica de las células HEK293 autobioluminescent se monitorizaron continuamente durante un período de 24 horas en respuesta a la exposición a antibióticos (Figura 1). Los efectos tóxicos de este antibiótico, que es un miembro de la familia bleomicina sabido para matar a las células vivas mediante la unión a, y escisión de ADN 13, se demostraron a través de una disminución en la producción bioluminiscente en comparación con las células no tratadas, que puede...

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

Las células de mamífero que expresan el casete del gen bioluminiscencia bacteriana (<em&gt; Lux</em&gt;) Producir luz autónoma. La dinámica bioluminiscentes resultantes tras la exposición química se han demostrado para reflejar los efectos de los tratamientos sobre el crecimiento celular y el metabolismo, haciendo que estas células una herramienta barata, continua, en tiempo real toxicidad de detección que puede ser fácilmente adaptado para la automatización de alto rendimiento.

Las células de mamíferos bioluminiscentes manera autónoma para la monitorización continua y en tiempo real de citotoxicidad

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 vistas.  Oak Ridge National Laboratory. Las células de mamífero que expresan el casete del gen bioluminiscencia bacteriana ( Lux) Producir luz autónoma. La dinámica bioluminiscentes resultantes tras la exposición química se han demostrado para reflejar los efectos de los tratamientos sobre el crecimiento celular y el metabolismo, haciendo que estas células una herramienta barata, continua, en tiempo real toxicidad de detección que puede ser fácilmente adaptado para la automatización de ...

Video: 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 Siembra y células de mamífero

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

Investigación

Biología

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.

MISIÓN esiRNA sobre el cribado del ARNi en células de mamífero

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.

Mutagénesis y análisis funcional de los canales iónicos heteróloga se expresa en células de mamífero

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.

Perfiles de Pre-micro ARN y microRNAs utilizando cuantitativa en tiempo real 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 ...

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.

Monitoreo en tiempo real de las interacciones ligando-receptor con transferencia de energía de resonancia fluorescente

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 Microscopía de Monitoreo en tiempo real de eventos de señalización en las células vivas Uso de biosensores unimoleculares

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.

Supervisión de la replicación del plásmido en células de mamíferos en vivo durante varias generaciones por microscopía de fluorescencia

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.

Medición de niveles hemo síntesis en células de mamífero

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.

PCR en tiempo real basada en sondeos Enfoques para la medición cuantitativa 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.

Monitoreo in situ de conjuntos de chaperona molecular formadas transitoriamente en bacterias, levaduras y células 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 ...