C. elegans transgénicos fueron proporcionados por el Centro de Genética Caenorhabditis (Universidad de Minnesota, Minneapolis, MN). Este trabajo fue apoyado por el NIH R21 conceder NS0667678-01 a KS. Todos los autores participaron en la recolección, análisis e interpretación de datos. CKL, KS, y KPC participaron en la redacción y revisión del manuscrito.

1. Preparación de comida de gusanos bacteriana Día 1 <ol><li> Añadir 5 ml de grasa saturada E. coli OP50 cultivo bacteriano a 500 ml de caldo fabuloso suplementado con 50 mg / ml de estreptomicina y crecer en una incubadora de agitación (225 rpm) durante la noche a 37 ° C. </li></ol> Día 2 <ol start="2"><li> Dividir el cultivo de bacterias durante la noche en diez tubos de 50 ml y bacterias centrífuga en una centrífuga refrigerada a 2.500 rcf durante 20 minutos. </li><li> Decantar el caldo LB y resuspender cada sedimento bacteriano en 10 ml de líquido de los medios de comunicación nematodo crecimiento (NGM). Agitar horizontalmente en el suelo agitador durante 15 minutos para volver a suspender las bacterias. Para hacer buffer NGM, añadir 3 g de NaCl en 1 l de agua desionizada y autoclave. Enfriar a 55 ° C y añadir con el fin de las siguientes soluciones estériles: 1 ml de 1 M de CaCl 2, 1 ml de 1 M MgSO 4, y 25 ml de 1 M de fosfato de potasio, pH 6,0. </li><li> Centrifugar el cultivo bacteriano en una centrífuga refrigerada a 2500 rcf durante 20 minutos. </li><li> Decantar buffer NGM y pesar el sedimento bacteriano. </li><li> Añadir un volumen igual de tampón de NGM para volver a suspender las pastillas, alícuota de 3 ml de bacterias se concentran en tubos de 15 ml y almacenar a -20 ° C. </li></ol> 2. A gran escala C. elegans de cultivo líquidos Nosotros usamos una línea transgénica VP596 (dvIs19 [pAF15 (gst-4:: GFP:: NLS)]; vsIs33 [dop-3:: RFP]) con dos construcciones fluorescentes: Pgst -4:: GFP 14 para controlar SKN-1 actividad y PDOP 3:: PP 17 para servir como un estándar para la normalización del gusano número. Día 1 <ol><li> Mezclar 150 ml NGM buffer, 1,5 ml de caldo de LB, 150 l de 5 mg / ml de colesterol (en etanol), y 75 l de 100 mg / ml de estreptomicina. Filtro a esterilizar la mezcla y añadir a un frasco estéril de 1 litro. NOTA: Agregar 1% caldo de LB para amortiguar NGM ayuda a evitar que los gusanos se pegue a la cristalería y utensilios de plástico, pero esta cantidad no es suficiente para apoyar el crecimiento de bacterias. </li><li> Sincronizar los gusanos con el procedimiento estándar de hipoclorito de 18 años. Hasta 0,5 ml de gusanos grávidos pueden ser procesados ​​en un tubo de 15 ml solo con 5 ml de solución de hipoclorito (3,75 ml de agua estéril, 1 ml de lejía de uso doméstico, y 250 l de NaOH 10 N). Lavado de los óvulos liberados con agua estéril y se resuspenden en 10 ml de solución tampón NGM. </li><li> Diluir una muestra de los huevos de 100 veces en NGM tampón (por ejemplo, 100 l en 10 ml) y contar el número de huevos en tres separadas 5 alícuotas. Varios el número promedio de 200.000 (100 x factor de dilución 2000) para estimar el número total de huevos. </li><li> Agregar 200.000 a 2 millones de huevos en el matraz con tampón NGM y agitar la cultura a 100 rpm a 20 ° C. </li></ol> Día 2 <ol start="5"><li> Por lo menos 16 horas después de añadir los huevos en el matraz, utilice una pipeta serológica estéril para retirar aproximadamente 0,5 ml de la lombricultura en suspensión. Pipeta de 5 gotas tres l en la tapa estéril de una placa de Petri. Coloque la tapa en un congelador a -20 ° C durante 1-2 minutos para paralizar los gusanos. Cuente el número promedio de gusanos vivos nacidos por cada 5 l, y luego varios en 30.000 (150 ml / 5 l) para estimar el número total de gusanos nacidos. </li><li> Descongelar un tubo de congelados OP50 cultivo bacteriano (Protocolo 1.6) y añadir 3 ml de 50% OP50 en la cultura del gusano por cada 500.000 nacidos gusanos. Agitar el matraz a 100 rpm a 20 ° C. Los gusanos se convierten en larvas L4 y adultos jóvenes en aproximadamente 51 horas. El OD600 de bacterias debe ser cerca de 2.200 desde el principio. Controlar la cantidad de bacterias mediante la absorción y añadir más si el OD 600 cae por debajo de 0.900. </li></ol> 3. Gusano de recogida y distribución Día 4 <ol><li> Use una pipeta serológica estéril para la transferencia de aproximadamente 0,5 ml de la cultura del gusano suspendido sobre una placa de agar NGM estándar. Ver los gusanos con un microscopio estereoscópico para asegurarse de que la mayoría son larvas L4 y adultos jóvenes. L4 larvas tienen una mancha ventral claro en la parte media del cuerpo. Los adultos jóvenes será un poco más grande y no tendrá un lugar despejado. </li><li> Vierta la lombricultura en el estéril tubos de 50 ml y se colocan los tubos en un bastidor de tubo de ensayo en el laboratorio. Permita que los gusanos que conformarse con unos 10 minutos. Eliminar el sobrenadante por aspiración o la pipeta. NOTA: Este paso puede ser importante para eliminar los huevos no eclosionados o gusanos que no se desarrolló debido a que se asentarán más lento que larvas L4 y adultos jóvenes. </li><li> Recoge todos los gusanos en un solo tubo de 50 ml y lavar con buffer NGM con caldo de LB 1% (NGM LB +) 3-4 veces para eliminar las bacterias. NOTA: Esto se hace mejor en una centrífuga de balanceo de cubo mediante la recopilación de los gusanos a 500 rcf durante 30 segundos y reemplazar el sobrenadante con frescos NGM + tampón LB. </li><li> Después del lavado, llenar el tubo con NGM + tampón LB a 50 ml y se vierte en un frasco de gusano personalizado de dosificación. Agregar una barra de poca agitación y revuelo para mantener a los gusanos en suspensión. Conde ªnúmero de gusanos de correo en tres 5 gotas l como se indica más arriba. Diluir con NGM LB + hasta llegar 12-15 gusanos por 5 gota l (~ 2.5-3.0 gusanos / l). NOTA: Un mínimo de 20 ml (60.000 gusanos) que se necesita para llenar el espacio muerto del frasco dispensador y dispensador de cinta. Cada placa de 384 y requiere cerca de 35.000 gusanos o 11,5 ml de suspensión gusanos. </li><li> Cargar un casete de 10 l de dosificación y esterilizar cebado con etanol al 70%. Enjuague el etanol mediante cebado con agua estéril. </li><li> Inserte el extremo de la cinta de distribución en el matraz de manera que es justo por encima de la barra de agitación sin interrumpir su movimiento. Asegúrese de que los gusanos permanecen suspendidas en todo el frasco entero. Programa del dispensador para dispensar a baja velocidad con dos pre-pulsos. Primer y ejecutar al menos 10 ml de suspensión gusanos. Llenar las placas, según sea necesario con rapidez para evitar que los gusanos se asiente en el tubo. </li><li> Sellar las placas con cinta adhesiva transpirable. </li><li> Coloque las placas en una plataforma de agitación en una incubadora a la temperatura adecuada para su análisis. No apile los platos. </li></ol> 4. El análisis de fluorescencia <ol><li> Coloque la placa de destino en un lector de microplacas para medir la intensidad de la fluorescencia de cada pozo con la longitud de onda apropiada de emisión y excitación (Filtros para nuestro ensayo: GFP 485/20ex 528/20em; RFP 540/25ex 590/35em). </li><li> Calcular la proporción de GFP / RFP y normalizar las lecturas de los pozos de control (sin compuesto activador) para determinar la diferencia exacta de veces la intensidad de fluorescencia derivado de los tratamientos individuales. </li></ol> 5. Los resultados representativos: Nuestra SKN-1 ensayo utiliza una cepa integrado cromosómicamente reportero dual (VP596). Como se muestra en la Figura 1, el número de gusanos se correlaciona bien con el volumen dispensado. Como se muestra en 2A y 2B, el total de las buenas prácticas agrarias y RFP cifras por fluorescencia y es altamente reproducible de un pozo a través de una placa de 384 pocillos. Como se muestra en la Figura 2C, sin inducida Pgst-4: la fluorescencia de GFP es linealmente correlacionada con PDOP-3:: Solicitud de Propuestas a través de 384 pozos. Cuando se expresa como una relación de buenas prácticas agrarias / RFP, la fluorescencia se convierte en altamente reproducible de un pozo a través de una placa de 384 pocillos (comparar el coeficiente de variación de la figura 2D a 2A) que demuestran la capacidad de la DPS-3:: reportero RFP para reducir la variabilidad. Como se muestra en la figura 3, la inducción de las buenas prácticas agrarias en relación a la RFP con un SKN-1 activar xenobióticos (38 M juglone) es sólido y altamente reproducible a través de una placa de 384 pocillos. <img src="/files/ftp_upload/2745/2745fig1.jpg" alt="Figura 1"/> Figura 1. El número de gusanos en función del volumen dispensado. Los gusanos se diluyeron hasta aproximadamente 2/μl y se distribuye en una placa de 24 pocillos para el conteo manual con un microscopio estereoscópico. N = 8 pozos por unidad de volumen. <img src="/files/ftp_upload/2745/2745fig2.jpg" alt="Figura 2"/> Figura 2. Fluorescencia total de gusanos se distribuye en una placa de 384 y es reproducible. Los gusanos se diluyeron a l aproximadamente 2.5/μl y 30 se distribuye en cada pocillo de una placa de 384 pocillos. GFP (A) y el PP (B) de fluorescencia de todos los pozos tenían un coeficiente de variación por debajo del 9%. (C) la fluorescencia de GFP está altamente correlacionada con la RFP de fluorescencia. (D) Cálculo de la relación de las buenas prácticas agrarias para reducir RFP el coeficiente de variación por debajo de 6% (A, B y D) Las líneas continuas indican los medios y las líneas discontinuas indican tres desviaciones estándar por encima o por debajo de la media. <img src="/files/ftp_upload/2745/2745fig3.jpg" alt="Figura 3"/> . Figura 3 La activación de Pgst-4:: GFP es robusta y consistente. Aproximadamente el 75 L4 gusanos fueron dispensadas en los pocillos de una placa de 384 pozos y 38 juglone M se añadió en cada columna. Las buenas prácticas agrarias y RFP de fluorescencia se midió a las 21 h de incubación. La media de ratios de fluorescencia relativa de todo control (1,0) y juglone pozos (8,9) están marcados con líneas continuas. Tres desviaciones estándar por encima de la media de control y por debajo de la media juglone están marcados con líneas discontinuas.

<ol>
	<li>Simeonov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+high-throughput+screen+identifies+inhibitors+of+the+Schistosoma+mansoni+redox+cascade.">Quantitative high-throughput screen identifies inhibitors of the Schistosoma mansoni redox cascade.</a> PLoS Negl Trop Dis. 2, e127-e127 (2008).</li><li>Giacomotto, J., Segalat, L. <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+screening+and+small+animal+models,+where+are+we.">High-throughput screening and small animal models, where are we.</a> Br J Pharmacol. 160, 204-216 (2010).</li><li>Artal-Sanz, M., de Jong, L., Tavernarakis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans:+a+versatile+platform+for+drug+discovery.">Caenorhabditis elegans: a versatile platform for drug discovery.</a> Biotechnol J. 1, 1405-1418 (2006).</li><li>An, J. H., Blackwell, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SKN-1+links+C.+elegans+mesendodermal+specification+to+a+conserved+oxidative+stress+response.">SKN-1 links C. elegans mesendodermal specification to a conserved oxidative stress response.</a> Genes Dev. 17, 1882-1893 (2003).</li><li>Choe, K., Przybysz, A., Strange, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+WD40+repeat+protein+WDR-23+functions+with+the+CUL4/DDB1+ubiquitin+ligase+to+regulate+nuclear+abundance+and+activity+of+SKN-1+in+Caenorhabditis+elegans.">The WD40 repeat protein WDR-23 functions with the CUL4/DDB1 ubiquitin ligase to regulate nuclear abundance and activity of SKN-1 in Caenorhabditis elegans.</a> Mol Cell Biol. 29, 2704-2715 (2009).</li><li>Hasegawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acrylamide-responsive+genes+in+the+nematode+Caenorhabditis+elegans.">Acrylamide-responsive genes in the nematode Caenorhabditis elegans.</a> Toxicol. Sci. 101, 215-225 (2008).</li><li>Tullet, J. M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+inhibition+of+the+longevity-promoting+factor+SKN-1+by+insulin-like+signaling+in+C.+elegans.">Direct inhibition of the longevity-promoting factor SKN-1 by insulin-like signaling in C. elegans.</a> Cell. 132, 1025-1038 (2008).</li><li>Oliveira, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Condition-adapted+stress+and+longevity+gene+regulation+by+Caenorhabditis+elegans+SKN-1/Nrf.">Condition-adapted stress and longevity gene regulation by Caenorhabditis elegans SKN-1/Nrf.</a> Aging Cell. , (2009).</li><li>Park, S. -. K., Tedesco, P. M., Johnson, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+and+longevity+in+Caenorhabditis+elegans+as+mediated+by+SKN-1.">Oxidative stress and longevity in Caenorhabditis elegans as mediated by SKN-1.</a> Aging Cell. 8, 258-269 (2009).</li><li>Hayes, J. D., McMahon, M., Chowdhry, S., Dinkova-Kostova, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+chemoprevention+mechanisms+mediated+through+the+Keap1-Nrf2+pathway.">Cancer chemoprevention mechanisms mediated through the Keap1-Nrf2 pathway.</a> Antioxid Redox Signal. , (2010).</li><li>Kwak, M. K., Kensler, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+NRF2+signaling+for+cancer+chemoprevention.">Targeting NRF2 signaling for cancer chemoprevention.</a> Toxicol Appl Pharmacol. 244, 66-76 (2010).</li><li>Leiser, S. F., Miller, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nrf2+signaling,+a+mechanism+for+cellular+stress+resistance+in+long-lived+mice.">Nrf2 signaling, a mechanism for cellular stress resistance in long-lived mice.</a> Mol Cell Biol. 30, 871-884 (2010).</li><li>Link, C. D., Johnson, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reporter+transgenes+for+study+of+oxidant+stress+in+Caenorhabditis+elegans.">Reporter transgenes for study of oxidant stress in Caenorhabditis elegans.</a> Methods Enzymol. 353, 497-505 (2002).</li><li>Vanduyn, N., Settivari, R., Wong, G., Nass, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SKN-1/Nrf2+inhibits+dopamine+neuron+degeneration+in+a+Caenorhabditis+elegans+model+of+methylmercury+toxicity.">SKN-1/Nrf2 inhibits dopamine neuron degeneration in a Caenorhabditis elegans model of methylmercury toxicity.</a> Toxicol Sci. , (2010).</li><li>Hasegawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+inexpensive+method+to+screen+for+common+foods+that+reduce+the+action+of+acrylamide,+a+harmful+substance+in+food.">A rapid and inexpensive method to screen for common foods that reduce the action of acrylamide, a harmful substance in food.</a> Toxicol Lett. 175, 82-88 (2007).</li><li>Chase, D. L., Pepper, J. S., Koelle, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+extrasynaptic+dopamine+signaling+in+Caenorhabditis+elegans.">Mechanism of extrasynaptic dopamine signaling in Caenorhabditis elegans.</a> Nature Neuroscience. 7, 1096-1103 (2004).</li><li>Hope, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> C. elegans, a Practical Guide. , (2005).</li><li>Pulak, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+analysis,+sorting,+and+dispensing+of+C.+elegans+on+the+COPAS+flow-sorting+system.">Techniques for analysis, sorting, and dispensing of C. elegans on the COPAS flow-sorting system.</a> Methods Mol Biol. 351, 275-286 (2006).</li></ol>

Se presenta un método para cultivar y distribuir grandes cantidades de nematodos transgénicos. El equipo utilizado para el cultivo de lombrices es el estándar para los laboratorios que realizan la clonación molecular y el manejo de líquidos y el equipo de fluorescencia es el estándar para los laboratorios de procesamiento de un gran número de microplacas. Otros métodos de dispensación de un gran número de vivir C. elegans requieren partículas caro equipo de clasificación 19. El Pgst-4...

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios (opcional) </th></tr><tr><td> LB caldo </td><td> Investigación de Productos International Corp. </td><td> L24066 </td><td></td></tr><tr><td> Excelente caldo de </td><td> Investigación de Productos International Corp. </td><td> T15100 </td><td></td></tr><tr><td> Synergy ™ HT Multi-modo de lector de microplacas </td><td> BioTek </td><td></td><td> Filtros: GFP 485/20ex 528/20em; RFP 540/25ex 590/35em </td></tr><tr><td> MicroFlo Seleccione dispensador </td><td> BioTek </td><td></td><td></td></tr><tr><td> Gusano de dispensación frasco </td><td> El sur de Scientific Inc., Micanopy, FL </td><td></td><td> Costumbre montado (352) 284-2531 </td></tr><tr><td> 384 microplacas </td><td> Greiner Bio-One </td><td> 5678-1209 </td><td></td></tr><tr><td> Cinta de sellado transpirable </td><td> Nunc </td><td> 241205 </td><td></td></tr><tr><td> (5-hidroxi-p-naphthoqinone) juglone </td><td> ACROS </td><td> 121640010 </td><td> Juglone se disuelve en DMSO </td></tr><tr><td> DMSO </td><td> Sigma-Aldrich </td><td> D-5879 </td><td></td></tr><tr><td> C. elegans transgénicos cepa </td><td> Autor de laboratorio </td><td> VP596 </td><td> Pgst-4:: GFP y PDOP 3:: SDP </td></tr></tbody></table>

high-throughput screening and biosensing with fluorescent c. elegans strains

Selección de alto rendimiento (HTS) es un poderoso método para la identificación de moduladores químicos de los procesos biológicos. Sin embargo, muchos de los compuestos identificados en las pantallas usando modelos de cultivo celular se encuentran a menudo que son tóxicos o inactivos farmacológicamente in vivo 1-2. Proyección en modelos animales todo puede ayudar a evitar estos escollos y agilizar el camino hacia el desarrollo de fármacos. C. elegans es un organismo multicelular modelo muy adecuado para HTS. Es pequeño (&lt;1 mm) y puede ser económicamente culta y se distribuye en los líquidos. C. elegans es también uno de los modelos animales más manejables experimentalmente que permitan la identificación rápida y detallada de las drogas el modo de acción 3. Se describe un protocolo para el cultivo y distribución de las cepas fluorescentes C. elegans para la selección de alto rendimiento de bibliotecas químicas o detección de contaminantes ambientales que alteran la expresión de un gen específico. Un gran número de gusanos de desarrollo sincronizado en un cultivo líquido, cosecha, lavado, y suspendió a una densidad definida. Los gusanos se añaden al negro, de fondo plano placas de 384 pocillos con un dispensador de líquidos peristáltica. Las moléculas pequeñas de una biblioteca de química o de las muestras de ensayo (por ejemplo, agua, alimentos o suelo) se pueden añadir a los pozos con los gusanos. En vivo y en tiempo real la intensidad de fluorescencia se mide con un lector de microplacas de fluorescencia. Este método se puede adaptar a cualquier gen inducible en C. elegans para que un periodista adecuados. Estrés inducible muchas vías de desarrollo y de la transcripción están bien definidos en C. elegans y las buenas prácticas agrarias reportero cepas transgénicas ya existen en muchos de ellos 4. Cuando se combina con los reporteros transgénicos corresponda, nuestro método puede ser utilizado para la detección de moduladores de la vía o el desarrollo de sólidas pruebas de biosensor de contaminantes ambientales. Demostramos nuestro C. elegans la cultura y el protocolo de dispensación con un ensayo HTS que hemos desarrollado para controlar el C. factor de transcripción collar &#39;n&#39; elegans tapa SKN-1. SKN-1 y su homólogo de mamíferos Nrf2 activar genes citoprotectores durante el estrés oxidativo y 10.5 xenobióticos. Nrf2 protege a los mamíferos a partir de numerosos trastornos relacionados con la edad como el cáncer, neurodegeneración y la inflamación crónica y se ha convertido en un importante objetivo de la quimioterapia 11-13. Nuestro análisis se basa en un reportero GFP transgénicos para la SKN-1 del gen objetivo -4 gst 14, que codifica un 6 glutatión-s-transferasa. El periodista gst -4 es también un biosensor para los productos químicos xenobióticos y de oxidación, que activan SKN-1 y se puede utilizar para detectar bajos niveles de contaminantes tales como la acrilamida y el metil-mercurio 15-16.

Watch this Scientific Journal Video about High-throughput Screening and Biosensing with Fluorescent C. elegans Strains at JoVE.com

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

Un procedimiento de cultivo a base de líquido y dispensación de<em&gt; C. elegans</em&gt; Cepas que expresan proteínas fluorescentes reportero se describe que no requiere de equipos de selección caro. Este enfoque puede aplicarse a numerosas inducible<em&gt; C. elegans</em&gt; Genes para la detección de drogas o biosensores de contaminantes.

Selección de alto rendimiento y biosensores fluorescentes con C. elegans Las cepas

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

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High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

17.9K vistas.  University of Florida. Un procedimiento de cultivo a base de líquido y dispensación de C. elegans Cepas que expresan proteínas fluorescentes reportero se describe que no requiere de equipos de selección caro. Este enfoque puede aplicarse a numerosas inducible C. elegans Genes para la detección de drogas o biosensores de contaminantes.

Video: High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

Cribado de alto rendimiento de la actividad endoglucanasa por hongos en Escherichia coli</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

Investigación

Biología

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi de detección para identificar fenotipos postembrionarios en C. elegans</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

Cuantitativos y automatizada de alto rendimiento del genoma completo de RNAi pantallas en C. elegans</em

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

De alto rendimiento de purificación de afinidad de proteínas marcadas con recombinantes

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

De alto rendimiento para los moduladores de molécula pequeña de tráfico de perfeccionamiento activo Canales rectificador de potasio

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

C. elegans Ensayo de quimiotaxis

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

Bioquímica y de Evaluación de Rendimiento microscópica de masa grasa en<em&gt; Caenorhabditis elegans</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

Selección de alto rendimiento funcional utilizando un doble resplandor de ensayo de luciferasa Homemade

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Selección de alto rendimiento para moduladores químicos de genes post-transcripcionalmente regulados

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

Identificación de los pares quinasa-sustrato Usando selección de alto rendimiento


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...