Damos las gracias a John Darga del MGH ADN Core para la preparación de la biblioteca de detección de ADN. Christopher Chigas de Perkin Elmer prestó apoyo invalorable para nuestra Wallac 1420 luminómetro. Steven Ciacco y Martin Thomae de Agilent proporcionó apoyo para el robot Bravo. Damos las gracias a Ron Johnson y Steve Tito en el NIH para ofrecer generosamente su protocolo de ensayo de luciferasa dual resplandor.

Para una visión general experimental, ver Figura 1. 1. Preparar reportero plásmidos que contienen la alfa-sinucleína Promotor Los elementos reguladores del gen de la alfa-sinucleína (Figura 2) abarcan aproximadamente 10 kb, desde el upstream NACP (A no componente beta amiloide) secuencia de repetición del dinucleótido 1,2 través intrón 2 3. Incluimos una parte de esta región en nuestro reportero construir. Encontramos que los plásmidos que contienen el intrón 2 se requieren procedimientos especiales para el crecimiento, que se detallan a continuación. Los plásmidos de luciferasa, pGL4.10 y pGL4.75 (Figura 3), están disponibles comercialmente de Promega y se pueden propagar a través de protocolos de biología molecular convencionales. <ol><li> Amplificar los 2 componentes de el promotor de alfa-sinucleína en las PCR separadas utilizando la receta en la Tabla 1 y los cebadores de la Tabla 2. </li><li> Verify productos de PCR en un gel de agarosa y se limpia utilizando el kit de purificación de PCR Qiaquick (Qiagen), eluyendo en 50 l de TE (Tris-EDTA). Almacenar el fragmento de 0,9 kb eluido a -20 ° C para su uso futuro. </li><li> Digerir todo el volumen del fragmento de PCR de 3,4 kb, así como 5 g de pGL4.10, con SacI y XhoI. Tratar el vector con fosfatasa alcalina de intestino de ternero (Roche) y gel de purificar utilizando el kit de PureLink Gel Extracción rápida (Invitrogen). </li><li> Se liga el fragmento y el vector utilizando ADN ligasa T4 (NEB). Limpiar la reacción completada usando el kit de PCR PureLink Micro (Invitrogen), eluyendo en 10 l de tampón TE. </li><li> Transformar 2 l de la limpiado, reacción de ligación en 20 l de células competentes ElectroMAX Stbl4 (Invitrogen) y electroporar de acuerdo con las instrucciones del fabricante. Agitar en un 30 ° C incubadora a 225 rpm durante 90 min. Spread 2 volúmenes en placas de LB que contenían 100 mg / ml de ampicilina y se incuba a 30 ° C durante 2 días. </li><li> Con un palillo, seleccione 4-6 pequeñas colonias de inoculación en 2 ml de TB (Terrific Broth). Cultivar estas culturas miniprep en un 30 ° C coctelera O / N. </li><li> Se purifica ADN plasmídico a partir de 1,5 ml de cada cultivo Miniprep utilizando el kit de PureLink HiPure plásmido Miniprep (Invitrogen). </li><li> Digerir las minipreparaciones con BamHI y a electroforesis en un gel de agarosa. El clon correcto debería producir fragmentos de 2,8 kb y 4,9 kb. </li><li> Restreak la cultura Miniprep restante del clon correcto en un LB (caldo de Luria) placa fresca y crecer a 30 ° C durante 2 días. </li><li> Seleccionar una pequeña colonia e inocular un cultivo iniciador 1,5 ml de TB. Agite O / N a 30 ° C. No deje que el cultivo iniciador crecer hasta la saturación. </li><li> Diluir todo el cultivo iniciador en 150 ml de TB precalentada y agitar a 30 º C durante 2 días. Antes de proceder al paso siguiente, utilizar 1,5 ml de este cultivo para una miniprep y la digestión adicional para confirmar que el clon no mutar durante replicati<html> en. </li><li> Se purifica ADN de plásmido a partir del cultivo restante utilizando el kit de PureLink HiPure plásmido maxiprep (Invitrogen). Los rendimientos esperados de este plásmido intermediario son de 50-150 mg por 150 ml de la cultura. </li><li> Descongelar el fragmento de 0,9 kb congelada en el paso 1.2. Repita los pasos 1.3 a 1.12 para clonar el fragmento de 0,9 kb en el plásmido intermedio, digiriendo lugar con KpnI y SacI. Ligar, transformar, seleccionar y crecer durante Maxipreps que el anterior. La digestión con BamHI debe producir fragmentos de 5,8 kb y 2,8 kb. Este es el plásmido que se utilizará para la pantalla. </li></ol> 2. Preparar las células 293T, el reportero plásmidos y ADN Library for Screening Células 293T se sembraron primero en placas de 96 pocillos y se transfectaron el día siguiente. Reportero plásmidos y ADN de biblioteca se diluyen en placas de 96 pocillos. Se obtuvieron placas de ADN biblioteca transfección de grado a partir del ADN del núcleo en el Hospital General de Massachusetts (obtener = &quot;_blank&quot;&gt; dnacore.mgh.harvard.edu). Este protocolo transfecta una placa única biblioteca en 2 placas idénticas de células 293T (Figura 1), por lo tanto, 2 placas de células deben ser sembradas para cada placa de la biblioteca que se proyectarán. Nota: Se cultivan las células en medio sin rojo fenol o antibióticos, ya que éstos interfieren con los ensayos de luciferasa y aumentan la toxicidad durante la transfección, respectivamente. <ol><li> Para cada placa de la biblioteca de ser controlados, volver a suspender las células 293T tratadas con tripsina a una concentración de 1,5 x 10 5 células / ml en DMEM que contenía 14% de FBS. Vierta la mezcla en un recipiente estéril (Axygen). </li><li> Coloque el depósito, 2 de fondo claro-, placas de 96 pocillos (Corning), y una caja de puntas de pipeta filtro (Agilent) en la cubierta del robot. Dispensar 100 l de células en cada pocillo de ambas placas, para una densidad de 1,5 x 10 4 células por pocillo. </li><li> Centrifugar las placas de las células a 50 xg durante 2 min, con bajas velocidades de rampa para garantizar la igualdaddensidad celular a lo largo de cada pocillo. Incubar a 37 ° C y 10% de CO 2 O / N. </li><li> Diluir la luciérnaga y Renilla reportero plásmidos a 5 ng / l en medio libre de suero. Combinar las diluciones en una proporción de 05:03 de luciérnaga a plásmido de Renilla (en volumen) en un tubo cónico de 15 ml. </li><li> Pipet los plásmidos combinados en cada pocillo de una placa de 96 pocillos, dosificar 17 l por placa de biblioteca, además de 2-10 l exceso, en cada pocillo. </li><li> Obtener las placas de la biblioteca de ADN transfección grado que el anterior y diluir a 13,3 ng / l de agua libre de endotoxinas. El volumen mínimo por pozo debe ser de 28 l. </li></ol> 3. Lleve a cabo transfecciones En el día 2 de la pantalla, reportero plásmidos y ADN de biblioteca se transfectaron en células 293T. <ol><li> Para cada placa de biblioteca, mezclar 107,5 l de RT Optifect (Invitrogen) con 4,3 ml de medio libre de suero (1:40 dilución) en un tubo de poliestireno. Incubar durante 5 min a TA, y elN prescindir de 44 l (por placa biblioteca) en cada pocillo de una placa de 96 pocillos, de fondo en V de poliestireno (Greiner). </li><li> Coloque el plato de diluida Optifect, reportero plásmidos (Paso 2.5), los ADN de la biblioteca (Paso 2.6), y una caja de puntas de pipeta en la cubierta del robot. </li><li> Aspirar 17 l de reportero plásmidos, 43 l de diluirse Optifect, y 26 l de ADN de la biblioteca, la inserción de un espacio de aire 5 l entre cada aspiración. El ADN de la biblioteca debe ser aspirado pasado para evitar la contaminación cruzada. Vierta el contenido de las puntas en una nueva placa de poliestireno V de fondo, mezclar, tapar y dejar de lado por 20 minutos para permitir que los complejos de lípido / ADN para formar. Si la transfección de múltiples placas de biblioteca, reemplace las puntas de pipeta y placa de biblioteca, y repetir. </li><li> Destape la placa de mezcla Optifect / ADN y lo coloca en la cubierta del robot con 2 placas de células 293T. El uso de una sola caja de puntas de pipeta, aspire 80 l de la Optifect: mezcla de ADN y lentamente dispensar 40 l en cada placa de células. Si transfecting múltiples placas de biblioteca, repita para todos Optifect: placas de ADN. Cubra las células. </li><li> Centrifugar las placas de células a 1200 xg durante 30 min. Cubrir y volver a la incubadora. </li><li> Se incuban las células durante 4-6 horas. Durante esta incubación, preparar medio fresco mediante la mezcla de 12 ml de DMEM que contiene 10% de FBS y 10 mg / ml de ciprofloxacina (CellGro) para cada placa de biblioteca transfectadas. Verter medio fresco en un depósito estéril. </li><li> Coloque 2 cajas de puntas de robots, una sola placa de las células, el depósito que contiene medio fresco, y un depósito de residuos en la cubierta del robot. Aspirar 100 l de medio de las células y dispensar en el depósito de residuos parcialmente lleno con etanol al 70%. Utilizando puntas frescas, aspirado de 100 l de medio fresco y se dispensa lentamente sobre las células. Repita el procedimiento para todas las placas de células. </li><li> Placas Volver a la incubadora durante 48 horas. </li></ol> 4. Realizar Dual-Luciferase ensayos El día 4 de la pantalla, lase llevan a cabo ensayos de luciferasa de luciérnaga y Renilla. Nota: El ensayo de doble luciferasa requiere la adición secuencial de 2 tampones de ensayo, 3X tampón de ensayo de luciérnaga y de Renilla 3X tampón de ensayo, como se describe en la Tabla 3 de la luciérnaga y de Renilla luciferasas no se secretan, por lo tanto las células deben ser lisadas antes de medir la actividad luciferasa. . Tampón de ensayo Firefly lisa células y proporciona sustrato para la luciferasa de luciérnaga; tampón de ensayo Renilla apaga la señal de luciérnaga y proporciona sustrato para la luciferasa de Renilla. <ol><li> Para cada placa de biblioteca, preparar 8 ml de 3X tampón de ensayo de luciérnaga a partir de soluciones madre como se describe en la Tabla 3, y dispensar 82 l en cada pocillo de una placa de 96 pocillos con fondo en V. </li><li> Para cada placa de biblioteca, también preparar 12 ml de tampón de ensayo 3X de Renilla y dispensar 122 l en cada pocillo de una placa de 96 pocillos con fondo en V. Ponga a un lado tanto elluciérnagas y Renilla tampones de ensayo. </li><li> Coloque una caja de puntas de pipeta, un depósito de residuos, y 2 placas de células (transfectadas del mismo plato biblioteca) en la cubierta del robot. </li><li> Aspirar 60 l de los medios de comunicación de cada placa y desechar en el depósito de residuos parcialmente lleno de etanol al 70%. Las placas de cubierta y reservar. Si la transfección de múltiples placas de biblioteca, repita para todos los conjuntos de placas. </li><li> Coloque 2 cajas de puntas de pipeta, la placa de 3X tampón luciérnaga ensayo, y un conjunto de placas de las células de la cubierta del robot. </li><li> Aspirar 40 l de tampón 3X luciérnaga ensayo y la agregan a la primera placa de células, mezclando bien. El cambio a las nuevas puntas de pipeta, repita el procedimiento para la segunda placa de la célula. Colocar las células a temperatura ambiente durante 10 minutos para completar la lisis. Si la transfección de múltiples placas de biblioteca, cambie las cajas de puntas y placas de las células, y repetir. </li><li> Luminiscencia Record de cada pocillo de cada placa de células en un luminómetro, grabar durante 1 segundo por pocillo. Placas Volver al robotcubierta. </li><li> Coloque 2 cajas de puntas de pipeta, la placa de tampón de ensayo 3X Renilla, y un conjunto de placas de las células de la cubierta del robot. </li><li> Aspirar 60 l de tampón de ensayo 3X Renilla, y agregarlo a la primera placa de células, mezclando bien. El cambio a una nueva caja de puntas de pipeta, repita el procedimiento para la segunda placa de la célula. Si la transfección de múltiples placas de biblioteca, repita para todos los conjuntos de placas de las células. </li><li> Luminiscencia Registro de todas las placas en un luminómetro, registrando cada pocillo durante 1 segundo el anterior. Deseche las placas. </li></ol> 5. Análisis de Datos <ol><li> Calcular la luciérnaga: relación Renilla luminiscencia (&quot;F: relación R&quot;) para cada bien dividiendo los condes de la señal de la luciérnaga por los condes de señal de Renilla. </li><li> Calcular el valor de inducción dividiendo la relación F: R de cada pocillo por el promedio de F: relación R de la placa, excluido el de mayor y más bajo 25% de los pozos. </li><li> La media de los valores de inducción a través de repeticionespara una placa de biblioteca única transfectadas. Los genes que inducen o reprimen la alfa-sinucleína más de tres pliegues son considerados &quot;hits&quot; en esta pantalla y están sujetas a una mayor validación y pantallas secundarias. </li></ol>

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Este trabajo fue apoyado por las regalías obtenidas por BacMamreagents licencia (patente EE.UU. 5.731.182).

Alfa-sinucleína se ha implicado en la enfermedad de Parkinson (EP) como un componente de los cuerpos de Lewy 4, inclusiones intracelulares considerados patognomónicos de la enfermedad. Numerosos estudios de asociación del genoma han relacionado polimorfismos de nucleótido único en la alfa-sinucleína con un mayor riesgo para los esporádicos PD 5,6,7. Aunque menos común que la enfermedad de Parkinson esporádica, PD familiar puede estar también causada por mutaciones en alfa-sinucleína <sup...

La capacidad de identificar elementos reguladores genéticos clave y los factores que actúan sobre ellas es fundamental para la exploración de numerosos procesos biológicos. Sin embargo, los factores que regulan la expresión de genes en los tipos de células raras, como la identificación de poblaciones neuronales específicas, puede ser un reto. Aquí, se presenta un protocolo para la identificación de nuevos reguladores transcripcionales de alfa-sinucleína (SNCA), un gen asociado con la enfermedad de Parkinson y expresado en las neuronas dopaminérgicas en la substantianigra pars compacta de la región del cerebro medio. Esto lo logramos mediante una pantalla reportero de alto rendimiento, doble luciferase, in vitro para deconstruir la expresión de alfa-sinucleína en las células 293T. El promotor de alfa-sinucleína se clona primero en un plásmido que contiene el gen reportero de luciferasa de luciérnaga. Un plásmido comercialmente disponible que contiene la luciferasa de Renilla bajo el control de un promotor constitutivamente activo sirve como un control interno. Thesconstrucciones e reportero se transfectan conjuntamente en células 293T en microplacas con plásmidos de una biblioteca de expresión de ADN, de tal manera que cada pocillo se transfectaron con un único plásmido de la biblioteca. Después de 48 horas, la actividad de la luciferasa para cada reportero se mide de forma secuencial utilizando un ensayo de doble resplandor. La expresión relativa de alfa-sinucleína en respuesta a cada gen biblioteca se deduce por la relación de luciérnaga: la actividad de luciferasa de Renilla en cada pocillo (F: relación R) después de la normalización a base de placa. Este protocolo proporciona la capacidad de detectar un gran número de genes (~ 2,500 por semana) por su capacidad para transactivar un gen reportero usando mano de obra mínima (1-2 personas) y un costo mínimo (unos 3 dólares el costo de reactivos por ensayo de microplacas). Reguladores de la transcripción de los genes neuronales (por ejemplo, alfa-sinucleína), que son difíciles de estudiar en neuronas cultivadas refractarios a la manipulación genética, se pueden comparar lado a lado en este ensayo funcional directa. Incluimos en nuestroprotocolo de un método detallado para la clonación y el crecimiento de plásmidos que contienen el promotor de alfa-sinucleína, ya que encontramos que los plásmidos que contienen esta región son inestables cuando se cultivan con procedimientos convencionales (véase la discusión). También incluimos una alternativa económica a los reactivos disponibles en el mercado dual de luciferasa para ensayos de alto rendimiento. Este protocolo se puede adaptar fácilmente para dirigirse a otros elementos reguladores de interés, o a cualquier proceso que requiere de alto rendimiento transfecciones transitorias.

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Material</td>
 </tr>
 <tr>
 <td>QIAQuick PCR Purification Kit</td>
 <td>Qiagen</td>
 <td>28106</td>
 <td></td>
 </tr>
 <tr>
 <td>PureLink Quick Gel Extraction Kit</td>
 <td>Invitrogen</td>
 <td>K2100-12</td>
 <td></td>
 </tr>
 <tr>
 <td>PureLink PCR Micro Kit</td>
 <td>Invitrogen</td>
 <td>K310250</td>
 <td></td>
 </tr>
 <tr>
 <td>PureLinkHiPure Plasmid Miniprep Kit</td>
 <td>Invitrogen</td>
 <td>K2100-03</td>
 <td></td>
 </tr>
 <tr>
 <td>PureLinkHiPure Plasmid Maxiprep Kit</td>
 <td>Invitrogen</td>
 <td>K2100-07</td>
 <td></td>
 </tr>
 <tr>
 <td>Bravo Robot</td>
 <td>Agilent</td>
 <td>-</td>
 <td></td>
 </tr>
 <tr>
 <td>Robot Pipet Tips with Filter</td>
 <td>Agilent</td>
 <td>19477-022</td>
 <td></td>
 </tr>
 <tr>
 <td>Robot Pipet Tips without Filter</td>
 <td>Agilent</td>
 <td>19477-002</td>
 <td></td>
 </tr>
 <tr>
 <td>Clear-bottomed 96-well Plates</td>
 <td>Corning</td>
 <td>3610</td>
 <td></td>
 </tr>
 <tr>
 <td>Reservoirs</td>
 <td>Axygen</td>
 <td>RES-SW96-HP-SI</td>
 <td></td>
 </tr>
 <tr>
 <td>Polystyrene V-bottomed 96-well Plate</td>
 <td>Greiner</td>
 <td>651101</td>
 <td></td>
 </tr>
 <tr>
 <td>Polypropylene V-bottomed 96-well Plate</td>
 <td>Greiner</td>
 <td>651201</td>
 <td></td>
 </tr>
 <tr>
 <td>Adhesive Plate Covers</td>
 <td>CryoStuff</td>
 <td>#FS100</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent</td>
 </tr>
 <tr>
 <td>Phusion DNA Polymerase</td>
 <td>New England Biolabs</td>
 <td>M0530L</td>
 <td></td>
 </tr>
 <tr>
 <td>BAC 2002-D6</td>
 <td>Invitrogen</td>
 <td>2002-D6</td>
 <td></td>
 </tr>
 <tr>
 <td>Calf Intestine Alkaline Phosphatase</td>
 <td>Roche</td>
 <td>10713023001</td>
 <td></td>
 </tr>
 <tr>
 <td>T4 DNA Ligase</td>
 <td>New England Biolabs</td>
 <td>M0202L</td>
 <td></td>
 </tr>
 <tr>
 <td>pGL4.10</td>
 <td>Promega</td>
 <td>E6651</td>
 <td></td>
 </tr>
 <tr>
 <td>pGL4.74</td>
 <td>Promega</td>
 <td>E6931</td>
 <td></td>
 </tr>
 <tr>
 <td>ElectroMAX Stbl4 Competent Cells</td>
 <td>Invitrogen</td>
 <td>11635-018</td>
 <td></td>
 </tr>
 <tr>
 <td>Subcloning Efficiency DH5&alpha; Competent Cells</td>
 <td>Invitrogen</td>
 <td>18265-017</td>
 <td></td>
 </tr>
 <tr>
 <td>DMEM without Phenol Red</td>
 <td>Invitrogen</td>
 <td>31053-028</td>
 <td></td>
 </tr>
 <tr>
 <td>Optifect</td>
 <td>Invitrogen</td>
 <td>12579017</td>
 <td></td>
 </tr>
 <tr>
 <td>Ciprofloxacin</td>
 <td>CellGro</td>
 <td>61-277-RF</td>
 <td></td>
 </tr>
 <tr>
 <td>Tris-Hydrochloride Powder</td>
 <td>Sigma</td>
 <td>93287</td>
 <td></td>
 </tr>
 <tr>
 <td>Tris-Base Powder </td>
 <td>Sigma</td>
 <td>93286</td>
 <td></td>
 </tr>
 <tr>
 <td>Triton X-100 Pure Liquid</td>
 <td>Fisher</td>
 <td>BP151-100</td>
 <td></td>
 </tr>
 <tr>
 <td>DTT</td>
 <td>Invitrogen</td>
 <td>15508-013</td>
 <td></td>
 </tr>
 <tr>
 <td>Coenzyme A</td>
 <td>Nanolight</td>
 <td>309</td>
 <td></td>
 </tr>
 <tr>
 <td>ATP</td>
 <td>Sigma</td>
 <td>A6419</td>
 <td></td>
 </tr>
 <tr>
 <td>Luciferin</td>
 <td>Invitrogen</td>
 <td>L2912</td>
 <td></td>
 </tr>
 <tr>
 <td>h-CTZ</td>
 <td>Nanolight</td>
 <td>301</td>
 <td></td>
 </tr>
 <tr>
 <td>PTC124</td>
 <td>SelleckBiochemicals</td>
 <td>S6003</td>
 <td></td>
 </tr>
 </tbody>
</table>

high-throughput functional screening using a homemade dual-glow luciferase assay

Se presenta un protocolo de cribado de alto rendimiento rápido y barato para identificar reguladores de la transcripción de la alfa-sinucleína, un gen asociado con la enfermedad de Parkinson. Células 293T se transfectaron de forma transitoria con plásmidos de una biblioteca de expresión ORF dispuestos, junto con plásmidos informadores de luciferasa, en un formato de microplaca de un gen-por-así. Actividad de la luciferasa de la luciérnaga se ensaya después de 48 horas para determinar los efectos de cada gen de la biblioteca después de la transcripción alfa-sinucleína, normalizada para la expresión de una construcción de control interno (un promotor de hCMV dirigir la luciferasa de Renilla). Este protocolo se ve facilitada por un robot de sobremesa encerrado en una cabina de bioseguridad, que realiza la manipulación de líquidos aséptica en formato de 96 pocillos. Nuestro protocolo de transfección automatizado es fácilmente adaptable a la producción de alto rendimiento biblioteca lentiviral u otros protocolos de cribado funcionales que requieren triples-transfecciones de un gran número de plásmidos de la biblioteca únicos en conjugadoFuncione con un conjunto común de plásmidos auxiliares. También presentamos una alternativa económica y validado para comercialmente disponibles reactivos de luciferasa duales, que emplea el PTC124, EDTA, y pirofosfato para suprimir la actividad luciferasa de luciérnaga antes de la medición de la luciferasa de Renilla. El uso de estos métodos, que exhibió 7.670 genes humanos y se identificaron 68 reguladores de la alfa-sinucleína. Este protocolo es fácilmente modificables para atacar otros genes de interés.

Luciferase valores típicos, F: R ratios y valores de inducción para un único medio de placas se muestran en la Figura 4 Nota del éxito en el pozo H2, un inductor de 32 veces.. Los genes que causan una toxicidad excesiva (por ejemplo, así E3), o los pozos que fueron mal transfectadas, producirán valores bajos para ambos luciérnaga y luciferasa Renilla, pero un valor medio de inducción. Los genes que causan la inducción no específica de la actividad de la luciferasa, tal vez a trav?...

Watch this Scientific Journal Video about High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay at JoVE.com

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

Se presenta un método de diagnóstico rápido y de bajo costo para la identificación de los reguladores transcripcionales utilizando alto rendimiento transfecciones robóticos y un ensayo de luciferasa dual brillo casero. Este protocolo genera rápidamente datos funcionales directos de lado a lado para miles de genes y es fácilmente modificable para apuntar cualquier gen de interés.

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-functional-screening-using-homemade-dual-glow

Research

JoVE Journal

Biology

23.3K vistas.  Massachusetts General Hospital.  Se presenta un método de diagnóstico rápido y de bajo costo para la identificación de los reguladores transcripcionales utilizando alto rendimiento transfecciones robóticos y un ensayo de luciferasa dual brillo casero. Este protocolo genera rápidamente datos funcionales directos de lado a lado para miles de genes y es fácilmente modificable para apuntar cualquier gen de interés. 

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

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

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

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

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

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 ...

Investigación

Biología

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 ...

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

De alto rendimiento de sacarificación de ensayo para materiales lignocelulósicos

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

El uso de un robot para la cristalización de alto rendimiento de proteínas de membrana en mesofases lipídicas

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

De Alto Rendimiento La titulación de luciferasa que expresan virus recombinantes

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

Un manual de la pantalla pequeña molécula Acercarse De Alto Rendimiento Utilizando embriones de pez cebra

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

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 ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Detection of miRNA Targets in High-throughput Using the 3&#039;LIFE Assay

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

La detección de los genes miARN objetivos en de alto rendimiento utilizando el ensayo 3&#39;LIFE

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

Análisis qPCRTag - Un alto rendimiento, Tiempo real ensayo PCR para Sc2.0 Genotipado

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

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 ...