SD was supported by a Sir Henry Wellcome Fellowship [092201/Z/10/Z]. This work was funded by Wellcome Trust grants [WT066839MA][104627/Z/14/Z][108445/Z/15/Z] to Professor Keith Gull. We would like to thank Professor Keith Gull for helpful conversations and insights.

1. 96 pocillos largo cebador de PCR <ol><li> Pre-congelación enfriador 96 pocillos PCR en -80 ° C congelador. </li><li> En un tubo de 10 ml, preparar Master Mix A: 2100 ddH l 2 O, 105 l PCR grado DMSO, 105 l dNTPs 10 mM, 105 l plantilla pPot (25 ng / l) 9, para un volumen total de 2415 l por placa . </li><li> Alícuota de 23 l en cada pocillo de una placa de 96 pocillos PCR. </li><li> Añadir 2 l de 100 mM agrupados cebadores para cada uno cargado también con una pipeta multicanal P20 de 12 canales. Sellar con la película, placa lugar en el enfriador de PCR previamente enfriado y dejar que se congele durante un mínimo de 20 min a -80 ° C. </li><li> En un tubo de 10 ml, preparar Master Mix B: 2,048 l ddH 2 O, 525 l de 10x tampón, 52,5 l de la polimerasa para un volumen total de 2626 l por placa. </li><li> Retire la placa y el refrigerador de PCR a partir del congelador a -80ºC. </li><li> Con la placa todavía en el enfriador de PCR, se añaden 25 l Master Mix B en la parte superior de la congelón Master Mix A para un volumen total de reacción de 50 l. Este es el tiempo crítico, y debe ser completado antes de la mezcla A puede descongelarse. Sellar la placa con película. </li><li> Conjunto termociclador de la siguiente manera: 94 ° C durante 10 min, luego 30 ciclos de 94 ° C durante 15 s, 65 ° C durante 30 seg, 72 ° C durante 2 min seguido de un periodo de extensión final de 72 ° C durante 7 min . </li><li> Cargar la placa de PCR que el bloque haya alcanzado 94 ° C. </li></ol> 2. Validación de 96 pocillos a largo cebador de PCR <ol><li> Reparto de un gran 1% (w / v) en gel de agarosa en Tris acetato EDTA (TAE) tampón de ejecución con peines de 4 x 28 pocillos para dar un gel con 4 filas de pocillos. Alinear los dientes suplentes del peine con la punta de una pipeta multicanal de 12 canales. Sumerja con cuidado el gel en TAE funcionamiento de amortiguación después de que los carriles están cargados. </li><li> Cargar escalera de ADN de 1 kb en la 1ª y 28ª pocillo de cada fila. </li><li> Con una pipeta multicanal P20 de 12 canales, transferir 2 l de producto de PCR dirrectamente a cada carril del gel (Figura 1A). Hacer esto de manera rápida para reducir la posibilidad de contaminación de los amplicones. <ol><li> Cargar los productos de PCR de la fila A de la placa de PCR en las pistas impares de la fila 1 del gel (A1 - carril 3, A2 - carril 5 ...... A11 - carril 23, A12 - carril 25) y cargar los productos de PCR de la fila B de la placa de PCR en los carriles pares del st fila del gel 1 (B1 - carril 4, A2 - carril 6 ...... B11 - carril 24, B12 - carril 26). </li><li> Cargar los productos de PCR de la fila C y D de la placa de PCR utilizando el mismo patrón que se ha descrito anteriormente, con la fila C en el carril numeradas y la fila D extraña en los carriles de número par. Continuar este patrón de carga hasta que cada pocillo de la placa de PCR se carga en el gel. tampón de carga de DNA no es necesaria para cargar este gel. </li></ol></li><li> Colocar el gel en el tanque de correr y llenar el tanque con TAE tampón de ejecución hasta que se sumergió el gel. Ejecutar a 100 V durante 30 min yvisualizar utilizando un transiluminador UV. Véase la Figura 1B para un gel de ejemplo. Nota: Los productos de PCR se pueden almacenar a -20 ° C durante varias semanas antes de la transfección </li></ol> 3. La transfección de 96 pocillos <ol><li> Utilice la T. brucei línea celular de forma procíclica SMOXP9 10 para este procedimiento y crecer en SDM-79 medio que contiene FCS al 10% 10. </li><li> Utilice 1 x 10 7 células por transfección (total de 1,1 x 10 9 células) cuando se marcan en el extremo N de la proteína y 2 x 10 7 células por transfección (un total de 2,2 x 10 9 células) cuando se marcan en el extremo C terminal de la proteína. Mantener las células en mid-log (1,2 x 10 6 -1 x 10 7 células / ml) durante varios días antes de la transfección y cosechar las células para la transfección a una densidad de 5-8 x 10 6 células / ml. </li><li> Permitir a los productos de la PCR para la transfección congelados se descongelen a temperatura ambiente. </li><li> Recuento de células usando un hemoocytometer o contador de células automático. </li><li> Pellet requerido número de células en múltiples tubos de 50 ml a 800 xg durante 10 min. </li><li> Después de sobrenadante de centrifugación de descarte y resuspender las células en 10 ml Cytomix modificado (EGTA mM 0,8 mM, KCl 24, 0,15 mM CaCl 2, 10 mM de tampón de fosfato de potasio pH 7,6, 25 mM HEPES-KOH pH 7,6, 2,6 mM MgCl 2, 0.5% (w / v) de glucosa, 100 mg / ml BSA, hipoxantina 1 mM, sacarosa 144 mM) por tubo. Transferencia de todas las soluciones de células a un solo tubo y girar de nuevo a 800 xg durante 10 min. </li><li> Después de la centrifugación, desechar el sobrenadante y resuspender las células en 23 ml de Cytomix modificado para una concentración final de 5 x 10 7 células / ml. </li><li> Mientras que las células están girando, añadir 1 ml de SDM-79 medios de comunicación a cada pocillo de placas de cultivo de tejido de 4 x 24 pocillos. Etiquetar las placas de AD y dibujar un anillo alrededor pocillo A1 en cada plato con un marcador permanente para ayudar orientación de la placa. </li><li> Conectar el controlador de placa a la electroporador. En la electroporaunidad tor ajustar el voltaje a 1500 V, la longitud del pulso de 100 microsegundos y el número de impulsos a 12 y el intervalo de impulso de 500 ms. En el controlador de placa, establecer el cómputo de impulsos a 1. Esto asegura que que electroporador se aplicará un pulso único a cada una de las 12 columnas de la placa de la electroporación. </li><li> El uso de un P200 12 pipeta multicanal canal, la transferencia de las reacciones de PCR de la placa de PCR a las placas de 96 pocillos de electroporación desechables, brecha 4 mm. </li><li> Cuando las células se han hecho girar y se resuspendieron a la concentración final de 5 x 10 7 células / ml (paso 3.7), transferir a un depósito de reactivo. Pipetear 200 l de la solución de células en cada pocillo de la placa de electroporación usando un P200 12 canal pipeta multicanal, mezclar con el producto PCR mediante pipeteo. </li><li> El uso de tejido a eliminar cualquier gota de la parte superior de la placa para evitar cortocircuitos. </li><li> Aplicar la película de sellado proporcionada en el envase placa a la parte superior de la placa de la electroporación, positioning que a fin de dejar los orificios de los electrodos no cubierto en la parte superior e inferior de cada columna. Evitar cubrir los puntos planteados utilizados para guiar la película. </li><li> Cargar la placa de electroporación en el controlador de la placa y cierre la tapa. Pulse &#39;Pulso&#39; en la unidad electroporador. </li><li> Después de la electroporación, transferir rápidamente las células de la placa de la electroporación de 96 pocillos a las placas de cultivo de tejido de 4 x 24 pocillos. Para transferir las células, utilizar una pipeta P200 12 canal multicanal con cada otra punta falta de tal manera que los 6 restantes puntas se alinean con los 6 filas en una placa de cultivo de tejidos de 24 pocillos. Transferencia de las células electroporadas en las placas electroporaciones de 96 pocillos en patrón predefinido a las placas de cultivo de tejidos de 24 pocillos (Figura 2A). </li><li> Preparar las puntas de pipeta P200 - para cada transfección de 96 pocillos preparar 2 cajas de 96 puntas con cada otra columna eliminado. <ol><li> pozos de transferencia A1 / A3 / A5 / A7 / A9 / A11 de la placa de electroporación de 96 pocillos aLa placa A la fila A de las placas de cultivo de tejidos de 24 pocillos, B1 / B3 / B5 / B7 / B9 / B11 en la fila B, C1 / C3 / C5 / C7 / C9 / C11 en la fila C y D1 / D3 / D5 / D7 / D9 / D11 en la fila D. Después de la transferencia de cada grupo de 6 pocillos de la placa de 96 pocillos a la placa de 24 pocillos, los pocillos en la placa de 96 pocillos se lavan a continuación con los medios de los pocillos correspondientes en el 24- así placa y el lavado se transfirieron de nuevo a los pozos de la placa de 24 pocillos. </li><li> pozos de transferencia E1 / E3 / E5 / E7 / E9 / E11 en la placa de electroporación de 96 pocillos a 24 pocillos placa B fila A, F1 / F3 / F5 / F7 / F9 / F11 en la fila B, G1 / G3 / G5 / G7 / G9 / G11 en la fila C y H1 / H3 / H5 / H7 / H9 / H11 en la fila D. Después de la transferencia de cada conjunto de 6 pozos de la placa de 96 pocillos de la placa de 24 pocillos, los pozos en el 96 placa -bien se lavan entonces con medios de los pocillos correspondientes en la placa de 24 pocillos y el lavado se transfiere de nuevo a los pocillos en la placa de 24 pocillos. </li><li> pozos de transferencia A2 / A4 / A6 / A8 / A10 / A12 de la placa de electroporación de 96 pocillos a 24 pocillos placa C la fila A, B2 / B4 / B6 / B8 / B10 / B12 en la fila B, C2 / C4 / C6 / C8 / C10 / C12 en la fila C y D2 / D4 / D6 / D8 / D10 / D12 en la fila D. Después de la transferencia de cada conjunto de 6 pozos desde el 96 -bien placa a la placa de 24 pocillos, los pozos de la placa de 96 pocillos se lavan a continuación con los medios de los pocillos correspondientes de la placa de 24 pocillos y el lavado se transfiere de nuevo a los pozos de la placa de 24 pocillos. </li><li> pozos de transferencia E2 / E4 / E6 / E8 / E10 / E12 en la placa de electroporación de 96 pocillos a 24 pocillos placa D fila A, F2 / F4 / F6 / F8 / F10 / F12 en la fila B, G2 / G4 / G6 / G8 / G10 / G12 en la fila C y H2 / H4 / H6 / H8 / H10 / H12 en la fila D. Después de la transferencia de cada conjunto de 6 pozos de la placa de 96 pocillos de la placa de 24 pocillos, los pozos en el 96 placa -bien se lavan entonces con medios de los pocillos correspondientes en la placa de 24 pocillos y el lavado se transfiere de nuevo a los pocillos en la placa de 24 pocillos. </li></ol></li><li> Utilizando un microscopio de contraste de fase invertida, así examinar 1 de cada columna para comprobar las células. Habrá una mezcla de células vivas y muertas con unacantidad sustancial de los restos celulares. Las células vivas se pueden distinguir de las células muertas, ya que será brillante bajo un microscopio de contraste de fase y se mueve. </li><li> Colocar las placas de 24 pocillos en una caja limpia, de plástico con una tapa que forma un sello hermético. Ponga la caja en un 28 ° C incubadora. </li><li> Entre 6-8 horas más tarde, añadir 1 ml de SDM-79 con 10% de FCS que contiene el antibiótico selectivo 2x a cada pocillo. En nuestra experiencia, las líneas celulares donde los genes etiquetados en el extremo N terminal son resistentes a concentraciones más altas de la droga selectiva. Por lo tanto, el uso de blasticidin como un ejemplo, el marcado en el extremo N terminal requiere una concentración final de 20 mg / ml de blasticidina y etiquetado en el extremo C terminal requiere una concentración final de 10 mg / ml de blasticidina. Nota: Las líneas celulares transgénicas serán visibles 9-10 días después de la transfección. </li><li> Passage al menos dos veces en fármaco fresco y medios de comunicación antes del análisis. La Figura 2B muestra los resultados de una típica transfe 96 pocilloscción para el marcado de 96 proteínas diferentes en el extremo N-terminal. Evaluar cada pocillo por microscopía de contraste de fase para determinar si contiene células sanas, resistentes a las drogas. Un bien sana contiene predominantemente (&lt;95%) las células altamente activos que son brillantes en microscopía de contraste de fase. se requiere un análisis posterior por microscopía de epifluorescencia o transferencia de western para determinar si la proteína ha sido etiquetado con éxito. </li></ol>

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The authors have nothing to disclose.

mejoras dramáticas en la sensibilidad de la proteómica y transcriptómica métodos en los últimos 5-10 años ha proporcionado valiosos datos sobre miles de genes y sus productos. Sin embargo, las herramientas para hacer frente a la función de estas proteínas no han avanzado al mismo ritmo. Etiquetar una proteína facilita numerosos experimentos para determinar su función. Por ejemplo, una proteína se puede fusionar a una proteína fluorescente en una variedad de diferentes colores para facilitar los estudios de localización y co-localización. Etiquetas desarrollados para microscopía electrónica, tales como APEX2 o miniSOG 11,12, permitir la localización ultraestructural de la proteína de la etiqueta. Etiquetas para la bioquímica, como la etiqueta TAP, y ProtC-TEV-ProtA (PTP) Marcar 7,13 permitir la purificación de complejos asociados con la proteína para la identificación de las parejas de unión o en ensayos bioquímicos in vitro. Los pasos específicos que son críticos para el éxito de la protocol son: la incorporación de DMSO en la PCR Master Mix 1, la congelación de la PCR Master Mix 1 antes de la adición de la mezcla maestra 2, el uso de la polimerasa comercial en Master Mix 2 y la modificación de la memoria intermedia de Cytomix electroporación. En nuestra experiencia, es necesario usar el doble del número de células para C transfecciones terminal de marcado como para N terminal de transfecciones de etiquetado con el fin de lograr una proporción similar de los pocillos positivos. Por lo tanto, todos los pasos deben realizarse como se ha descrito. Esta técnica sólo es probable que tenga éxito cuando la transfección de la forma de insecto tripanosoma procíclico. Formas sanguíneas tienen una menor eficiencia de transfección 14, por otra parte son propensos a morir durante la selección debido a la toxicidad dependiente de la densidad que no está relacionada con el fármaco selectivo. Por lo tanto, nuestro protocolo anterior representa la mejor tecnología actual para etiquetar de forma tripanosomas torrente sanguíneo 9. También es probable que el transeficiencia infección variará dependiendo del aislado tripanosoma específico. Este protocolo se ha optimizado el uso de 927 SMOX procíclico formas - otras cepas pueden requerir una optimización adicional. Medidas que pueden aumentar la probabilidad de éxito incluyen: el aumento de la cantidad de amplicón de PCR, el aumento del número de células incluidas en la transfección. Se presenta un método donde cientos de proteínas se pueden etiquetar de forma paralela. Esto facilitará estudios a gran escala sobre la localización y la interacción, como complemento de grandes conjuntos de datos existentes y proporcionar información de gran valor para la comunidad. plantillas de cebadores están disponibles bajo petición y una lista de plantillas de plásmidos está disponible a partir de: http://www.sdeanresearch.com/

Trypanosoma brucei es un parásito protozoario que causa trypanosomasis africana humana y nagana en el ganado. T. brucei es un organismo ideal para el análisis de la función de la proteína debido a la combinación de un genoma de alta calidad, numerosos proteómica y conjuntos de datos transcriptómica y herramientas moleculares bien desarrollados 1-3. Los avances en la proteómica y la secuenciación han dado lugar a grandes conjuntos de datos que ponen de relieve los genes potencialmente interesantes 4-6; sin embargo, muchos genes tienen un mínimo de información asociada con ellos en las bases de datos existentes. Por tanto, existe una necesidad de un método de alto rendimiento para ayudar a la caracterización funcional de proteínas. La expresión de una proteína marcada puede dar una multiplicidad de conocimientos sobre la función de una proteína. Por ejemplo, una proteína marcada con una proteína fluorescente o epítopo puede ser localizada mediante microscopía de fluorescencia, lo que da información acerca de dónde la protein podría estar ejerciendo su efecto biológico. Alternativamente, una proteína marcada con un TAP 7, 8 o HaloTag His se puede purificar para los ensayos bioquímicos y la identificación de sus compañeros de interacción. Recientemente hemos desarrollado una metodología robusta para el etiquetado T. brucei 9. Este utilizado durante mucho tiempo cebador de PCR para generar el ADN para la transfección y permitió que el marcaje de proteínas de docenas en paralelo - una mejora importante de los protocolos existentes. Ahora hemos mejorado la escalabilidad de este protocolo en formas procíclicas en un orden de magnitud. A continuación, presentamos nuestro método en el que llevamos a cabo la PCR y la transfección en placas de 96 pocillos, con la recuperación y la selección se produce en placas de 24 pocillos. Como cientos de proteínas ahora se pueden etiquetar de forma paralela este método proporciona un método rentable y viable para etiquetar todo el genoma tripanosoma.

<table border="0" cellpadding="0" cellspacing="0" width="617">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Oligonucleotide</td>
			<td style="width:115px;">Life technologies</td>
			<td style="width:119px;">na</td>
			<td style="width:167px;">desalt only (do not use additional purification)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">DMSO</td>
			<td style="width:115px;">Roche</td>
			<td style="width:119px;">Included with the Expand HiFi pack</td>
			<td>Must be PCR grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dNTP</td>
			<td style="width:115px;">any reputable</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Expand HiFi polymerase</td>
			<td style="width:115px;">Roche</td>
			<td style="width:119px;">11759078001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BTX ECM830</td>
			<td style="width:115px;">Harvard Apparatus</td>
			<td style="width:119px;"></td>
			<td>Electroporator</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HT-200 plate handler</td>
			<td style="width:115px;">Harvard Apparatus</td>
			<td style="width:119px;">HT-200</td>
			<td>Handles the 96-well eletroplates</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MOS 96 ELECTROPLATE 4mm</td>
			<td style="width:115px;">Harvard Apparatus</td>
			<td style="width:119px;">45-0452</td>
			<td>Electroplate for the 96-well electroporation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blasticidin S Hydrochloride</td>
			<td>Melford</td>
			<td style="width:119px;">3513-03-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hygromycin b Gold</td>
			<td>Invivogen</td>
			<td style="width:119px;">ant-hg-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phleomycin</td>
			<td style="width:115px;">Melford</td>
			<td style="width:119px;">P0187</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">225cm TC Flask, canted neck, phenolic cap</td>
			<td style="width:115px;">Appletonwoods</td>
			<td style="width:119px;">BC006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">24 well culture plates</td>
			<td style="width:115px;">Appletonwoods</td>
			<td style="width:119px;">BC017</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Eppendorf&#174; PCR Cooler, iceless cold storage system for 96 well plates and PCR tubes</td>
			<td style="width:115px;">Sigma Aldrich</td>
			<td style="width:119px;">Z606634-1EA</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">96-well Multiply&#174; PCR plate with lateral skirt</td>
			<td style="width:115px;">Sarstedt</td>
			<td style="width:119px;">72.1979.203</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-throughput gene tagging in trypanosoma brucei

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

En este transfección representativa, los cebadores se diseñaron utilizando la secuencia de comandos Perl TagIt 9 y sintetizados comercialmente. El 96 pocillos PCR se llevó a cabo y validó como se describe (Figura 1A); en este ejemplo, 95/96 PCR tuvieron éxito (Figura 1B). En nuestra experiencia, la repetición de las reacciones fallidas ya sea con los cebadores originales o re-sintetizado cebadores no da lugar a una PCR con éxito. Los amplicones se transfirieron a placas de 96 pocillos de electroporación para la electroporación (Figura 2A) y después se transfirieron a placas de cultivo de 24 pocillos para la recuperación y selección. Después de tiempo suficiente para permitir la selección que se produzca, los pocillos se marcaron para la supervivencia antes de un análisis adicional. En este ejemplo, pozos 88/96 (92%) fueron positivos después de la selección de los 15 días. <img alt = &quot;Figura 1&quot; src = &quot;/ files / ftp_upload / 54342 / 54342fig1.jpg&quot; /&gt; Figura 1: Validación de PCR del patrón largo de imprimación (A) para la transferencia de productos de PCR de 96 pocillos placa de PCR para el gel de agarosa para la validación de PCR.. (B) A 96 pocillos gel representante validación de PCR para el marcado de 95 genes en el terminal N, y 1 amplicón no contiene &#39;homología 5 de orientación para actuar como un control negativo en la transfección. Las muestras se cargan en un 1% (w / v) en gel de agarosa y se resolvieron a 100 V durante 30 minutos. <a href="https://www.jove.com/files/ftp_upload/54342/54342fig1large.jpg" target="_blank">Por favor, haga clic aquí para ver una versión más grande de esta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/54342/54342fig2.jpg" /> Figura 2: de 96 pocillos electroporación y selección (A) Modelo para la transferencia de las células sometidas a electroporación de 96 pocillos plat electroporación.e a placas de cultivo de tejidos de 24 pocillos. (B) Un diagrama esquemático que muestra una puntuación típica en vivo / muerto después de 15 días de selección en placas de 24 pocillos. El verde representa una transfección exitosa, gris representa un pozo con sólo las células muertas. En este ejemplo, 88/96 (92%) de los pozos contenían parásitos transfectadas con éxito. <a href="https://www.jove.com/files/ftp_upload/54342/54342fig2large.jpg" target="_blank">Por favor, haga clic aquí para ver una versión más grande de esta figura.</a>

Watch this Scientific Journal Video about High-throughput Gene Tagging in Trypanosoma brucei at JoVE.com

High-throughput Gene Tagging in Trypanosoma brucei

Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

De Alto Rendimiento de genes de marcado en las<em&gt; Trypanosoma brucei</em

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

high-throughput-gene-tagging-in-trypanosoma-brucei

Research

JoVE Journal

Immunology and Infection

High-throughput Gene Tagging in Trypanosoma brucei

7.9K vistas.  University of Oxford. Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

Video: High-throughput Gene Tagging in Trypanosoma brucei

Examination of the Telomere G-overhang Structure in Trypanosoma brucei

The telomere G-overhang structure has been identified in many eukaryotes including yeast, vertebrates, and Trypanosoma brucei. It serves as the substrate for telomerase for de novo telomere DNA synthesis and is therefore important for telomere maintenance. T. brucei is a protozoan parasite that causes sleeping sickness in humans and nagana in cattle. Once infected mammalian host, T. brucei cell regularly switches its surface antigen to evade the host's immune attack. We have recently demonstrated that the T. brucei telomere structure plays an essential role in regulation of surface antigen gene expression, which is critical for T. brucei pathogenesis. However, T. brucei telomere structure has not been extensively studied due to the limitation of methods for analysis of this specialized structure. We have now successfully adopted the native in-gel hybridization and ligation-mediated primer extension methods for examination of the telomere G-overhang structure and an adaptor ligation method for determination of the telomere terminal nucleotide in T. brucei cells. Here, we will describe the protocols in detail and compare their different advantages and limitations.

Examination of the Telomere G-overhang Structure in Trypanosoma brucei

Telomeres are essential for chromosome stability and the telomere G-overhang structure is essential for telomerase-mediated telomere maintenance. We have recently adopted two methods for detecting the telomere G-overhang structure in Trypanosoma brucei, which are native in-gel hybridization and ligation-mediated primer extension, which will be described.

El examen de los telómeros G-Estructura en voladizo Trypanosoma brucei

The telomere G-overhang structure has been identified in many eukaryotes including yeast, vertebrates, and Trypanosoma brucei. It serves as the substrate ...

Investigación

Inmunología e Infección

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

De alto rendimiento Método de detección de virus de la influenza

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Cuantitativo de alto rendimiento de una sola célula de ensayo de citotoxicidad de las células T

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

Escalable de alto rendimiento seleccion de bibliotecas de anticuerpos sintéticos fagos

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) cycles between a tsetse vector and a mammalian host. It evades the mammalian host immune system by periodically switching the dense, variant surface glycoprotein (VSG) that covers its surface. The detection of antigenic variation in Trypanosoma brucei can be both cumbersome and labor intensive. Here, we present a method for quantifying the number of parasites that have 'switched' to express a new VSG in a given population. The parasites are first stained with an antibody against the starting VSG, and then stained with a secondary antibody attached to a magnetic bead. Parasites expressing the starting VSG are then separated from the rest of the population by running the parasites over a column attached to a magnet. Parasites expressing the dominant, starting VSG are retained on the column, while the flow-through contains parasites that express a new VSG as well as some contaminants expressing the starting VSG. This flow-through population is stained again with a fluorescently labeled antibody against the starting VSG to label contaminants, and propidium iodide (PI), which labels dead cells. A known number of absolute counting beads that are visible by flow cytometry are added to the flow-through population. The ratio of beads to number of cells collected can then be used to extrapolate the number of cells in the entire sample. Flow cytometry is used to quantify the population of switchers by counting the number of PI negative cells that do not stain positively for the starting, dominant VSG. The proportion of switchers in the population can then be calculated using the flow cytometry data.

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

African trypanosomes grown in vitro undergo antigenic variation at a low rate, such that populations are made up of parasites expressing a dominant variant surface glycoprotein (VSG) type and a small population of "switched" variants. This protocol describes a fast method for detecting and quantifying these populations.

Detección de<em&gt; Trypanosoma brucei</em&gt; Variant glicoproteína de superficie de conmutación por Magnetic Activated Cell Sorting y Citometría de Flujo

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

La detección de alto rendimiento de los patógenos respiratorios de especímenes animales por PCR nanoescala

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Building Up a High-throughput Screening Platform to Assess the Heterogeneity of HER2 Gene Amplification in Breast Cancers

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast cancers. However, a minority of cases displays a heterogeneous distribution of HER2-positive cells, which generates major clinical challenges. To date, no reliable and standardized protocols for the characterization and quantification of HER2 heterogeneous gene amplification in large cohorts have been proposed. Here, we present a high-throughput methodology to simultaneously assess the HER2 status across different topographic areas of multiple breast cancers. In particular, we illustrate the laboratory procedure to construct enhanced tissue microarrays (TMAs) incorporating a targeted mapping of the tumors. All TMA parameters have been specifically optimized for the silver in situ hybridization (SISH) of formalin-fixed paraffin-embedded (FFPE) breast tissues. Immunohistochemical analysis of the prognostic and predictive biomarkers (i.e., ER, PR, Ki67, and HER2) should be performed using automated procedures. A customized SISH protocol has been implemented to allow a high-quality molecular analysis across multiple tissues that underwent different fixation, processing, and storage procedures. In this study, we provide a proof-of-principle that specific DNA sequences could be localized simultaneously in distinct topographic areas of multiple and heterogeneously processed breast cancers using an efficient and cost-effective method.

Heterogeneous distribution of HER2-positive cells can be observed in a subset of breast cancers and generates clinical dilemmas. Here, we introduce a reliable and cost-effective protocol to define, quantify, and compare HER2 intra-tumor genetic heterogeneity in a large series of heterogeneously processed breast cancers.

Construcción de una plataforma de proyección de alto rendimiento para evaluar la heterogeneidad de la amplificación del gen HER2 en cáncer de mama

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast ...

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to combat multiple drug resistance. This has led to the conclusion that researchers do not only need to find new drugs, but also need to develop new ways of finding them. Amongst the most promising candidate methods are whole-organism, in vivo assays that use high-throughput, phenotypic readouts and hosts that range from Caenorhabditis elegans to Danio rerio. These hosts have several powerful advantages, including dramatic reductions in false positive hits, as compounds that are toxic to the host and/or biounavailable are typically dropped in the initial screen, prior to costly follow up.
Here we show how our assay has been used to interrogate host variation in the well-documented C. elegans&#8212;Pseudomonas aeruginosa liquid killing pathosystem. We also demonstrate several extensions of this well-worked out technique. For example, we are able to carry out high-throughput genetic screens using RNAi in 24- or 96-well plate formats to query host factors in this host-pathogen interaction. Using this assay, whole genome screens can be completed in only a few months, which can dramatically simplify the task of identifying drug targets, potentially without the need for laborious biochemical purification approaches.
We also report here a variation of our method that substitutes the gram-positive bacterium Enterococcus faecalis for the gram-negative pathogen P. aeruginosa. Much as is the case for P. aeruginosa, killing by E. faecalis is time-dependent. Unlike previous C. elegans&#8212;E. faecalis assays, our assay for E. faecalis does not require preinfection, improving its safety profile and reducing the chances of contaminating liquid-handling equipment. The assay is highly robust, showing ~95% death rates 96 h post infection.

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

Here we describe a protocol that is an adaptable, whole host, high-content screening tool that can be utilized to study host-pathogen interactions and be used for drug discovery.

Un alto rendimiento, alto contenido líquido y basada en C. elegans patosistema

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to ...

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In response to these damages, complex molecular machineries rapidly reseal the plasma membrane to restore its barrier function and maintain cell survival. Despite 60 years of research in this field, we still lack a thorough understanding of the cell resealing machinery. With the goal of identifying cellular components that control plasma membrane resealing or drugs that can improve resealing, we have developed a fluorescence-based high-throughput assay that measures the plasma membrane resealing efficiency in mammalian cells cultured in microplates. As a model system for plasma membrane damage, cells are exposed to the bacterial pore-forming toxin listeriolysin O (LLO), which forms large 30-50 nm diameter proteinaceous pores in cholesterol-containing membranes. The use of a temperature-controlled multi-mode microplate reader allows for rapid and sensitive spectrofluorometric measurements in combination with brightfield and fluorescence microscopy imaging of living cells. Kinetic analysis of the fluorescence intensity emitted by a membrane impermeant nucleic acid-binding fluorochrome reflects the extent of membrane wounding and resealing at the cell population level, allowing for the calculation of the cell resealing efficiency. Fluorescence microscopy imaging allows for the enumeration of cells, which constitutively express a fluorescent chimera of the nuclear protein histone 2B, in each well of the microplate to account for potential variations in their number and allows for eventual identification of distinct cell populations. This high-throughput assay is a powerful tool expected to expand our understanding of membrane repair mechanisms via screening for host genes or exogenously added compounds that control plasma membrane resealing.

Here we describe a high-throughput fluorescence-based assay that measures the plasma membrane resealing efficiency through fluorometric and imaging analyses in living cells. This assay can be used for screening drugs or target genes that regulate plasma membrane resealing in mammalian cells.

Medida de alto rendimiento de membrana plasmática resellado eficacia en células de mamíferos

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...

High Throughput In Vitro Assessment of Latency Reversing Agents on HIV Transcription and Splicing

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune system and is not targeted by the current antiretroviral therapy (cART). Transcription and splicing have been shown to reinforce HIV-1 latency in resting CD4+ T cells. Reversal of latency by the use of latency reversal agents (LRAs) in the &#34;shock and kill&#34; approach has been studied extensively in an attempt to purge this reservoir but has thus far not shown any success in clinical trials due to the lack of development of adequate small molecules that can efficiently perturb this reservoir. The protocol presented here provides a method for reliably and efficiently assessing latency reversal agents (LRAs) on HIV transcription and splicing. This approach is based on the use of an LTR-driven dual color reporter that can simultaneously measure the effect of an LRA on transcription and splicing by flow cytometry. The protocol described here is adequate for adherent cells as well as the cells in suspension. It is useful for testing a large number of drugs in a high throughput system. The method is technically simple to implement and cost-effective. In addition, the use of flow cytometry allows the assessment of cell viability and thus drug toxicity at the same time.

A high throughput protocol for functional assessment of HIV efficient reactivation and clearance of latent proviruses is described and applied by testing the impact of interventions on HIV transcription and splicing. Representative results of the effect of latency reversing agents on LTR-driven transcription and splicing are provided.

Alto rendimiento en la evaluación de Vitro de latencia inversión a en VIH transcripción y Splicing

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune ...