Este trabajo fue financiado por el NIH subvenciones para la investigación K01AR053210, R01AR061484 y una beca de la Fundación Nacional de Psoriasis.

1. Información general La levadura de dos sistema híbrido es una poderosa técnica genética utilizada para descubrir las interacciones proteína-16, 17. Varias clases de dos híbridos de sistemas, tales como la serie LEXA-los sistemas basados ​​en el sistema de contratación Sos, y las bacterias o células de mamíferos base-2-híbridos, son comerciales disponibles, en este documento se centra específicamente en las modificaciones de las más utilizadas GAL4 base levadura de dos sistema híbrido. En resumen, el método se basa en las propiedades de la proteína GAL4 de levadura, que consiste en separar los dominios responsables de la activación de unión al ADN y la transcripción. El cebo de proteína se expresa como una fusión de la GAL4 dominio de unión a ADN (DNA-BD), mientras que la presa proteínas se expresan como fusiones al dominio de activación GAL4 (AD). La interacción entre el cebo y presa proteínas de fusión lleva a la activación transcripcional de GAL4 sitios de unión que contiene genes reporteros que se integran en el g de levaduraenome. El principio de Y2H se ilustra en la figura. 1 y el procedimiento experimental se resume en la figura. 2. 2. Los materiales necesarios y las soluciones <ol><li> Medio de crecimiento YPD (una mezcla de peptona, extracto de levadura y dextrosa en proporciones óptimas para el cultivo de la mayoría de las cepas de Saccharomyces cerevisiae). </li><li> Bases mínimas SD (mínimo sintéticos definidos (SD) son las bases de una base nitrogenada de levadura, sulfato de amonio y una fuente de carbono, dextrosa. Deserción (DO) los suplementos pueden ser añadidos a la base mínima SD para hacer un medio sintético, que se define que carecen de la especificada nutrientes). </li><li> Leu /-Trp de deserción (DO) suplemento (que contienen todos los aminoácidos esenciales excepto leucina y triptófano) </li><li> -His/-Leu/-Trp/-Ura De deserción (DO) Suplemento (que contiene todos los aminoácidos esenciales excepto leucina, triptófano, histidina y uracilo) </li><li> Caldo Luria (LB) (triptona 10 g / L, extracto de levadura 5 g / L, NaCl 5 g / L) </li><li> X-Gal (5-bromo-4-cloro-3-indolil-β-D-galactopiranósido): Se prepara como una de 20 mg / ml de solución de archivo de N, N-dimetilformamida </li><li> TE 10X (100 mM Tris-HCl (pH 7,5), 10 mM EDTA, autoclave) </li><li> Arenque sonicado o ADN de esperma de salmón, hervida (10 mg / ml) </li><li> LiAc 10X (1 M de acetato de litio, autoclave) </li><li> 50% de PEG-3350 una solución, esterilizada por filtración </li><li> 3-amino-1, 2, 4-triazol (3AT) </li><li> Kanamicina </li><li> Ampicilina </li><li> Electromax DH5a células </li><li> Z buffer: 16,1 g Na2HPO4 7H2O (8,52 g, o anhidro), 5,5 g de NaH2PO4 H2O (o 4,8 ganhydrous), 0,75 g de KCl, 0,246 g MgSO4 7H2O (o 0,12 g anhidro), disuelto en 1 litro de agua en autoclave, destilada y se ajusta a pH 7,0. </li></ol> 3. Cebo generación (pDBLeu-PGRN) Un fragmento de ADNc que codifica PGRN que carecen de péptido señal (aa21-588) se clonó direccionalmente en el Sal I-No me los sitios del vector pDBLeu (ProQuest el sistema de dos híbridos, Invitrogen),mantener el marco de la traducción de lectura que el dominio de unión a ADN GAL4 generar pDBLeu-PGRN. <ol><li> Amplificar el fragmento de cDNA de PGRN descrito anteriormente mediante PCR utilizando oligonucleótidos diseñados para contener sitios de restricción (Sal I en el extremo 5 &#39;y no yo en el extremo 3&#39;) para que dentro del marco de la fusión. </li><li> Gel purificar el producto de PCR, digestión con endonucleasas de restricción Sal I y Not I. Prepare la base de datos vector pDBLeu restricción de la digestión doble con las endonucleasas de restricción misma. </li><li> Ligar el fragmento PGRN restricción en el vector linealizado pDBLeu y se transforman en DH5a con la selección de LB + kanamicina a 25 mg / ml. </li><li> Comprobar la corrección de la construcción de la secuenciación del ADN. </li></ol> 4. La transformación a pequeña escala de cebo plásmido <ol><li> Inocular 5 ml de YPD con una colonia de Mav203 (Invitrogen), agitar la noche a 30 ° C. </li><li> Diluir el cultivo de una noche en 50 ml de YPD. Crecer un complementoitional 04.02 horas. </li><li> Que sedimenten las células a 3000 rpm durante 5 min a temperatura ambiente. Resuspender el precipitado en 40 ml de agua esterilizada, destilada. </li><li> Re-precipitar las células. Resuspender en 2 ml de solución I, se incuban a temperatura ambiente 10 min. Solución I: 0.5 ml 10xLiAc, 0,5 ml 10xTE, 4 ml de H 2 O </li><li> Dispensar el ADN de los tubos: 3.2 l de pDBLeu-PGRN (0.25μg/μl) y 10 l de ADN desnaturalizado, esquilada de esperma de salmón (10μg/μl). Agregar 100μl células de levadura, mezclar bien. </li><li> Agregar 700μl solución II, mezclar bien. Se incuba a 30 ° C durante 30 min. Solución II: 0,2 ml 10xLiAc, 0,2 ml 10xTE, 1,6 ml de 50% PEG3350. </li><li> Choque térmico a 42 ° C durante 15 min. </li><li> Pellet durante 2 min. Eliminar el sobrenadante. Resuspender el precipitado en 200 l agua esterilizada, destilada. </li><li> Placa de la suspensión en SD-Leu placas con diluciones seriadas. Incubar la placa a 30 ° C durante 2-3 días. </li></ol> 5. Cebo de validación Antes de realizar síst dos híbridos de pantalla, prueba pDBLeu-PGRN para la auto-activación y determinar los niveles de expresión basal del gen reportero HIS3. Esta prueba determina si los cebos activar la transcripción y si el auto-activación puede ser neutralizado por los inhibidores. 3-amino-1, 2, 4-triazol (3-AT) es un inhibidor competitivo del producto HIS3-gen y puede ser utilizado para valorar el nivel mínimo de HIS3 expresión necesaria para el crecimiento en la histidina deficiencia de los medios de comunicación. <ol><li> Transformar el pDBLeu-PGRN en la levadura Mav203 cepa, la placa de la transformación en SD-Leu placas e incubar durante 48-72 horas a 30 ° C. </li><li> Colonias de parches de cada transformación con palillos de dientes en autoclave SD-Leu-Sus placas que contienen 3-AT a concentraciones de 10 mm, 25 mm, 50 mm, 75 mm, y 100. 3-AT es un inhibidor competitivo de la enzima HIS3 y los cebos solo que muestran auto-activación será capaz de crecer en presencia de la 3-AT. </li><li> Cepas de cebo que crecen en las placas de containing 100 mM 3-AT no son adecuados para su uso en la pantalla de dos híbridos. Para los cebos que se pueden utilizar, el uso más bajo de 3 a una concentración que inhibe el crecimiento celular. En muchos casos, 25 mM de 3-A se utiliza para la detección. </li></ol> 6. selección de la biblioteca de cDNA El plásmido pDBLeu-PGRN se introduce en MaV203 mediante una transformación a pequeña escala como se describió anteriormente. Introducir pPC86-biblioteca (Invitrogen) en MaV203 (pDBLeu-PGRN), el procedimiento descrito a continuación normalmente da ~ 4 x 10 4 colonias con 0,5 mg de ADN plásmido biblioteca. Por lo tanto, 2,5 x 10 6 transformantes de levadura se requieren ~ 30,0 mg pPC86-cDNA biblioteca de ADN plásmido, las transformaciones de 25 años, y cincuenta platos de 10 cm (SD-Leu-Trp-His-Ura 3 AT). <ol><li> Preparar el número adecuado de 10 cm SD-Leu-Trp-His-Ura 3 en las placas (50 para el siguiente procedimiento). También hay que preparar por lo menos cuatro de 10 cm SD-Leu-Trp placas para estimar el número de transformantes. </li><li> Transformar la biblioteca enla MaV203 contiene pDBLeu-PGRN de acuerdo con el procedimiento descrito a continuación. <ol><li> Suspender varias colonias aisladas de MaV203 (pDBLeu-PGRN) en ~ 100μl agua esterilizada, destilada, y las tendían en una de 10 cm SD-Leu plato. Repita el procedimiento con una segunda placa SD-Leu. Incubar ambas placas durante 18-24 horas a 30 ° C. </li><li> Raspe y suspender completamente las células en 10 ml de agua en autoclave, destilada. Añadir un volumen suficiente de la suspensión celular a 500 ml de medio líquido YPD en un frasco para dar una OD 600 de ~ 0.1. </li><li> Verifique que el OD es de ~ 0.1 después de la inoculación. </li><li> Agitar a 30 ° C hasta los 600 OD alcanza ~ 0,4. </li><li> Preparar fresca: <ol type="a"><li> 110 ml 1X TE / LiAc mediante la combinación de 11 ml de 10X TE, 11 ml LiAc 10 veces, y 88 ml de agua esterilizada. </li><li> 16 ml de PEG / LiAc mediante la combinación de 1,6 ml de 10X TE, 1,6 ml LiAc 10X, y 12,8 ml de 50% de PEG-3350. </li></ol></li><li> Que sedimenten las células a 3000g durante 5 min a temperatura ambiente. </li><li> Deseche tque sobrenadantes y suavemente resuspender el precipitado pipeteando arriba y hacia abajo en 100 ml de agua en autoclave, destilada a temperatura ambiente. </li><li> Centrifugar a 3000 g por 5 min a temperatura ambiente. </li><li> Descartar el sobrenadante de las células se centrifugaron y resuspender el botón celular en 50 ml de 1X TE / LiAc solución. </li><li> Centrifugar a 3000 g por 5 min a temperatura ambiente. </li><li> Eliminar el sobrenadante y resuspender el precipitado en un volumen final de 2,5 ml 1X TE / LiAc solución. </li><li> Realiza 25 transformaciones. Combinar celdas 2.5 ml, 125 l (10μg/μl) desnaturalizada, el ADN de esperma de salmón cortado, y 30 mg de la biblioteca de ADNc. Mezclar suavemente con la pipeta hacia arriba y abajo. Añadir 15 ml de PEG / LiAc solución y mezclar suavemente. Alícuota en 25 autoclave de 1,5 ml tubos de microcentrífuga de 700 l cada uno. </li><li> Incubar durante 30 minutos en un baño de 30 ° C el agua. </li><li> Choque térmico durante 15 minutos en un baño de 42 ° C el agua. </li><li> Se centrifuga a 6.000 g durante 1 minuto a temperatura ambiente. Retire con cuidado el súpersobrenadante. Resuspenda suavemente cada sedimento en 400 l agua esterilizada, destilada con la pipeta hacia arriba y abajo. </li></ol></li><li> Placa de la mezcla de 200 l de transformación de cada transformación en solo 10 cm SD-Leu-Trp-His-Ura 3 AT (3AT concentración: 25 mm) placas con una barra de propagación, por lo que 25 autoclave de 1,5 ml tubos de microcentrífuga puede hacer 50 planchas (SD-Leu-Trp-His-Ura 3 AT). </li><li> Incubar las placas durante 5-10 días a 30 ° C. </li><li> Para estimar la eficiencia de transformación de la reacción, diluciones en serie de una reacción (1:40; 1:400 y 1:4000) se colocan en SD-Leu-Trp plato. Después de incubar durante 3 días a 30 ° C, el número de colonias contadas y es el número total de transformantes calculado. </li></ol> 7. X-gal ensayo <ol><li> La transferencia de las colonias de la placa de YPD, se incuba a 30 ° C durante la noche. </li><li> Lugar redondeado membrana de nitrocelulosa sobre la placa de YPD, asegúrese de que no haya burbujas entre la membrana y la placa de YPD. Después de 1-2 minutos, make seguro de todas las colonias se transfirieron a membranas (papel de nitrocelulosa). </li><li> Coloque el lado de la membrana colonia en barco hecho con papel de aluminio. </li><li> Poner la membrana en nitrógeno líquido, esperar veinte segundos, y se sumergen en nitrógeno líquido durante un par de minutos. </li><li> Saque la membrana que se descongele. </li><li> Mientras tanto, mezclar el reactivo siguiente: 1,5 ml buffer Z 20μl X-gal (20 mg / ml) </li><li> Caída de buffer Z / X-gal en una placa de Petri, lugar de papel Whatman encima de eso. </li><li> Con cuidado, coloque papel de nitrocelulosa en la parte superior del papel Whatman. </li><li> Se incuba a 37 ° hasta que las colonias azules aparecen. </li></ol> 8. Retransformación ensayo Presa proteínas de fusión (AD-Y) aisladas a partir del examen de la biblioteca debe mantener la interacción con la proteína de fusión cebo (DB-X) para inducir los genes informe, y la retransformación de clones de la presa y el cebo en la construcción de la levadura puede además eliminar los falsos positivos y facilitar añadiranálisis itional. <ol><li> Aislar el ADN de plásmido a partir de cepas de levaduras que pueden contener proteínas que interactúan. </li><li> Transformar el ADN en las células de E. coli DH5a, la placa de las transformaciones en el LB 100 mg / ml ampicilina placas para aislar selectivamente la biblioteca pPC86-cDNA, y se incuba durante la noche a 37 ° C. </li><li> Elige varias colonias de la placa para poner en LB 100 mg / ml de caldo a la ampicilina. </li><li> Prepare minipreparación ADN y examinar mediante análisis de restricción doble con Sal I y I. No </li><li> Co-transformar la presa plásmido y el cebo plásmido en MaV203, la placa de las mezclas de transformación en el SC-Leu-Trp placas y se incuban durante 2-3 días a 30 ° C. </li><li> Escoger tres diferentes colonias de la placa SC-Leu-Trp, lleve a cabo X-gal de ensayo. </li></ol> 9. Secuenciación y análisis bioinformático La secuencia del ADN del plásmido que se aisló de los clones positivos probablemente cierto, comparar estas secuencias con las del GenBank utilizando el BLAPrograma de ST, e identificar los clones de dos híbridos que corresponden a los genes conocidos. La secuencia de datos mostró que dos de los 12 positivos obtenidos anteriormente eran de la superficie celular TNFR2 (TNFRSF1B/CD120b; adhesión # NM_130426). Además, la interacción entre PGRN y TNFR se verifica por medio de diversos ensayos de proteína-proteína interacción, incluyendo in vitro en fase sólida ensayo de unión, Co-inmunoprecipitación, análisis de resonancia de plasmones de superficie, y el ensayo de citometría de flujo 14. 10. Resultados representante El diagrama de flujo de la proyección se resume en la figura. 3. Por lo general 50-100 candidatos clon positivo se obtuvo en este paso. Inicialmente se aisló 54 candidatos clon positivo entre los 2,5 millones transformantes seleccionados con el cebo PGRN. Candidatos clon positivo fueron posteriormente comprobados por la realización del ensayo X-gal. Por lo general, aproximadamente el 50% de falsos positivos clones se eliminan a través de X-gal ensayo. Se obtuvieron 23 clones positivos franca<html> ates en este paso de la carnada PGRN (Fig. 4). Retransformación de los clones y la construcción de la presa de cebo en la levadura aún eliminar los falsos positivos y los clones que aún activan los genes reportero probablemente representan los verdaderos positivos. Por lo general, aproximadamente el 50% positivo clon candidatos serán eliminados. Finalmente, aislados 12 clones positivos que interactúan con PGRN en la levadura. <img alt="Figura 1" src="/files/ftp_upload/3562/3562fig1.jpg" /> Figura 1. Principio de la levadura sistema híbrido de dos <img alt="Figura 2" src="/files/ftp_upload/3562/3562fig2.jpg" /> Figura 2. Pipeline de la identificación de socios de unión a proteínas utilizando la levadura sistema híbrido de dos <img alt="Figura 3" src="/files/ftp_upload/3562/3562fig3.jpg" /> Figura 3. Diagrama de flujo de la levadura de dos híbridos de selección de la biblioteca.rge.jpg &quot;target =&quot; _blank &quot;&gt; Haga clic aquí para ver una versión en tamaño completo de esta imagen. <img alt="Figura 4" src="/files/ftp_upload/3562/3562fig4.jpg" /> Figura 4. Beta-galactosidasa de ensayo de los candidatos clon positivo. A los clones, positivos obtenidos en la pantalla de la biblioteca fueron trasladados a la placa de YPD y se incuba a 30 ° C durante la noche, B, todas las colonias en la placa de YPD se transfirieron a membranas de nitrocelulosa y se realiza la beta-galactosidasa ensayo.

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Levadura de dos híbridos de cribado ha demostrado ser una herramienta eficaz en la identificación de la interacción de proteínas 16, 17. En comparación con otros métodos para la identificación de la proteína de unión de la pareja, tales como la purificación bioquímica de proteínas y co-chips, dos híbridos de levadura es un enfoque sensible genética que puede ser utilizado para la detección de un número muy alto de las secuencias codificadoras de un experimento relativamente sencillo, en Ademá...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> ProQuest sistema híbrido de dos </td><td> Invitrogen </td><td> 10835 </td></tr><tr><td> YPD medio de crecimiento </td><td> Clontech </td><td> 630409 </td></tr><tr><td> YPD medio Agar </td><td> Clontech </td><td> 630410 </td></tr><tr><td> Mínimo SD Base de agar </td><td> Clontech </td><td> 630412 </td></tr><tr><td> -Leu se complementa </td><td> Clontech </td><td> 630414 </td></tr><tr><td> -Leu/-Trp Se complementa </td><td> Clontech </td><td> 630417 </td></tr><tr><td> -His/-Leu/-Trp/-Ura Se complementa </td><td> Clontech </td><td> 630425 </td></tr><tr><td> 3-amino-1 ,2,4-triazol (3AT) </td><td> Sigma-Aldrich </td><td> A8056 </td></tr><tr><td> Caldo Luria (LB) </td><td> Sigma-Aldrich </td><td> L3022 </td></tr><tr><td> X-Gal </td><td> Invitrogen </td><td> 15520-034 </td></tr><tr><td> Salmón sonicado ADN de los espermatozoides </td><td> Stratagene </td><td> 201190 </td></tr><tr><td> Ampicilina </td><td> Amresco </td><td> 0339 </td></tr><tr><td> Kanamicina sulfato </td><td> Invitrogen </td><td> 11815-024 </td></tr><tr><td> Eficiencia subclonación ™ ™ DH5a células competentes </td><td> Invitrogen </td><td> 18265-017 </td></tr><tr><td> Trizma ® base </td><td> Sigma-Aldrich </td><td> T6066 </td></tr><tr><td> Acetato de litio </td><td> Sigma-Aldrich </td><td> L4158 </td></tr><tr><td> Ácido etilendiaminotetraacético </td><td> Sigma-Aldrich </td><td> ED </td></tr><tr><td> Membrana de nitrocelulosa </td><td> Bio-Rad </td><td> 162-0115 </td></tr><tr><td> 10 cm de una caja de petri </td><td> ITI Científico </td><td> CT-903 </td></tr><tr><td> Incubadora (30 ° C) </td><td> ATR (Ecotron) </td><td></td></tr></table>

modified yeast-two-hybrid system to identify proteins interacting with the growth factor progranulin

Progranulina (PGRN), también conocido como precursor granulina epithelin (GEP), es un factor de crecimiento autocrino 593-amino-ácido. PGRN se sabe que juega un papel crítico en una variedad de procesos fisiológicos y enfermedades, incluyendo principios de la embriogénesis, cicatrización de heridas 1, la inflamación 2, 3 y 4 de defensa del huésped. PGRN también funciona como un factor neurotrófico 5, y las mutaciones en el gen PGRN resultando en la pérdida parcial de la demencia frontotemporal causa PGRN proteínas 6, 7. Nuestros estudios recientes han conducido al aislamiento de PGRN como un importante regulador del desarrollo del cartílago y la degradación de 8-11. A pesar de PGRN, descubierto hace casi dos décadas, juega un papel crucial en varias condiciones fisiológicas y patológicas, los esfuerzos para aprovechar las acciones de PGRN y entender los mecanismos implicados han sido considerablemente obstaculizada por nuestra incapacidad para identificar su unión al receptor (s). Para solucionar este problema, hemos desarrollado una modiFIED dos híbridos de levadura (MY2H) enfoque basado en el más utilizado GAL4 basada en dos sistema híbrido. En comparación con la levadura convencional de dos híbridos de pantalla, MY2H reduce drásticamente el proceso de la pantalla y reduce el número de falsos positivos clones. Además, este método es reproducible y confiable, y hemos empleado con éxito este sistema en el aislamiento de las proteínas de unión de diversos cebos, incluidos los canales de iones 12, proteína de la matriz extracelular 10, 13, 14 y factor de crecimiento. En este trabajo se describe el procedimiento experimental MY2H en detalle utilizando PGRN como ejemplo, que condujo a la identificación de TNFR2 como el primero conocido PGRN asociada a los receptores 14, 15.

Watch this Scientific Journal Video about Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin at JoVE.com

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Hemos modificado la levadura de dos híbridos convencionales de detección, una herramienta eficaz en la identificación genética interacción de proteínas. Esta modificación notable acorta el proceso, reduce la carga de trabajo, y lo más importante, reduce el número de falsos positivos. Además, este método es reproducible y confiable.

Modificado levadura sistema híbrido de dos para identificar proteínas que interactúan con el factor de crecimiento progranulina

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

modified-yeast-two-hybrid-system-to-identify-proteins-interacting

Research

JoVE Journal

Biology

28.6K vistas.  NYU Hospital for Joint Diseases. Hemos modificado la levadura de dos híbridos convencionales de detección, una herramienta eficaz en la identificación genética interacción de proteínas. Esta modificación notable acorta el proceso, reduce la carga de trabajo, y lo más importante, reduce el número de falsos positivos. Además, este método es reproducible y confiable.

Video: Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Growth Factor-Coated Bead Placement on Dorsal Forebrain Explants

This video demonstrates two methods for preparing and placing beads, which have been coated with growth factor, on explants of the developing cerebral cortex. These beads can be used to induce spatially restricted gene expression on developing neural tissue such as forebrain explants. Methods are given for using both Affi-Gel beads and heparin acryllic beads.

Factor de crecimiento-Coated colocación de bolas de explantes cerebro anterior dorsal

Investigación

Biología

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

The neural tube expresses many proteins in specific spatiotemporal patterns during development. These proteins have been shown to be critical for cell fate determination, cell migration, and formation of neural circuits. Neuronal induction and patterning involve bone morphogenetic protein (BMP), sonic hedgehog (SHH), fibroblast growth factor (FGF), among others. In particular, the expression pattern of Fgf8 is in close proximity to regions expressing BMP4 and SHH. This expression pattern is consistent with developmental interactions that facilitate patterning in the telencephalon.

Here we provide a visual demonstration of a method in which an in ovo preparation can be used to test the effects of Fgfs in the formation of the forebrain. Beads are coated with protein and placed in the developing neural tube to provide sustained exposure. Because the procedure uses small, carefully placed beads, it is minimally invasive and allows several beads to be placed within a single neural tube. Moreover, the method allows for continued development so that embryos can be analyzed at a more mature stage to detect changes in anatomy and in neural patterning. This simple but useful protocol allows for real time imaging. It provides a means to make spatially and temporally limited changes to endogenous protein levels.

A variety of growth factors and proteins interact to induce cells to take on different cell fates during development. Here we demonstrate the use of an in ovo preparation to address possible interactions between different proteins in development by placing beads on E2.5 chick embryos.

La colocación de crecimiento recubierto Factor-bolas en los embriones tempranos de la etapa de pollo

The neural tube expresses many proteins in specific spatiotemporal patterns during development.   These proteins have been shown to be critical for cell ...

Preparing T Cell Growth Factor from Rat Splenocytes

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion 1,2. Addition of interleukin 2 to the culture media during the expansion phase is necessary to prevent cell death and sufficient to maintain short-term T cell lines but has been shown to increase Th1 polarization 3. Replacement of interleukin 2 by T cell growth factor (TCGF) which contains a mix of cytokines is more effective than interleukin 2 in maintaining long-term T cell lines in vitro 3. Moreover, TCGF can easily be prepared in large amounts in the laboratory and is much cheaper than recombinant interleukin 2. 
Here, we show how to prepare TCGF from rat splenocyte culture supernatants. For this procedure, we harvest spleens from naive Lewis rats euthanized for thymus and blood collection. We prepare single-cell suspensions from the spleens, lyze the red blood cells by osmotic shock, and seed the splenocytes in culture medium. The cells are stimulated with concanavalin A, a mitogen that non-selectively activates all rat T lymphocytes, inducing the production of cytokines. The culture supernantant is collected 48 hours later andexcess concanavalin A is bound to alpha methyl mannoside to prevent it from activating T cell lines to which TCGF will be added. The TCGF is then sterile-filtered, aliquoted, and stored at -20&deg;C.

We describe the preparation of T cell growth factor used for the in vitro expansion of antigen-specific rat T lymphocyte lines.

La preparación de Factor de Crecimiento Celular T a partir de esplenocitos de rata

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion 1,2. ...

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys loop, the glutamate-gated and the P2X receptor channels2. In each case binding of transmitter leads to the opening of a pore through which ions flow down their electrochemical gradients. Many ligand-gated channels are also permeable to calcium ions3, 4, which have downstream signaling roles5 (e.g. gene regulation) that may exceed the duration of channel opening. Thus ligand-gated channels can signal over broad time scales ranging from a few milliseconds to days. Given these important roles it is necessary to understand how ligand-gated ion channels themselves are regulated by proteins, and how these proteins may tune signaling. Recent studies suggest that many, if not all, channels may be part of protein signaling complexes6. In this article we explain how to identify the proteins that bind to the C-terminal aspects of the P2X2 receptor cytosolic domain.
P2X receptors are ATP-gated cation channels and consist of seven subunits (P2X1-P2X7). P2X receptors are widely expressed in the brain, where they mediate excitatory synaptic transmission and presynaptic facilitation of neurotransmitter release7. P2X receptors are found in excitable and non-excitable cells and mediate key roles in neuronal signaling, inflammation and cardiovascular function8. P2X2 receptors are abundant in the nervous system9 and are the focus of this study. Each P2X subunit is thought to possess two membrane spanning segments (TM1 &amp; TM2) separated by an extracellular region7 and intracellular N and C termini (Fig 1a)7. P2X subunits10 (P2X1-P2X7) show 30-50% sequence homology at the amino acid level11. P2X receptors contain only three subunits, which is the simplest stoichiometry among ionotropic receptors. The P2X2 C-terminus consists of 120 amino acids (Fig 1b) and contains several protein docking consensus sites, supporting the hypothesis that P2X2 receptor may be part of signaling complexes. However, although several functions have been attributed to the C-terminus of P2X2 receptors9 no study has described the molecular partners that couple to the intracellular side of this protein via the full length C-terminus. In this methods paper we describe a proteomic approach to identify the proteins which interact with the full length C-terminus of P2X2 receptors.

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

Proteómica para identificar proteínas que interactúan con los canales de cationes P2X2 ligando

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

Split-ubiquitina levadura membrana base de dos híbridos (MITO) del sistema: Una poderosa herramienta para identificar interacciones proteína-proteína

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR-Clip - un método para identificar transcriptoma en todo los sitios de unión de las proteínas de unión a ARN

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

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

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

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

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

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

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking metabolism and inflammation (e.g.&#160;hepatic steatosis)1,2. There has been much progress in the identification of agonist ligands for PXR, however, there are limited descriptions of drug-like antagonists and their binding sites on PXR3,4,5. A critical barrier has been the inability to efficiently purify full-length protein for structural studies with antagonists despite the fact that PXR was cloned and characterized in 1998. Our laboratory developed a novel high throughput yeast based two-hybrid assay to define an antagonist, ketoconazole&#39;s, binding residues on PXR6. Our method involves creating mutational libraries that would rescue the effect of single mutations on the AF-2 surface of PXR expected to interact with ketoconazole. Rescue or &#34;gain-of-function&#34; second mutations can be made such that conclusions regarding the genetic interaction of ketoconazole and the surface residue(s) on PXR are feasible. Thus, we developed a high throughput two-hybrid yeast screen of PXR mutants interacting with its coactivator, SRC-1. Using this approach, in which the yeast was modified to accommodate the study of the antifungal drug, ketoconazole, we could demonstrate specific mutations on PXR enriched in clones unable to bind to ketoconazole. By reverse logic, we conclude that the original residues are direct interaction residues with ketoconazole. This assay represents a novel, tractable genetic assay to screen for antagonist binding sites on nuclear receptor surfaces. This assay could be applied to any drug regardless of its cytotoxic potential to yeast as well as to cellular protein(s) that cannot be studied using standard structural biology or proteomic based methods. Potential pitfalls include interpretation of data (complementary methods useful), reliance on single Y2H method, expertise in handling yeast or performing yeast two-hybrid assays, and assay optimization.

Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.

Invierta levadura de dos sistema híbrido para identificar mamíferos Receptor Nuclear Residuos que interactúan con ligandos y / o antagonistas

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

Captura compuesto Espectrometría de Masas - Una herramienta de gran alcance para identificar nuevos c-di-GMP efectoras Proteínas

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

Xenopus laevis como modelo para identificar Traducción Deterioro

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...