This study was supported by grants to M.A. from NCI (1RO1CA155091), NHLBI (1R21HL114074) and the Leukemia Research Foundation. M.A. is a recipient of V-Scholar award from the V- Foundation. Authors are thankful to Dr. Sara Rohrabaugh for editing.

NOTA: Todos los procedimientos de este protocolo se llevaron a cabo de acuerdo con el Instituto Nacional de Salud directrices para el tratamiento ético y cuidado de los animales, y de acuerdo con un protocolo de uso de los animales aprobado IACUC. Línea 1. Mantenimiento de la celda <ol><li> Cultura células BAF3 en medio RPMI-1640 suplementado con suero bovino fetal al 10% y penicilina / estreptomicina (100 unidades / ml y 100 mg / ml) y medio de cultivo gastado de las células Wehi. Se cultivan las células HEK293T en medio DMEM suplementado con suero bovino fetal al 10% y penicilina / estreptomicina (100 unidades / ml y 100 mg / mL). Mantener las células a 37 ° C en un ambiente humidificado contiene 5% de CO2. </li></ol> 2. Construcción de plásmidos <ol><li> Clonar el ratón JAK2 y su oncogénico isoforma JAK2 V617F-en pMSCV-puro-GW por LR Clonase usando el procedimiento de clonación de recombinación estándar. </li><li> Para la construcción del vector de expresión de luciferasa (pMSCV-Luc-cereza-GW) que se utiliza en el in-viimágenes vo, amplificar la luciferasa de pLVX-Tet-ON vector mediante PCR utilizando cebadores (LUC-SD / RP TTACAATTTGGACTTTCCG y LUC-SD / FP-CACCATGGAAGACGCCAAAAAC) siguieron con la clonación en pENTR-SD-TOPO para generar pENTR-Luc, que se utiliza para transferir el gen de la luciferasa en el vector retroviral, pMSCV-cereza-GW por recombinación mediada por clonación usando LR clonasa. </li></ol> 3. Preparación de Random Mutación Biblioteca, detección e identificación de las mutaciones 3.1) Random Mutagenesis <ol><li> Clon de longitud completa de JAK2 tipo salvaje y la mutación en pMSCV-puro-GW vector retroviral V617F utilizando una tecnología de clonación de recombinación comercial. </li><li> Utilice 1 g de ADN plásmido JAK2 V617F-transformar, XL-1 Rojo, E. coli células, lo que es defectuoso en los mecanismos de reparación del ADN lo que les permite incorporar mutaciones aleatorias durante la replicación. Más específicamente, mezclar 50 - 100 ng de ADN (más de 100 ng de ADN disminuye la eficiencia de transformación) con 100 l de células competentes en un tubo de polipropileno pre-enfriada y se incubaron en hielo durante 30 min, agitando suavemente cada 5 min. Para una buena cobertura de la biblioteca, utilice cinco y cincuenta y seis tubos de células competentes. NOTA: Más de 100 ng de ADN disminuye la eficiencia de transformación </li><li> Sumerja el tubo en un baño de agua a 42 ° C durante un choque térmico de 45 segundos e incubar en hielo durante 2 min. A continuación, añadir 1 ml de medio SOC (2,0% de triptona, extracto de levadura al 0,5%, 0,05% de NaCl, MgCl2 10 mM, MgSO4 10 mM, y 0,4% de D-glucosa). Incubar el tubo a 37 ° C con agitación a 225-250 rpm durante 90 min. </li><li> Células de la placa de cada tubo en cuatro placas de 10 cm de agar LB que contienen 100 mg / ml de ampicilina. Incubar las placas durante 16-24 horas a 37 ° C. </li><li> Después de colonias visibles, ellos recoger raspando las placas con un raspador de placa estéril. Piscina células de cada placa y el plásmido aislado de ADN usando un kit de extracción de plásmido comercial. NOTA: Por lo general, las colonias son más pequeños y toman 18-24 horas para servisible porque estos son cepas bacterianas de crecimiento lento. En esta etapa, evaluar la heterogeneidad de las mutaciones en la biblioteca por digestión de restricción con un cortador frecuentes, tales como Sau3A1 o Taq1 o secuenciación. </li></ol> 3.2) Producción de sobrenadantes retrovirales y la transducción <ol><li> Un día antes de la transfección, placa de 4 x 10 6 células HEK 293T sobre 100 cm de platos en DMEM que contenía 10% de FCS, penicilina / estreptomicina y 2 mM L-glutamina. </li><li> Al día siguiente, reemplace el medio y transfectar las células con mutagenized biblioteca pMSCV-JAK2-V617F. Para cada placa de 10 cm, mezclar 10 g de ADN (5 g de la biblioteca de plásmido y 5 g de plásmido de empaquetamiento retroviral, pCLEco) con 40 l de FuGENE a un volumen total de 400 l de suero utilizando medio DMEM libre. Incubar la mezcla de ADN y lípidos durante 20 min a temperatura ambiente. Después de 20 minutos añadir el complejo de abandono ADN lípidos sabio en la parte superior de las células HEK293T. </li><li> Después de seis horas, cambiar el medio de transfección wITH medio fresco y se incuban las placas durante 48 horas a 37 ° C para la producción de virus. Después de 48 horas de incubación, recoger los sobrenadantes virales filtrados a través de un filtro de 0,45 micras acrodisco. </li></ol> 3.3) La selección de clones resistentes in vitro <ol><li> Para mutantes resistentes JAK2 V617F-drogas, transducir 100 millones de células BAF3 con 100 ml de sobrenadantes de virus que tienen un título viral de 0,5-1 x10 6. Más específicamente, mezclar 10 8 células con 100 ml de sobrenadante de virus a un volumen total de 300 ml utilizando medio RPMI, conteniendo 10% de suero y 24 mg / ml de polyberene (concentración final de ployberene es de 8 mg / ml). Distribuir estos mezcladores de células en múltiples placas de seis pocillos (4-5 ml por pocillo). </li><li> Centrifugar las placas a 1250 xg durante 90 minutos a 25 ° C. Después de la centrifugación, se incuban las placas a 37 ° C durante 24 horas. </li><li> Al día siguiente, en común las células juntos y mezclar con ruxolitinib y medio con agar blando y chapada en six placas de pocillos. Para cada concentración de fármaco, mezclar 10 ml de células BAF3 transducidas (10-20 millones de células) con la cantidad apropiada de ruxolitinib en un tubo de 50 ml. Completar el volumen a 40 ml usando RPMI que contiene 20% de suero. Añadir 10 ml de 1,2% de agar a las células, mixcarefully y la placa en placas de seis pocillos. </li><li> Incubar las placas a 37 ° C durante dos semanas. Tome las colonias resistentes utilizando puntas de pipeta de 0,2 ml y subcultura individualmente en placas de 24 pocillos durante 3-4 días. </li><li> Recoger las células BAF3 resistentes por centrifugación a 450 xg durante cinco minutos a temperatura ambiente. Aislar el ADN genómico usando un kit de aislamiento de ADN genómico comercial. PCR amplificar ADNc completo JAK2 de 100 ng de ADN utilizando cebadores genómicos (mJAK2FP-ATGGCCTGCCTTACAATGACAG y mJAK2RP-GGTTCTGGAAGTCTGCTTGG) y largo plantilla ampliar los sistemas de PCR de alta fidelidad. </li><li> Separar los productos de PCR mediante electroforesis en gel de agarosa. Escindir el 3,4 kb de banda de ADN que representan JAK2 trama de codificación y aislar el ADNutilizando un kit de extracción de gel comercial. Secuencia de la longitud total de cDNA utilizando 8 cebadores internos (P1 - P8) que abarca la secuencia de codificación y analizar secuencias utilizando el software comercial. </li></ol> 4. En Vitro Validación de mutaciones resistentes Muchos clones de células llevan más de una mutación, Para probar la contribución de cada mutación en el fenotipo de resistencia, generar variantes seleccionadas por mutagénesis dirigida al sitio utilizando el plásmido pMSCV-JAK2V-617F como plantilla. <ol><li> Realizar mutagénesis dirigida al sitio en el plásmido pMSCV-JAK2-V617F utilizando un kit de mutagénesis comercial y oligonucleótidos diseñados para crear mutaciones puntuales. Confirmar la identidad de los clones mutantes por secuenciación. </li><li> Transducir células BAF3 con retrovirus hacen usando plásmidos mutantes seguidos con la selección utilizando puromicina de 1 g / ml. </li><li> Plate 10 4 células BAF3-JAK2-V617F (50 l de RPMI con 10% de FCS) en cada pocillo de una placa de 96 pocillos. Separadoly añadir 50 l de los medios de comunicación ruxolitinib que contiene a los pozos a través de la placa (concentración final: 0, 1, 3, 5, 10 y 20 mM), e incubar las placas durante 60 horas a 37 ° C. </li><li> Evaluar la viabilidad celular mediante la adición de 10 l de reactivo WST-1 a cada pocillo seguido con incubación a 37 ° C durante 2-4 h. Absorbancia Record en A450 usando un lector de placas. Realizar todos los ensayos por triplicado y parcela promedio de absorbancia contra INCB018424 concentraciones. Realice una curva sigmoidal de ajuste óptimo usando un algoritmo de ajuste de curva no lineal. Puntuación de la concentración de fármaco resultante en la viabilidad celular 50% como el IC50 celular. </li><li> A continuación, la placa de seis millones de células que expresan variantes BAF3 JAK2 en placas de seis pocillos en medio RPMI que contenía 10% de suero con concentraciones crecientes de ruxolitinib y se incubaron durante 4-6 horas a 37 ° C. </li><li> Recoger las células por centrifugación a 450 g durante cinco minutos a 4 ° C. Lavar las células una vez con PBS, y se lisan en 20 mM Tris-Cl (pH 7,5), 50mM de NaCl, 1% NP-40, 0,1% SDS, EDTA 5 mM, EGTA 1 mM, fluoruro de sodio 1 mM, 2 mM de sodio vanadato, y 5% de glicerol suplementado con inhibidores de cóctel inhibidor de proteasa y fosfatasa. Sonicar la suspensión de células durante 1 min seguido por adición de 5x tampón de carga de gel (350 mM Tris-HCl [pH 6,8], DTT 500 mM, 15% de SDS, benzamidina 10 mM, EDTA 5 mM, 5 mM EGTA, 5 mM de sodio vanadato , cóctel de proteasa, 50% de glicerol, y 0,001% de azul de bromofenol) y desnaturalización a 70 ° C durante 5-10 min. </li><li> Resolver las proteínas en un gel de poliacrilamida SDS 8% en condiciones desnaturalizantes. Realizar inmunotransferencia con anticuerpo monoclonal de ratón anti-phopsho STAT5. Pele los borrones y reprobed con un anticuerpo policlonal de conejo anti-SATA5. Visualice las bandas utilizando reactivos ECL según la recomendación del proveedor. </li></ol> 5. En Vivo Validación <ol><li> Transducir células BAF3 expresan luciferasa y cereza (transducidas con retrovirus hechos de pMSCV-Luc-cherry GW) con virus que expresan JAK2-V617F y variantes resistentes. Seleccionar las células que sobreviven a la selección de puromicina para la expresión de JAK2 y sus variantes de resistencia. </li><li> Inyectar dos millones de células en crecimiento activo / cereza BAF3-Luc llevan JAK2-V617F y sus variantes resistentes en 200 l de PBS a 6 ratones Balb / C a través de la inyección de la cola de la vena. </li><li> Después de tres días de trasplantes de células, inyectar a los ratones con ruxolitinib (100 mg / kg) dos veces al día durante dos semanas. </li><li> Realizar imágenes de bioluminiscencia basado en la luciferasa con un sistema de imagen. . <ol><li> En resumen, preparar la solución de luciferina por disolución de 300 mg a 25 ml de agua desionizada estéril, la alícuota en volúmenes más pequeños y tienda. NOTA: luciferina tienda a -80 ° C y protegido de la luz hasta su uso. </li><li> Inyectar 250 l a cada ratón por IP para alcanzar 125 mg / kg en cada ratón. Anestesiar a los ratones de un mismo grupo juntos utilizando isoflurano inhalado (1,5% -2,0%) en una caja sellada. Aplique un ungüento veterinario en el the ojo para prevenir la sequedad. NOTA: El tiempo necesario para que los ratones dormir (10-15 min) permitió la actividad luciferina a pico. Mantenga el tiempo constante entre los grupos para evitar la variación. </li></ol></li><li> Transferir los ratones inmediatamente a la etapa de cámara de imágenes y mantener la anestesia en el 1,5-2% isofluorano durante los procedimientos de imagen. Ratones imagen secuencial con 0,1, 0,5, 1, 5, 30, 60, y 300 segundos de exposición. Captura imágenes y cuantificar la intensidad de la bioluminiscencia usando adquisición de imágenes y software de análisis. Siguiendo imágenes devolver los ratones de nuevo a su jaula. </li><li> Para bonemarrow totales, células in vivo cosecha quimerismo. La eutanasia a los ratones con CO 2 durante 5 min seguido con dislocación cervical. Cosecha de los huesos y aplastar en 4 ml de PBS frío para recoger la médula ósea. Analizar las células positivas por ciento de cerezo bajo el microscopio de fluorescencia y cuantificar mediante FACS. Recoge veinte mil eventos de cada muestra. </li></ol>

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No conflicts of interest declared.

El éxito clínico de imatinib en el tratamiento de la LMC demostró no sólo la posibilidad de dirigirse a las rouge quinasas por inhibidores de moléculas pequeñas, pero las limitaciones también al descubierto de la terapia dirigida: recaída clínica y la aparición de mutaciones de resistencia a fármacos en el gen diana. Identificación de mutaciones de resistencia ayuda a una mejor gestión clínica y el desarrollo de inhibidores de la próxima generación. Este protocolo describe una metodología para identific...

Las proteínas quinasas son enzimas reguladoras clave de las vías de transducción de señales intracelulares que aparentemente modulan cada función celular. Un adecuado control de la señalización mediada por la quinasa es crucial para la homeostasis y el desarrollo, que se basa principalmente en una adecuada regulación de quinasas, fosfatasas y su degradación por UPS (sistema ubiquitina proteasoma). Quinasas desreguladas están en el centro del escenario de muchos tipos de cáncer e implicado en multitud de enfermedades humanas 1. Genoma humano codifica más de 500 proteínas quinasas que se han relacionado, directa o indirectamente, a ~ 400 enfermedades humanas 2. Estas observaciones apoyaron el concepto de focalización terapéutica de quinasas por los inhibidores de moléculas pequeñas de 3-5. La demostración de inhibidores de la quinasa ABL, tales como Imatinib, en el tratamiento de la leucemia mieloide crónica (CML) proporcionó la prueba de concepto para este enfoque 6,7. Esta observación no solo revolucionó la hormigai-quinasa terapia, pero también hace cumplir la idea de identificar las lesiones genéticas en otras enfermedades neoplásicas para el enfoque terapéutico, que conducen al descubrimiento de mutaciones oncogénicas en el JAK2 de la policitemia vera (PV) y los pacientes con neoplasias mieloproliferativas (MPN). Este descubrimiento generó gran interés en el tratamiento de las MPN apuntando JAK2 con inhibidores de la quinasa de moléculas pequeñas. Ahora, casi una docena de inhibidores de JAK2 están en ensayos clínicos y uno de ellos ha sido aprobado recientemente para el tratamiento de la mielofibrosis. Si bien la orientación específica de quinasas oncogénicas por inhibidores de moléculas pequeñas en los cánceres traer resultados prometedores, que también sufre de desarrollar resistencia al tratamiento. De hecho, hasta ahora, los pacientes tratados con inhibidores de la quinasa, como Imatinib, Gefitinib, erlotinib y dasatinib desarrollaron mutaciones de resistencia en su mayoría mediante la adquisición de mutaciones en el dominio quinasa de qué fármaco se dirige a 08.10. Resistencia como resultado de la mutación del gen pone de relieve las limitaciones of dirigido monoterapia contra las quinasas oncogénicas, y representa el siguiente reto en el desarrollo de la quimioterapia del cáncer cada vez más éxito. Consecuencias mecánicas y funcionales de resistencia a los medicamentos deben proporcionar una justificación para la selección y el diseño de compuestos de cortesía para el desarrollo de fármacos. Las mutaciones identificadas a través de pantallas in vitro, han demostrado un alto grado de correlación con las que se encuentran en los pacientes. Por lo tanto, la detección in vitro de mutaciones que confieren resistencia a las drogas para un determinado objetivo pares de drogas en asistencias de desarrollo clínico o preclínico en la identificación de los patrones de resistencia que puedan causar la recaída clínica. La identificación de estas formas mutantes no sólo es útil en el seguimiento de los pacientes para la respuesta a los fármacos y la recaída, pero también es esencial para el diseño de inhibidores de próxima generación más robustos. Por ejemplo, el desarrollo de inhibidores de BCR / ABL de próxima generación, nilotinib y Ponatinib, fueron posibles debido a la mayor mecentendimientos hanistic ganaron de mutagénesis, estructurales y estudios funcionales. Anteriormente, hemos informado de los resultados de nuestra pantalla usando mutagénesis aleatoria de BCR / ABL para revelar el espectro de mutaciones que confieren resistencia a los inhibidores como el Imatinib 11,12, PD166326 12 y 13 AP24163. Los resultados no sólo identificaron las mutaciones que confieren resistencia y la enfermedad recaída clínica, sino que también proporcionan la comprensión mecanicista de la resistencia y de los principios que rigen la función quinasa 11,14 drogas. Aquí proporcionamos detalles metodológicos adicional, usando ruxolitinib y JAK2 como un par de objetivos farmacológicos, para permitir una aplicación más amplia de esta estrategia de cribado.

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			<td height="21" style="height:21px;width:344px;">name of Materials/Equipment</td>
			<td style="width:161px;">Company</td>
			<td style="width:116px;">Catalog Number</td>
			<td style="width:453px;">Comments/ Description</td>
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			<td height="21" style="height:21px;">Cell and Tissue culture&#160;</td>
			<td></td>
			<td></td>
			<td></td>
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			<td height="21" style="height:21px;">BaF3 Cells</td>
			<td>ATCC</td>
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			<td>ATCC</td>
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			<td height="21" style="height:21px;">pMSCV-JAK2-V617F-puro.GW</td>
			<td>A gift from Ross Levine</td>
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			<td height="21" style="height:21px;">pMSCV-JAK2-V617F/Y931C.GW</td>
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			<td height="21" style="height:21px;">pMSCV-JAK2-V617F/L983F.GW</td>
			<td>Made in house</td>
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			<td height="21" style="height:21px;">pMSCV-JAK2-V617F/P58A.GW</td>
			<td>Made in house</td>
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			<td height="21" style="height:21px;">pMSCV-V617F-Cherry.GW</td>
			<td>Made in house</td>
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			<td height="21" style="height:21px;">pMSCV-JAK2-V617F/Y931C-cherry.GW</td>
			<td>Made in house</td>
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			<td height="21" style="height:21px;">pMSCV-JAK2-V617F/L983F-cherry.GW</td>
			<td>Made in house</td>
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			<td height="21" style="height:21px;">pMSCV-Luciferase-puro.GW</td>
			<td>Made in house</td>
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			<td height="21" style="height:21px;">RPMI</td>
			<td>Cellgro (corning)</td>
			<td>15-040-CV</td>
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			<td height="21" style="height:21px;">DMEM</td>
			<td>Cellgro (corning)</td>
			<td>15-013-CV</td>
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			<td height="21" style="height:21px;">Penn/Strep</td>
			<td>Cellgro (corning)</td>
			<td>30-002-CI</td>
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			<td height="21" style="height:21px;">FBS</td>
			<td>Atlanta biological</td>
			<td>S11150</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Trypsin EDTA 1X</td>
			<td>Cellgro (corning)</td>
			<td>25-052-CI</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">1XPBS</td>
			<td>Cellgro (corning)</td>
			<td>21-040-CV</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">L-Glutamine</td>
			<td>Cellgro (corning)</td>
			<td>25-005-CL</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Puromycin</td>
			<td>Gibco (life technologies)</td>
			<td>A11138-03</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Protamine sulfate</td>
			<td>Sigma</td>
			<td>P3369</td>
			<td>5mg/ml stock in water</td>
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			<td height="21" style="height:21px;">Trapan Blue solution (0.4%)</td>
			<td>Sigma</td>
			<td>T8154</td>
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			<td>Cellgro (corning)</td>
			<td>25-950-CQC</td>
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			<td height="21" style="height:21px;">INCB018424 (Ruxolitinib)</td>
			<td>ChemieTeK</td>
			<td>941678-49-5</td>
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		<tr height="21">
			<td height="21" style="height:21px;">WST-1</td>
			<td>Roche</td>
			<td>11644807001</td>
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		<tr height="21">
			<td height="21" style="height:21px;">0.45uM acro disc filter</td>
			<td>PALL</td>
			<td>2016-10</td>
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			<td height="21" style="height:21px;">70um nylon cell stariner</td>
			<td>Becton Dickinson</td>
			<td>352350</td>
			<td></td>
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			<td height="21" style="height:21px;">Bacterial Culture</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">XL-1 red E.Coli cells</td>
			<td>Agilent Tech</td>
			<td>200129</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">SOC</td>
			<td>New England Biolabs</td>
			<td>B90920s</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Ampicillin</td>
			<td>Sigma</td>
			<td>A0166</td>
			<td>100mg/ml stock solution&#160;</td>
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			<td height="21" style="height:21px;">Bacto agar</td>
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			<td>214050</td>
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			<td height="21" style="height:21px;">Terrific broth</td>
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			<td>243820</td>
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			<td height="21" style="height:21px;">Agarose</td>
			<td>Genemate</td>
			<td>E-3119-500</td>
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			<td height="21" style="height:21px;">Kits</td>
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			<td>69506</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Expand long template PCR system</td>
			<td>Roche</td>
			<td>1168134001</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Wizard Sv gel and PCR clean up system</td>
			<td>Promega</td>
			<td>A9282</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Pure Yield plasmid mini prep system</td>
			<td>Promega</td>
			<td>A1222</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR Cloning System with Gateway Technology with pDONR 221 &#38; OmniMAX 2 Competent Cells</td>
			<td>Invitrogen</td>
			<td>12535029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gateway&#160; LR Clonase Enzyme mix&#160;</td>
			<td>Invitrogen</td>
			<td>11791019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vivo-Glo Luciferin in-vivo Grade</td>
			<td>Promega</td>
			<td>P1043</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1/2cc Lo-Dose u-100 insulin syringe 28 G1/2</td>
			<td>Becton Dickinson</td>
			<td>329461</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mortor pestle</td>
			<td>Coor tek&#160;</td>
			<td colspan="2">60316 and 60317</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflorane (Isothesia TM)</td>
			<td>Butler Schien</td>
			<td>29405</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NAPCO series 8000 WJ CO2 incubator</td>
			<td>Thermo scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Swing bucket rotor centrifuge 5810R</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TC-10 automated cell counter</td>
			<td>Bio-RAD</td>
			<td></td>
			<td>This is not necessary, one can use standard hemocytomemetr for cell counting</td>
		</tr>
	</tbody>
</table>

a method for screening and validation of resistant mutations against kinase inhibitors

El descubrimiento de BCR / ABL como un oncogén conductor en la leucemia mieloide crónica (CML) dio como resultado el desarrollo de Imatinib, que, de hecho, demostró el potencial de focalizar la quinasa en los cánceres por tratar eficazmente los pacientes con LMC. Esta observación revolucionó el desarrollo de fármacos para orientar las quinasas oncogénicas implicado en varios otros tumores malignos, tales como, EGFR, B-RAF, KIT y PDGFR. Sin embargo, una desventaja importante de las terapias anti-quinasa es la aparición de mutaciones de resistencia a fármacos que prestan el objetivo de haber reducido o perdido afinidad por la droga. La comprensión de los mecanismos empleados por variantes resistentes no sólo ayuda en el desarrollo de los inhibidores de la próxima generación, sino también da impulso a la gestión clínica usando la medicina personalizada. Nos informó de una estrategia de cribado retroviral vector basado para identificar el espectro de resistencia que confiere mutaciones en BCR / ABL, lo que ha ayudado en el desarrollo de la próxima generación de BCR / ABL inhibidores. Utilizando Ruxolitinib y JAK2 como un par de objetivos farmacológicos, aquí nos describen en los métodos de cribado in vitro que utiliza las células BAF3 ratón que expresan la biblioteca mutación aleatoria de JAK2 quinasa.

La aparición de mutaciones genéticas plantea un gran desafío para la terapia anti-quinasa específica. Mutacional estudios, además de proporcionar conocimientos sobre mecánica y funcionales que son instrumentales en la selección y el diseño de desarrollo de fármacos de nueva generación, que también permite una mejor gestión clínica y puedan en el futuro ser más útil para el tratamiento personalizado. En este experimento, se muestra la detección de mutaciones de resistencia ruxolitinib en JAK2 V617F-quinas...

Watch this Scientific Journal Video about A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors at JoVE.com

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

Aparición de resistencia genética contra la terapia con inhibidores de quinasa plantea reto significativo para la terapia del cáncer eficaz. Identificación y caracterización de mutaciones resistentes contra un fármaco recientemente desarrollado ayuda a una mejor gestión clínica y el diseño de fármacos de próxima generación. A continuación, describimos nuestro protocolo para la detección in vitro y la validación de las mutaciones resistentes.

Un método para la detección y validación de mutaciones de resistencia contra la quinasa Inhibidores

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the ...

a-method-for-screening-validation-resistant-mutations-against-kinase

Research

JoVE Journal

Biology

14.8K vistas.  Cincinnati Children's Hospital Medical Center.  Aparición de resistencia genética contra la terapia con inhibidores de quinasa plantea reto significativo para la terapia del cáncer eficaz. Identificación y caracterización de mutaciones resistentes contra un fármaco recientemente desarrollado ayuda a una mejor gestión clínica y el diseño de fármacos de próxima generación. A continuación, describimos nuestro protocolo para la detección in vitro y la validación de las mutaciones resistentes. 

Video: A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

Injection of An. stephensi Embryos to Generate Malaria-resistant Mosquitoes

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This video protocol demonstrates a method used by the James laboratory to microinject DNA constructs into Anopheles stephensi embryos for the generation of transformed mosquitoes. Techniques for preparing microinjection needles, collecting and preparing embryos and performing the microinjection are illustrated.

Anopheles stephensi mosquitoes are vectors for malaria inhabiting India and throughout Asia. This video demonstrates the technique for performing microinjections of this species with transgenes that will confer resistance to the malaria to the mosquito. Much of the methodology demonstrated in this video is applicable to microinjection techniques of other mosquito species.

La inyección de una. Los embriones stephensi para generar la malaria resistente a los mosquitos

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This ...

Investigación

Biología

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations at risk. In the process of attempting to identify novel genetic factors associated with PD, scientists have generated many lists of candidate genes, polymorphisms, and proteins that represent important advances, but these leads remain mechanistically undefined. Our work is aimed toward significantly narrowing such lists by exploiting the advantages of a simple animal model system. While humans have billions of neurons, the microscopic roundworm Caenorhabditis elegans has precisely 302, of which only eight produce dopamine (DA) in hemaphrodites. Expression of a human gene encoding the PD-associated protein, alpha-synuclein, in C. elegans DA neurons results in dosage and age-dependent neurodegeneration.
Worms expressing human alpha-synuclein in DA neurons are isogenic and express both GFP and human alpha-synuclein under the DA transporter promoter (Pdat-1). The presence of GFP serves as a readily visualized marker for following DA neurodegeneration in these animals. We initially demonstrated that alpha-synuclein-induced DA neurodegeneration could be rescued in these animals by torsinA, a protein with molecular chaperone activity 1. Further, candidate PD-related genes identified in our lab via large-scale RNAi screening efforts using an alpha-synuclein misfolding assay were then over-expressed in C. elegans DA neurons. We determined that five of seven genes tested represented significant candidate modulators of PD as they rescued alpha-synuclein-induced DA neurodegeneration 2. Additionally, the Lindquist Lab (this issue of JoVE) has performed yeast screens whereby alpha-synuclein-dependent toxicity is used as a readout for genes that can enhance or suppress cytotoxicity. We subsequently examined the yeast candidate genes in our C. elegans alpha-synuclein-induced neurodegeneration assay and successfully validated many of these targets 3, 4.
Our methodology involves generation of a C. elegans DA neuron-specific expression vector using recombinational cloning of candidate gene cDNAs under control of the Pdat-1 promoter. These plasmids are then microinjected in wild-type (N2) worms, along with a selectable marker for successful transformation. Multiple stable transgenic lines producing the candidate protein in DA neurons are obtained and then independently crossed into the alpha-synuclein degenerative strain and assessed for neurodegeneration, at both the animal and individual neuron level, over the course of aging.

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson&#039;s Disease Genes

This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.

Aplicación de un ensayo de C. elegans la degeneración neuronal de dopamina para la validación del potencial de la enfermedad de Parkinson genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

The MODS method for diagnosis of tuberculosis and multidrug resistant tuberculosis

Patients with active pulmonary tuberculosis (TB) infect 10-15 other persons per year, making diagnosing active TB essential to both curing the patient and preventing new infections.  Furthermore, the emergence of multidrug resistant tuberculosis (MDRTB) means that detection of drug resistance is necessary for stopping the spread of drug-resistant strains.  The microscopic-observation drug-susceptibility (MODS) assay is a low-cost, low-tech tool for high-performance detection of TB and MDRTB.  The MODS assay is based on three principles: 1) mycobacterium tuberculosis (MTB) grows faster in liquid media than on solid media 2) microscopic MTB growth can be detected earlier in liquid media than waiting for the macroscopic appearance of colonies on solid media, and that growth is characteristic of MTB, allowing it to be distinguished from atypical mycobacteria or fungal or bacterial contamination 3) the drugs isoniazid and rifampicin can be incorporated into the MODS assay to allow for simultaneous direct detection of MDRTB, obviating the need for subculture to perform an indirect drug susceptibility test. Competing current diagnostics are hampered by low sensitivity with sputum smear, long delays until diagnosis with solid media culture, prohibitively high cost with existing liquid media culture methods, and the need to do subculture for indirect drug susceptibility testing to detect MDRTB.  In contrast, the non-proprietary MODS method has a high sensitivity for TB and MDRTB, is a relatively rapid culture method, provides simultaneous drug susceptibility testing for MDRTB, and is accessible to resource-limited settings at just under $3 for testing for TB and MDRTB.

The microscopic-observation drug-susceptibility (MODS) assay is a low-cost, low-tech tool for high-performance detection of tuberculosis (TB) and multidrug-resistant tuberculosis (MDRTB). This video describes the MODS liquid media culture method.

La MODS método para el diagnóstico de la tuberculosis y la tuberculosis multirresistente

Patients with active pulmonary tuberculosis (TB) infect 10-15 other persons per year, making diagnosing active TB essential to both curing the patient and ...

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

Ensayo de la actividad quinasa de LRRK2 In vitro</em

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life science research and clinical diagnostics. However, ELISA has limitations. It is typically performed in a 96-well microplate, and the wells are coated with capture antibody, requiring a relatively large amount of sample to capture an antigen of interest . The large surface area of the wells and the hydrophobic binding of capture antibody can also lead to non-specific binding and increased background. Additionally, most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity. Such amplification is not always linear and can thus skew results.
In the past 15 years, a new technology has emerged that offers the benefits of the ELISA, but also enables higher throughput, increased flexibility, reduced sample volume, and lower cost, with a similar workflow 1, 2. Luminex xMAP Technology is a microsphere (bead) array platform enabling both monoplex and multiplex assays that can be applied to both protein and nucleic acid applications 3-5. The beads have the capture antibody covalently immobilized on a smaller surface area, requiring less capture antibody and smaller sample volumes, compared to ELISA, and non-specific binding is significantly reduced. Smaller sample volumes are important when working with limiting samples such as cerebrospinal fluid, synovial fluid, etc. 6. Multiplexing the assay further reduces sample volume requirements, enabling multiple results from a single sample.
Recent improvements by Luminex include: the new MAGPIX system, a smaller, less expensive, easier-to-use analyzer; Low-Concentration Magnetic MagPlex Microspheres which eliminate the need for expensive filter plates and come in a working concentration better suited for assay development and low-throughput applications; and the xMAP Antibody Coupling (AbC) Kit, which includes a protocol, reagents, and consumables necessary for coupling beads to the capture antibody of interest. (See Materials section for a detailed list of kit contents.)
In this experiment, we convert a pre-optimized ELISA assay for TNF-alpha cytokine to the xMAP platform and compare the performance of the two methods 7-11. TNF-alpha is a biomarker used in the measurement of inflammatory responses in patients with autoimmune disorders.
We begin by coupling four candidate capture antibodies to four different microsphere sets or regions. When mixed together, these four sets allow for the simultaneous testing of all four candidates with four separate detection antibodies to determine the best antibody pair, saving reagents, sample and time. Two xMAP assays are then constructed with the two most optimal antibody pairs and their performance is compared to that of the original ELISA assay in regards to signal strength, dynamic range, and sensitivity.

An ELISA can be easily converted to a Luminex xMAP assay and, through the benefits of multiplexing, several antibodies can be screened simultaneously to identify an optimum antibody pair, resulting in increased sensitivity and dynamic range, while reducing assay cost.

La conversión de un ELISA de captura para un ensayo de Luminex xMAP utilizando un anticuerpo Multiplex Método de detección

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life ...

RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes

Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been made in identifying the components of the editosome complex that catalyze RNA editing. However, it is still not clear how those proteins work together. Chemical compounds obtained from a high-throughput screen against the editosome may block or affect one or more steps in the editing cycle. Therefore, the identification of new chemical compounds will generate valuable molecular probes for dissecting the editosome function and assembly. In previous studies, in vitro editing assays were carried out using radio-labeled RNA. These assays are time consuming, inefficient and unsuitable for high-throughput purposes. Here, a homogenous fluorescence-based &#8220;mix and measure&#8221; hammerhead ribozyme in vitro reporter assay to monitor RNA editing, is presented. Only as a consequence of RNA editing of the hammerhead ribozyme a fluorescence resonance energy transfer (FRET) oligoribonucleotide substrate undergoes cleavage. This in turn results in separation of the fluorophore from the quencher thereby producing a signal. In contrast, when the editosome function is inhibited, the fluorescence signal will be quenched. This is a highly sensitive and simple assay that should be generally applicable to monitor in vitro RNA editing or high throughput screening of chemicals that can inhibit the editosome function.

A highly sensitive ribozyme-based assay, applicable to high-throughput screening of chemicals targeting the unique process of RNA editing in trypanosomatid pathogens, is described in this paper. Inhibitors can be used as tools for hypothesis-driven analysis of the RNA editing process and ultimately as therapeutics.

RNA catalizador como reportero para rastrear fármacos contra ARN de edición en tripanosomas

Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been ...

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

Eficiente iPS Generación Celular de sangre usando episomas y HDAC inhibidores

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

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

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

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

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

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

Identification of Kinase-substrate Pairs Using High Throughput Screening

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

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

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


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

A Step-By-Step Method to Detect Neutralizing Antibodies Against AAV using a Colorimetric Cell-Based Assay

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health conditions in both the laboratory and the clinic. However, pre-existing neutralizing antibodies (NAbs) against AAV capsids pose an ongoing challenge for the successful administration of gene therapies in both large animal experimental models and human populations. Preliminary screening for host immunity against AAV is necessary to ensure the efficacy of AAV-based gene therapies as both a research tool and as a clinically viable therapeutic agent. This protocol describes a colorimetric in vitro assay to detect neutralizing factors against AAV serotype 6 (AAV6). The assay utilizes the reaction between an AAV encoding an alkaline phosphatase (AP) reporter gene and its substrate NBT/BCIP, which generates an insoluble quantifiable purple stain upon combination. 
In this protocol, serum samples are combined with an AAV expressing AP and incubated to permit potential neutralizing activity to occur. Virus serum mixture is subsequently added to cells to allow for viral transduction of any AAVs that have not been neutralized. The NBT/BCIP substrate is added and undergoes a chromogenic reaction, corresponding to viral transduction and neutralizing activity. The proportion of area colored is quantitated using a free software tool to generate neutralizing titers. This assay displays a strong positive correlation between coloration and viral concentration. Assessment of serum samples from sheep before and after administration of a recombinant AAV6 led to a dramatic increase in neutralizing activity (125 to &gt;10,000-fold increase). The assay displayed adequate sensitivity to detect neutralizing activity in &gt;1:32,000 serum dilutions. This assay provides a simple, rapid, and cost-effective method to detect NAbs against AAVs.

A comprehensive laboratory protocol and analysis workflow are described for a rapid, cost-effective, and straightforward colorimetric cell-based assay to detect neutralizing elements against AAV6.

Un método paso a paso para detectar anticuerpos neutralizantes contra AAV utilizando un ensayo colorimétrico basado en células

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health ...