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 os procedimentos deste protocolo foram conduzidos de acordo com o Instituto Nacional de diretrizes de saúde para o tratamento ético e tratamento dos animais, e de acordo com um protocolo de uso de animais IACUC aprovado. 1. Linha celular de manutenção <ol><li> Culturas de células BAF3 em meio RPMI-1640 suplementado com 10% de soro bovino fetal e penicilina / estreptomicina (100 unidades / ml e 100 ug / ml) e meio de cultura gasto de células WEHI. Cultivar células HEK293T em DMEM suplementado com 10% de soro bovino fetal e penicilina / estreptomicina (100 unidades / ml e 100 ug / ml). Manter as células a 37 ° C numa atmosfera humidificada contendo 5% de CO2. </li></ol> 2. Construção de plasmídeo <ol><li> Clonar o rato JAK2 e JAK2 sua isoforma oncogénica-V617F em pMSCV-puro-GW por clonase LR, utilizando um processo de recombinação de clonagem padrão. </li><li> Para a construção do vector de expressão de luciferase (Luc-pMSCV-Cherry-GW) utilizado na in-viimagiologia vo, amplificar a luciferase de pLVX-Tet-ON vector por PCR utilizando iniciadores (LUC-SD / RP-LUC TTACAATTTGGACTTTCCG e SD / FP-CACCATGGAAGACGCCAAAAAC) seguido pela clonagem em pENTR-SD-TOPO para gerar pENTR-Luc, que é usada para transferir o gene de luciferase em vector retroviral, pMSCV-Cherry-GW de clonagem por recombinação mediada usando clonase LR. </li></ol> 3. Preparação da Random Mutation Library, Triagem e identificação das Mutações 3.1) A mutagénese aleatória <ol><li> Comprimento total Clone de JAK2 tipo selvagem e a mutação V617F em pMSCV-puro-GW retroviral vector usando uma tecnologia de recombinação clonagem comercial. </li><li> Use 1 ug de ADN plasmídeo de JAK2 V617F-se transformar, XL-1 Vermelho, E. coli, que é defeito nos mecanismos de reparo do DNA permitindo-lhes incorporar mutações aleatórias durante a replicação. Mais especificamente, mistura 50 - 100 ng de ADN (mais do que 100 ng de ADN diminui a eficiência de transformação) com 100 ul de células competentes de um tubo de polipropileno pré-gelada e incubadas em gelo durante 30 min, agitando suavemente a cada 5 min. Para uma boa cobertura a biblioteca, utilizam 4-6 tubos de células competentes. NOTA: mais do que 100 ng de ADN diminui a eficiência de transformação </li><li> Mergulha-se o tubo num banho de água a 42 ° C durante um choque térmico de 45 seg e incubar em gelo durante 2 min. Em seguida, adicionar 1 ml de meio SOC (2,0% de triptona, 0,5% de extrato de levedura, 0,05% de NaCl, 10 mM de MgCl2, 10 mM de MgSO4, e 0,4% de D-glucose). Incubar o tubo a 37 ° C enquanto agitando a 225-250 rpm durante 90 min. </li><li> Placa células de cada tubo em quatro placas de 10 cm de LB-ágar contendo 100 ug / ml de ampicilina. Incubar as placas durante 16-24 horas a 37 ° C. </li><li> Seguindo colónias visíveis, coletá-los raspando as placas com uma placa raspador estéril. Piscina células de cada placa e isolar o ADN de plasmídeo utilizando um kit de extracção de plasmídeo comercial. NOTA: Normalmente, as colônias são menores e tomar 18-24 horas para servisível, porque estes são crescentes estirpes bacterianas lentas. Nesta fase, avaliar a heterogeneidade de mutações na biblioteca por digestão de restrição com um cortador frequente, tais como Sau3A1 ou Taq1 ou sequenciação. </li></ol> 3.2) A produção de retrovirais sobrenadantes e transdução <ol><li> Um dia antes da transfecção, placa de 4 x 10 6 células HEK 293T Onto 100 centímetros pratos em DMEM contendo 10% de FCS, penicilina / estreptomicina e 2 mM de L-glutamina. </li><li> No dia seguinte, substituir o meio e transfecção das células com mutado biblioteca pMSCV-JAK2-V617F. Para cada placa de 10 cm, misturar 10 ug de ADN (5 ug da biblioteca de plasmídeo e 5 ug de plasmídeo de empacotamento retroviral, pCLEco) com 40 ul de FuGENE para um volume total de 400 ul utilizando meio DMEM livre de soro. Incubar a mistura de ADN e de lípido durante 20 minutos à temperatura ambiente. Após 20 min adicionar a queda complexo DNA-lipídico sábio em cima de células HEK293T. </li><li> Após seis horas, mudar o meio de transfecção wom meios frescos e incubar as placas durante 48 horas a 37 ° C para a produção de vírus. Após 48 horas de incubação, recolher os sobrenadantes virais filtradas através de um filtro de 0,45 um Acrodisc. </li></ol> 3.3) Seleção de clones resistentes In Vitro <ol><li> Para mutantes resistentes à JAK2 V617F-droga, a transdução de 100 milhões de células BAF3 com 100 ml de sobrenadantes de vírus com um título virai de 0,5-1 x 10 6. Mais especificamente, misturar 10 8 células com 100 ml de sobrenadante do vírus para um volume total de 300 ml, utilizando meio RPMI, contendo soro a 10% e 24 ug / ml de polyberene (concentração final de ployberene é de 8 ug / ml). Distribuir estes misturadores de células em placas de seis poços múltiplos (4-5 ml por poço). </li><li> Centrifuga-se as placas a 1.250 xg durante 90 min a 25 ° C. Após centrifugação, incubar as placas a 37 ° C durante 24 horas. </li><li> No dia seguinte, reunir as células e misture com Ruxolitinib e meio contendo agar macio e banhado em six placas de poços. Para cada concentração de fármaco, misturar 10 mL de células BAF3 transduzidas (10-20 milhões de células) com a quantidade adequada de Ruxolitinib em um tubo de 50 ml. Completar o volume de 40 ml, utilizando RPMI contendo 20% de soro. Adicionar 10 ml de 1,2% de agar para as células, e mixcarefully placa em placas de seis poços. </li><li> Incubar as placas a 37 ° C durante duas semanas. Escolha as colónias resistentes, utilizando 0,2 ml pontas para pipetas e sub-cultura-los individualmente em placas de 24 poços para 3-4 dias. </li><li> Colher as células BAF3 resistentes por centrifugação a 450 xg durante cinco minutos à temperatura ambiente. Isolar o ADN genómico utilizando um kit comercial de isolamento de ADN genómico. PCR amplificar comprimento total JAK2 cDNA a partir de 100 ng de DNA genômico utilizando primers (mJAK2FP-ATGGCCTGCCTTACAATGACAG e mJAK2RP-GGTTCTGGAAGTCTGCTTGG) e longa-template expandir sistemas de PCR de alta fidelidade. </li><li> Separa-se os produtos de PCR por electroforese em gel de agarose. Extirpar o 3.4 kb de banda DNA representando JAK2 quadro de codificação e isolar o DNAutilizando um kit de extracção de gel comercial. Sequência de todo o comprimento do cDNA utilizando 8 primers internos (P1 - P8) que mede a sequência de codificação e analisar seqüências usando software comercial. </li></ol> 4. Validação in vitro de mutações resistentes Muitos clones de células transportar mais do que uma mutação, Para testar a contribuição de cada mutação no fenótipo de resistência, gerar variantes seleccionadas por mutagénese dirigida a um local utilizando pMSCV-JAK2V-617F plasmídeo como modelo. <ol><li> Realizar mutagénese dirigida em pMSCV-JAK2 V617F-plasmídeo utilizando um kit de mutagénese comercial e oligonucleótidos concebidos para criar mutações pontuais. Confirme as identidades dos clones mutantes por sequenciação. </li><li> Transdução de células BAF3 com retrovírus feita usando plasmídeos mutantes seguiram com a seleção usando ï¿½puromicina em 1 ug / ml. </li><li> Placa 4 10 células BAF3-JAK2 V617F (50-ul de meio RPMI com FCS a 10%) em cada poço de uma placa de 96 poços. Separadoly adicionar 50 ul de meio contendo o ruxolitinib aos poços ao longo da placa (concentração final: 0, 1, 3, 5, 10, e 20 uM), e incuba-se as placas durante 60 horas a 37 ° C. </li><li> Avaliar a viabilidade das células por adição de 10 ul de reagente WST-1 a cada poço, seguido de incubação com a 37 ° C durante 2-4 horas. Ficha absorvância a A450 usando um leitor de placas. Realizar todos os ensaios em triplicado e enredo média absorção contra concentrações INCB018424. Realize uma curva sigmoidal melhor ajuste usando um algoritmo de ajuste de curva não-linear. Conseguir a concentração de droga que resulta em 50% de viabilidade celular como a IC50 celular. </li><li> Em seguida, placa seis milhões de células BAF3 que expressam variantes de JAK2 em placas de seis poços em meio RPMI contendo 10% de soro com concentrações crescentes de ruxolitinib e incubadas durante 4-6 horas a 37 ° C. </li><li> Recolher as células por centrifugação a 450 xg durante cinco minutos a 4 ° C. Lavar as células uma vez com PBS, e lise em 20 mM Tris-Cl (pH 7,5), 50mM de NaCl, 1% de NP-40, 0,1% de SDS, 5 mM de EDTA, 1 mM EGTA, 1 mM de fluoreto de sódio, 2 mM de vanadato de sódio, e 5% de glicerol suplementado com inibidores de cocktail inibidor de protease e fosfatase. Sonicar a suspensão de células durante 1 min, seguido por adição de tampão de carregamento de gel 5x (350 mM Tris-HCl [pH 6,8], 500 mM de DTT, 15% de SDS, 10 mM de benzamidina, 5 mM de EDTA, 5 mM de EGTA, 5 mM de vanadato de sódio , Protease Cocktail, 50% de glicerol, e azul de bromofenol a 0,001%) e desnaturação a 70 ° C durante 5-10 min. </li><li> Resolver as proteínas em um gel de poliacrilamida SDS 8% em condições de desnaturação. Execute immunoblotting com anticorpo monoclonal anti-phopsho STAT5. Tira as manchas e sondado com um anticorpo policlonal de coelho anti-SATA5. Visualize as bandas que utilizam reagentes ECL de acordo com a recomendação do fornecedor. </li></ol> 5. In Vivo de validação <ol><li> Transdução de células que expressam BAF3 luciferase e cereja (transduzidas com retrovírus feitos de pMSCV-Luc-cherry GW) com vírus que expressam JAK2-V617F e variantes resistentes. Seleccionar células que sobrevivem puromicina selecção para a expressão de JAK2 e suas variantes de resistência. </li><li> Injectar dois milhões de células em crescimento activo / cereja BAF3-Luc-JAK2 V617F que transportam e as suas variantes resistentes em 200 ul de PBS a 6 ratinhos Balb / c por via injecção venosa da cauda. </li><li> Depois de três dias de transplantes de células, injectar os ratinhos com Ruxolitinib (100 mg / kg) duas vezes por dia durante duas semanas. </li><li> Execute-base luciferase bioluminescência de imagem com um sistema de imagem. . <ol><li> Em resumo, preparar solução luciferina dissolvendo 300 mg a 25 ml de água deionizada estéril, da alíquota em volumes e loja de menores. NOTA: luciferina Armazenar a -80 ° C e protegido da luz até à utilização. </li><li> Injectar 250 ul a cada rato por IP para conseguir 125 mg / kg em cada rato. Anestesiar os ratos do mesmo grupo em conjunto, utilizando isoflurano inalado (1,5% -2,0%) em uma caixa selada. Aplicar vet pomada em the olho para evitar ressecamento. NOTA: O tempo necessário para que os ratos de dormir (10-15 min) permitiu atividade luciferina a pico. Mantenha o tempo consistente entre os grupos para evitar variação. </li></ol></li><li> Transferir os ratos imediatamente a fase câmara de imagem e manter a anestesia em 1,5-2% de isofluorano durante os procedimentos de imagiologia. Ratinhos imagem sequencial com 0,1, 0,5, 1, 5, 30, 60, e 300 segundos de exposição. Capturar imagens e quantificar a intensidade de bioluminescência usando aquisição de imagem e software de análise. Na sequência de imagens devolver os ratos volta a sua jaula. </li><li> Para, células de medula óssea no total in vivo quimerismo colheita. Eutanásia os ratos com CO 2 para 5 min seguido com luxação cervical. Colher os ossos e esmagar em 4 ml de PBS frio para recolher a medula óssea. Analisar as células positivas por cento de cereja sob microscópio de fluorescência e quantificar utilizando FACS. Recolha vinte mil eventos de cada amostra. </li></ol>

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No conflicts of interest declared.

O sucesso clínico de imatinib no tratamento da CML demonstrado não só o potencial de atacar as quinases rouge por inibidores de moléculas pequenas, mas também descoberto limitações de terapia direcionada: recaída clínica e aparecimento de mutações de resistência a drogas no gene alvo. Identificação de mutações de resistência ajuda na melhor gestão clínica e desenvolvimento de inibidores de próxima geração. Este protocolo descreve uma metodologia para a identificação de mutações resistentes a dr...

As proteína-quinases são enzimas reguladoras-chave de vias intracelulares de transdução de sinal que aparentemente modulam cada função celular. Um controle adequado da quinase mediada sinalização é fundamental para a homeostase e desenvolvimento, que se baseia principalmente na regulamentação adequada de quinases, fosfatases e sua degradação por UPS (sistema ubiquitina proteassoma). Quinases desregulados estão no centro do palco de muitos cânceres e envolvida em série de doenças humanas 1. Genoma humano codifica mais de 500 proteínas cinases que foram ligadas, directa ou indirectamente, para ~ 400 doenças humanas 2. Estas observações suportado o conceito para direccionamento terapêutico de cinases por pequenas moléculas inibidoras 3-5. A demonstração de inibidores da quinase ABL, tais como imatinib, no tratamento de leucemia mielóide crónica (LMC) forneceu a prova de conceito para esta abordagem 6,7. Esta observação não só revolucionou a formigai-quinase terapêutica mas também aplicada a ideia para identificar as lesões genéticas em outras doenças neoplásicas para direccionamento terapêutico, que levam à descoberta de mutações oncogénicas em JAK2 de policitemia vera (PV) e os pacientes com neoplasias mieloproliferativas (NMP). Esta descoberta gerou grande interesse no tratamento MPNs alvejando JAK2 com inibidores da quinase de moléculas pequenas. Agora, quase uma dúzia de inibidores de JAK2 estão em ensaios clínicos, e um deles foi aprovado recentemente para o tratamento de mielofibrose. Enquanto direccionamento específico de quinases oncogénicas por pequenas moléculas inibidoras em cancros trazer resultados promissores, também sofre de desenvolvimento de resistência ao tratamento. De facto, até agora, os pacientes tratados com inibidores de cinase, tais como imatinib, gefitinib, erlotinib e dasatinib mutações de resistência desenvolvida principalmente através da aquisição de mutações no domínio de cinase de droga que tem como alvo 8-10. Resistência como resultado de mutação genética destaca as limitações de of monoterapia alvo contra as quinases oncogénicas, e representa o seguinte desafio para o desenvolvimento de quimioterapia do cancro cada vez mais bem sucedidos. Consequências mecanicistas e funcionais de resistência aos medicamentos deve fornecer uma justificação para a selecção e design de compostos de cortesia para o desenvolvimento de drogas. As mutações identificadas via telas in vitro, mostraram um alto grau de correlação com os encontrados em pacientes. Portanto, rastreio in vitro para mutações que conferem resistência aos fármacos para um dado alvo pares droga auxilia em desenvolvimento clínico ou pré-clínicos em identificar os padrões de resistência que são susceptíveis de causar recaída clínica. A identificação dessas formas mutantes não só é útil no acompanhamento dos pacientes para a resposta à droga e recaída, mas também essencial para a concepção de inibidores mais robustos de próxima geração. Por exemplo, o desenvolvimento de inibidores BCR / ABL próxima geração, o nilotinib e ponatinibe, foram possíveis por causa da maior mecentendimentos hanistic adquirida com mutagênese, estrutural, e estudos funcionais. Anteriormente, relataram os resultados de nossa tela usando mutagênese aleatória de BCR / ABL para revelar o espectro de mutações de resistência aos inibidores tais como Imatinib 11,12, PD166326 12 e 13 AP24163. Os resultados não só identificou as mutações que conferem resistência e recidiva da doença clínica, mas também forneceu a compreensão mecanicista da resistência e dos princípios que regem a função quinase 11,14 drogas. Aqui nós fornecemos detalhes metodológicos adicionais, usando Ruxolitinib e JAK2 como um par alvo da droga, para permitir uma aplicação mais ampla dessa estratégia de triagem.

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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 height="21" style="height:21px;">pMSCV-JAK2-V617F-puro.GW</td>
			<td>A gift from Ross Levine</td>
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			<td>Made in house</td>
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			<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>Made in house</td>
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			<td>Cellgro (corning)</td>
			<td>15-040-CV</td>
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			<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>
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			<td height="21" style="height:21px;">Trypsin EDTA 1X</td>
			<td>Cellgro (corning)</td>
			<td>25-052-CI</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>
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			<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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			<td height="21" style="height:21px;">WST-1</td>
			<td>Roche</td>
			<td>11644807001</td>
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			<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>
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			<td height="21" style="height:21px;">Bacterial Culture</td>
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			<td></td>
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			<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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			<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>E-3119-500</td>
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			<td height="21" style="height:21px;">Expand long template PCR system</td>
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			<td>A9282</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>
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		<tr height="21">
			<td height="21" style="height:21px;">Gateway&#160; LR Clonase Enzyme mix&#160;</td>
			<td>Invitrogen</td>
			<td>11791019</td>
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		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Mouse reagents</td>
			<td></td>
			<td></td>
			<td></td>
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		<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>
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		<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

A descoberta de BCR / ABL como um oncogene condutor na leucemia mielóide crónica (LMC), resultou no desenvolvimento de Imatinib, o qual, na verdade, demonstraram o potencial do alvo a quinase em cancros por tratar eficazmente os pacientes de CML. Esta observação revolucionou o desenvolvimento de drogas para alvejar as quinases oncogénicas implicados em várias outras doenças malignas, tais como, o EGFR, B-RAF, KIT e PDGFRs. No entanto, uma grande desvantagem de terapias anti-quinase é o aparecimento de mutações de resistência a drogas que tornam o alvo ter reduzido ou perdido afinidade para a droga. A compreensão dos mecanismos empregados pela variantes resistentes não só ajuda no desenvolvimento dos inibidores de nova geração, mas também dá impulso à gestão clínica usando a medicina personalizada. Nós relatou uma estratégia de rastreamento retroviral vector base para identificar o espectro de resistência conferindo mutações no BCR / ABL, o que tem ajudado no desenvolvimento da próxima geração BCR / ABL inibidores. Usando Ruxolitinib e JAK2 como um par alvo da droga, aqui descrevemos em métodos de rastreio in vitro que utiliza as células do rato BAF3 expressam a biblioteca mutação aleatória de JAK2 quinase.

Surgimento de mutações genéticas coloca grande desafio para a terapia anti-quinase alvo. Estudos de mutação, além de proporcionar idéias mecanicistas e funcionais, que são fundamentais para a seleção e projeto de desenvolvimento de medicamentos de última geração, também permite uma melhor gestão clínica e pode, no futuro, ser mais útil para o tratamento personalizado. Neste experimento, mostramos triagem para mutações de resistência ruxolitinib em JAK2 V617F-quinase (Figura 1). Const...

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

Surgimento de resistência genética a terapia com inibidor da quinase poses desafio significativo para a terapia do cancro eficaz. Identificação e caracterização de mutações resistentes contra uma droga recém-desenvolvido ajuda na melhor gestão clínica e concepção de medicamentos próxima geração. Aqui, descrevemos o nosso protocolo de rastreio in vitro e validação das mutações resistentes.

Um método de triagem e Validação de mutações resistentes aos inibidores da quinase

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 visualizações.  Cincinnati Children's Hospital Medical Center.  Surgimento de resistência genética a terapia com inibidor da quinase poses desafio significativo para a terapia do cancro eficaz. Identificação e caracterização de mutações resistentes contra uma droga recém-desenvolvido ajuda na melhor gestão clínica e concepção de medicamentos próxima geração. Aqui, descrevemos o nosso protocolo de rastreio in vitro e validação das mutações resistentes. 

Vídeo: 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.

Uma injeção de. Embriões stephensi para gerar malária resistente a mosquitos

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

Biologia

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.

Aplicação de um elegans dopamina C. Ensaio degeneração dos neurônios de Validação do Potencial Doença 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.

O método MODS para o diagnóstico da tuberculose e da tuberculose resistente a múltiplas drogas

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.

Doseamento do atividade da quinase 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.

A conversão de um ELISA de captura para um ensaio de xMAP Luminex usando um método de rastreio de anticorpos Multiplex

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 Catalisador como repórter para Triagem Drogas contra edição de RNA em Tripanossomas

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 Geração células iPS a partir de sangue, utilizando epissomas e HDAC Inibidores

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.

Rastreio de alto rendimento para moduladores químicos de Genes Post-transcriptionally Regulamentados

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.

Identificação de pares Kinase-substrato utilizando 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 ...

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.

Um método passo-a-passo para detectar anticorpos neutralizantes contra a AAV usando um ensaio baseado em células coloridas

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