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: Tutte le procedure di questo protocollo sono stati condotti secondo l&#39;Istituto Nazionale di Salute linee guida per il trattamento etico e la cura degli animali, e secondo un protocollo di uso animale IACUC approvato. 1. Cella Linea Manutenzione <ol><li> Culture cellule BAF3 in mezzo RPMI-1640 supplementato con 10% siero bovino fetale e penicillina / streptomicina (100 unità / ml e 100 mg / ml) e terreno di coltura di cellule WEHI trascorso. Grow HEK293T cellule in DMEM supplementato con 10% siero bovino fetale e penicillina / streptomicina (100 unità / ml e 100 mg / ml). Mantenere cellule a 37 ° C in atmosfera umidificata contenente il 5% di CO2. </li></ol> 2. plasmidi Construction <ol><li> Clonare il mouse JAK2 e la sua oncogenica isoforma JAK2-V617F in pMSCV-puro-GW da LR clonase utilizzando procedura di clonazione ricombinazione standard. </li><li> Per la costruzione di espressione luciferasi vettoriale (pMSCV-Luc-Cherry-GW) utilizzato nella-viimmagini vo, amplificare luciferasi da pLVX-Tet-ON vettore mediante PCR utilizzando primer (LUC-SD / RP TTACAATTTGGACTTTCCG e LUC-SD / FP-CACCATGGAAGACGCCAAAAAC) seguita con la clonazione in pENTR-SD-TOPO per generare pENTR-Luc, che viene utilizzato per trasferire il gene della luciferasi in vettore retrovirale, pMSCV-Cherry-GW da ricombinazione clonazione mediata con LR clonase. </li></ol> 3. Preparazione di Random Mutazione Biblioteca, riciclaggio e identificare le mutazioni 3.1) casuale Mutagenesi <ol><li> Clone lunghezza della JAK2 wild type e la mutazione V617F in pMSCV-puro-GW vettore retrovirale utilizzando una tecnologia di clonazione ricombinazione commerciale. </li><li> Utilizzare 1 mg di JAK2-V617F plasmide DNA per trasformare, XL-1 Red, E. coli, che è difettoso in meccanismi di riparazione del DNA permettendo loro di integrare le mutazioni casuali durante la replica. Più specificamente, mix 50 - 100 ng di DNA (più di 100 ng di DNA diminuisce l&#39;efficienza della trasformazione) con 100 ml di cellule competenti in un tubo di polipropilene pre-raffreddata e incubate in ghiaccio per 30 minuti, rimestando delicatamente ogni 5 min. Per una buona copertura libreria, utilizzare 5:56 tubi di cellule competenti. NOTA: Più di 100 ng di DNA diminuisce l&#39;efficienza di trasformazione </li><li> Immergere la provetta in un bagno d&#39;acqua a 42 ° C per uno shock termico di 45 sec e incubare su ghiaccio per 2 min. Avanti, aggiungere 1 ml di mezzo SOC (2,0% triptone, estratto di lievito 0,5%, 0,05% di NaCl, MgCl2 10 mM, MgSO4 10 mm, e il 0,4% D-glucosio). Incubare il tubo a 37 ° C agitando a 225-250 rpm per 90 min. </li><li> Cellule piastra di ciascun tubo su quattro 10 centimetri piatti LB-agar contenenti 100 mg / ml di ampicillina. Incubare le piastre per 16-24 ore a 37 ° C. </li><li> A seguito di colonie visibili, raccogliendoli raschiando le piastre con un piatto raschietto sterile. Cellule Pool di ogni piatto e plasmide isolare il DNA utilizzando un kit di estrazione plasmide commerciale. NOTA: Di solito colonie sono più piccoli e prendere 18-24 ore per esserevisibile perché questi sono lenti ceppi batterici crescita. In questa fase, di valutare l&#39;eterogeneità di mutazioni nella libreria da digestione di restrizione con un cutter frequente come Sau3A1 o Taq1 o sequenziamento. </li></ol> 3.2) Produzione di retrovirali surnatante e trasduzione <ol><li> Un giorno prima trasfezione, piastra 4 x 10 6 cellule HEK 293T su 100 centimetri piatti in DMEM contenente 10% FCS, penicillina / streptomicina e 2 mM L-glutammina. </li><li> Il giorno dopo, sostituire il medio e trasfettare le cellule con mutagenizzato biblioteca pMSCV-JAK2-V617F. Per ogni piastra 10 cm miscela 10 mg di DNA (5 ug del plasmide libreria e 5 ug del plasmide retrovirale imballaggi, pCLEco) con 40 ml di FuGENE ad un volume totale di 400 microlitri di siero utilizzando supporti DMEM libera. Incubare la miscela DNA e lipidi per 20 min a temperatura ambiente. Dopo 20 minuti aggiungere il DNA-lipide complesso goccia a goccia in cima HEK293T cellule. </li><li> Dopo sei ore, cambiare il supporto trasfezione wesimo mezzi freschi e incubare le piastre per 48 ore a 37 ° C per la produzione di virus. Dopo 48 ore di incubazione, raccogliere surnatanti virali filtrati attraverso un filtro da 0,45 micron Acrodisc. </li></ol> 3.3) Selezione dei cloni resistenti in vitro <ol><li> Per resistenti ai farmaci mutanti JAK2-V617F, trasduzione 100 milioni BAF3 cella con 100 ml di surnatanti virus hanno un titolo virale di 0,5-1 x10 6. Più specificamente, mescolare 10 8 cellule con 100 ml di surnatante virus ad un volume totale di 300 ml con mezzi RPMI contenente siero, 10% e 24 ug / ml di polyberene (concentrazione finale di ployberene è 8 mg / ml). Distribuire questi mixer cellulari in più sei pozzetti (4-5 ml per pozzetto). </li><li> Centrifugare le piastre a 1.250 xg per 90 min a 25 ° C. Dopo centrifugazione, incubare le piastre a 37 ° C per 24 ore. </li><li> Il giorno dopo, in comune le cellule insieme e mescolare con Ruxolitinib e terreno contenente agar morbido e placcato in six piatti pure. Per ogni concentrazione del farmaco, miscelare 10 ml di cellule trasdotte BAF3 (10-20 milioni di cellule) con la quantità appropriata di Ruxolitinib in un tubo da 50 ml. Portare al volume di 40 ml con RPMI contenente siero del 20%. Aggiungere 10 ml di 1,2% agar alle cellule, mixcarefully e piastra in sei pozzetti. </li><li> Incubare le piastre a 37 ° C per due settimane. Scegli le colonie resistenti con 0,2 ml puntali e sub-cultura singolarmente in 24 tavole e per 3-4 giorni. </li><li> Raccogliere le cellule resistenti BAF3 mediante centrifugazione a 450 xg per cinque minuti a temperatura ambiente. Isolare il DNA genomico utilizzando un kit di isolamento DNA genomico commerciale. PCR amplifica full length JAK2 cDNA da 100 ng di DNA genomico utilizzando primer (mJAK2FP-ATGGCCTGCCTTACAATGACAG e mJAK2RP-GGTTCTGGAAGTCTGCTTGG) e lungo template espandere sistemi PCR ad alta fedeltà. </li><li> Separare i prodotti di PCR mediante elettroforesi su gel di agarosio. Accise 3,4 kb di banda DNA rappresentano JAK2 telaio codifica e isolare il DNAutilizzando un kit di estrazione gel commerciale. Sequenza tutta la lunghezza del cDNA usando 8 primer interni (P1 - P8) che attraversa la sequenza codificante e analizzare sequenze utilizzando software commerciale. </li></ol> 4. In Vitro convalida di mutazioni resistenti Molti cloni cellulari trasportare più di una mutazione, per verificare il contributo di ogni mutazione in resistenza fenotipo, generare varianti selezionati da mutagenesi sito-specifica utilizzando pMSCV-JAK2V-617F plasmide come modello. <ol><li> Eseguire mutagenesi sito-specifica in pMSCV-JAK2-V617F plasmide utilizzando un kit commerciale mutagenesi e oligonucleotidi progettati per creare mutazioni puntiformi. Confermare l&#39;identità dei cloni mutanti mediante sequenziamento. </li><li> Trasdurre cellule BAF3 con retrovirus realizzati con plasmidi mutanti seguita con selezione con puromicina a 1 ug / ml. </li><li> Tavola 10 4 cellule BAF3-JAK2-V617F (50 ml di RPMI con 10% FCS) in tutti i pozzetti di una piastra ben 96. Separatoly aggiungere 50 ml di ruxolitinib contenente supporti ai pozzetti attraverso la piastra (concentrazione finale: 0, 1, 3, 5, 10, e 20 mM), e incubare le piastre per 60 ore a 37 ° C. </li><li> Valutare vitalità cellulare aggiungendo 10 ml di reagente WST-1 ad ogni pozzetto seguito con incubazione a 37 ° C per 2-4 ore. Record assorbanza a A450 con un lettore di piastre. Eseguire tutti i test in triplice copia e trama in media assorbanza contro INCB018424 concentrazioni. Eseguire una curva sigmoidale best-fit utilizzando un algoritmo di curve fitting non lineare. Nota la concentrazione del farmaco con conseguente vitalità cellulare del 50% come IC50 cellulare. </li><li> Successivamente, piastra sei milioni BAF3 cellule esprimenti varianti JAK2 in sei pozzetti in terreno RPMI contenente 10% di siero con concentrazioni crescenti di ruxolitinib ed incubate per 4-6 ore a 37 ° C. </li><li> Raccogliere le cellule per centrifugazione a 450 g per cinque minuti a 4 ° C. Lavare le cellule una volta con PBS, e lisare in 20 mM Tris-Cl (pH 7,5), 50mM NaCl, 1% NP-40, 0.1% SDS, EDTA 5 mM, 1mM EGTA, 1 mM fluoruro di sodio, 2 mM sodio Vanadate, e il 5% glicerina integrato con inibitori della proteasi cocktail e fosfatasi. Sonicare la sospensione cellulare per 1 min seguita da aggiunta di tampone 5x gel loading (350 mM Tris-HCl [pH 6,8], 500 mM DTT, 15% SDS, 10 mM Benzamidine, 5 mM EDTA, EGTA 5 mM, 5 mM Sodio vanadato , proteasi cocktail, 50% glicerolo, e 0,001% blu di bromofenolo) e denaturazione a 70 ° C per 5-10 min. </li><li> Risolvere le proteine ​​su un gel di poliacrilammide SDS dell&#39;8% in condizioni denaturanti. Eseguire immunoblotting con il mouse monoclonale anti-phopsho STAT5. Spellare le macchie e reprobed con un anticorpo anti-SATA5 policlonale di coniglio. Visualizzare le bande utilizzando reagenti ECL secondo le raccomandazioni del fornitore. </li></ol> 5. In Vivo Validation <ol><li> Trasdurre cellule BAF3 esprimono luciferasi e ciliegia (trasdotte con retrovirus realizzati pMSCV-Luc-cherry GW) con i virus che esprimono JAK2-V617F e varianti resistenti. Selezionare cellule che sopravvivono selezione puromicina per l&#39;espressione di JAK2 e le sue varianti resistenza. </li><li> Iniettare due milioni di crescita attiva BAF3-Luc cellule / ciliegia che trasportano JAK2-V617F e le sue varianti resistenti in 200 ml di PBS a 6 Balb / C di topo mediante iniezione-coda vena. </li><li> Dopo tre giorni di trapianti di cellule, iniettare i topi con Ruxolitinib (100 mg / kg) due volte al giorno per due settimane. </li><li> Eseguire base luciferasi imaging bioluminescenza con un sistema di imaging. . <ol><li> In breve, preparare la soluzione luciferina sciogliendo 300 mg per 25 ml di acqua deionizzata sterile, un&#39;aliquota di minori volumi e negozio. NOTA: Conservare luciferina a -80 ° C e proteggere dalla luce fino al momento dell&#39;uso. </li><li> Iniettare 250 microlitri per ogni mouse IP per ottenere 125 mg / kg in ciascun topo. Anestetizzare i topi dal gruppo stesso insieme con isoflurano inalato (1,5% -2,0%), in una scatola sigillata. Applicare pomata veterinario su the occhio per prevenire la secchezza. NOTA: Il tempo necessario per i topi a dormire (10-15 minuti) ha permesso l&#39;attività luciferina a picco. Tenere il tempo coerente tra i gruppi per evitare variazioni. </li></ol></li><li> Trasferire il mouse immediatamente fase camera di imaging e mantenere l&#39;anestesia al 1,5-2% isofluorane durante le procedure di imaging. Topi fotografia con sequenziale 0,1, 0,5, 1, 5, 30, 60, e 300 sec esposizione. Cattura immagini e quantificare l&#39;intensità bioluminescenza utilizzando l&#39;acquisizione delle immagini e software di analisi. A seguito di imaging restituire i topi alla sua gabbia. </li><li> Per, in vivo chimerismo raccolto cellule bonemarrow totali. Eutanasia i topi con CO 2 per 5 minuti, seguito con dislocazione cervicale. Raccogliere le ossa e schiacciare in 4 ml PBS freddo per raccogliere il midollo osseo. Analizzare i cento ciliegi cellule positive al microscopio a fluorescenza e quantificare con FACS. Raccogliere ventimila eventi da ogni campione. </li></ol>

<ol>
	<li>Huse, M., Kuriyan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+conformational+plasticity+of+protein+kinases.">The conformational plasticity of protein kinases.</a> Cell. 109, 275-282 (2002).</li><li>Melnikova, I., Golden, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+protein+kinases.">Targeting protein kinases.</a> Nat Rev Drug Discov. 3, 993-994 (2004).</li><li>Cohen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinases--the+major+drug+targets+of+the+twenty-first+century.">Protein kinases--the major drug targets of the twenty-first century.</a> Nat Rev Drug Discov. 1, 309-315 (2002).</li><li>Dancey, J., Sausville, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Issues+and+progress+with+protein+kinase+inhibitors+for+cancer+treatment.">Issues and progress with protein kinase inhibitors for cancer treatment.</a> Nat Rev Drug Discov. 2, 296-313 (2003).</li><li>Noble, M. E., Endicott, J. A., Johnson, L. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+inhibitors:+insights+into+drug+design+from+structure.">Protein kinase inhibitors: insights into drug design from structure.</a> Science. 303, 1800-1805 (2004).</li><li>Druker, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+of+a+specific+inhibitor+of+the+BCR-ABL+tyrosine+kinase+in+the+blast+crisis+of+chronic+myeloid+leukemia+and+acute+lymphoblastic+leukemia+with+the+Philadelphia+chromosome.">Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome.</a> N Engl J Med. 344, 1038-1042 (2001).</li><li>Druker, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+a+selective+inhibitor+of+the+Abl+tyrosine+kinase+on+the+growth+of+Bcr-Abl+positive+cells.">Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells.</a> Nat Med. 2, 561-566 (1996).</li><li>Gorre, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+resistance+to+STI-571+cancer+therapy+caused+by+BCR-ABL+gene+mutation+or+amplification.">Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification.</a> Science. 293, 876-880 (2001).</li><li>Shah, N. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+BCR-ABL+kinase+domain+mutations+confer+polyclonal+resistance+to+the+tyrosine+kinase+inhibitor+imatinib+(STI571)+in+chronic+phase+and+blast+crisis+chronic+myeloid+leukemia.">Multiple BCR-ABL kinase domain mutations confer polyclonal resistance to the tyrosine kinase inhibitor imatinib (STI571) in chronic phase and blast crisis chronic myeloid leukemia.</a> Cancer Cell. 2, 117-125 (2002).</li><li>Branford, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+frequency+of+point+mutations+clustered+within+the+adenosine+triphosphate-binding+region+of+BCR/ABL+in+patients+with+chronic+myeloid+leukemia+or+Ph-positive+acute+lymphoblastic+leukemia+who+develop+imatinib+(STI571)+resistance.">High frequency of point mutations clustered within the adenosine triphosphate-binding region of BCR/ABL in patients with chronic myeloid leukemia or Ph-positive acute lymphoblastic leukemia who develop imatinib (STI571) resistance.</a> Blood. 99, 3472-3475 (2002).</li><li>Azam, M., Latek, R. R., Daley, G. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+autoinhibition+and+STI-571/imatinib+resistance+revealed+by+mutagenesis+of+BCR-ABL.">Mechanisms of autoinhibition and STI-571/imatinib resistance revealed by mutagenesis of BCR-ABL.</a> Cell. 112, 831-843 (2003).</li><li>Azam, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+of+dual+SRC-ABL+inhibitors+highlights+the+role+of+BCR/ABL+kinase+dynamics+in+drug+resistance.">Activity of dual SRC-ABL inhibitors highlights the role of BCR/ABL kinase dynamics in drug resistance.</a> Proc Natl Acad Sci U S A. 103, 9244-9249 (2006).</li><li>Azam, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AP24163+inhibits+the+gatekeeper+mutant+of+BCR-ABL+and+suppresses+in+vitro+resistance.">AP24163 inhibits the gatekeeper mutant of BCR-ABL and suppresses in vitro resistance.</a> Chem Biol Drug Des. 75, 223-227 (2010).</li><li>Azam, M., Seeliger, M. A., Gray, N. S., Kuriyan, J., Daley, G. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+tyrosine+kinases+by+mutation+of+the+gatekeeper+threonine.">Activation of tyrosine kinases by mutation of the gatekeeper threonine.</a> Nat Struct Mol Biol. 15, 1109-1118 (2008).</li><li>Soverini, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implications+of+BCR-ABL1+kinase+domain-mediated+resistance+in+chronic+myeloid+leukemia.">Implications of BCR-ABL1 kinase domain-mediated resistance in chronic myeloid leukemia.</a> Leukemia research. 38, 10-20 (2014).</li><li>Deshpande, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinase+domain+mutations+confer+resistance+to+novel+inhibitors+targeting+JAK2V617F+in+myeloproliferative+neoplasms.">Kinase domain mutations confer resistance to novel inhibitors targeting JAK2V617F in myeloproliferative neoplasms.</a> Leukemia. 26, 708-715 (2012).</li><li>Koppikar, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterodimeric+JAK-STAT+activation+as+a+mechanism+of+persistence+to+JAK2+inhibitor+therapy.">Heterodimeric JAK-STAT activation as a mechanism of persistence to JAK2 inhibitor therapy.</a> Nature. 489, 155-159 (2012).</li><li>Marit, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Random+mutagenesis+reveals+residues+of+JAK2+critical+in+evading+inhibition+by+a+tyrosine+kinase+inhibitor.">Random mutagenesis reveals residues of JAK2 critical in evading inhibition by a tyrosine kinase inhibitor.</a> PLoS One. 7, 43437 (2012).</li><li>Weigert, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+resistance+to+JAK2+enzymatic+inhibitors+is+overcome+by+HSP90+inhibition.">Genetic resistance to JAK2 enzymatic inhibitors is overcome by HSP90 inhibition.</a> The Journal of experimental medicine. 209, 259-273 (2012).</li><li>Kesarwani, M., Huber, E., Azam, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overcoming+AC220+resistance+of+FLT3-ITD+by+SAR302503.">Overcoming AC220 resistance of FLT3-ITD by SAR302503.</a> Blood cancer journal. 3, 138 (2013).</li></ol>

No conflicts of interest declared.

Il successo clinico di Imatinib nel trattamento CML ha dimostrato non solo il potenziale di colpire chinasi rouge da piccole molecole inibitrici, ma anche le limitazioni allo scoperto di terapia mirata: ricaduta clinica e comparsa di mutazioni di resistenza ai farmaci nel gene bersaglio. L&#39;identificazione di mutazioni di resistenza aiuta a migliorare la gestione clinica e lo sviluppo di inibitori di nuova generazione. Questo protocollo descrive un metodo per identificare mutazioni farmaco-resistenti nel gene bersagl...

Proteina chinasi sono enzimi chiave di regolamentazione degli intracellulari di trasduzione del segnale che apparentemente modulano ogni funzione cellulare. Un adeguato controllo della chinasi mediata segnalazione è fondamentale per l&#39;omeostasi e sviluppo, che si basa principalmente su una corretta regolamentazione delle chinasi, fosfatasi e la sua degradazione da UPS (sistema ubiquitina proteasoma). Chinasi deregolamentati sono al centro della scena di molti tumori e coinvolti in serie di malattie umane 1. Genoma umano codifica più di 500 proteine ​​chinasi che sono stati collegati, direttamente o indirettamente, a ~ 400 malattie umane 2. Queste osservazioni hanno sostenuto il concetto per terapeutico mirati chinasi da piccole molecole inibitrici 3-5. La dimostrazione di inibitori della chinasi ABL, come Imatinib nel trattamento della leucemia mieloide cronica (LMC) ha fornito la prova del concetto di questo approccio 6,7. Questa osservazione ha rivoluzionato non solo la formicai-chinasi terapia, ma anche applicata l&#39;idea di identificare le lesioni genetiche in altre malattie neoplastiche per il targeting terapeutico, che portano alla scoperta di mutazioni oncogeniche in JAK2 da policitemia vera (PV) e nei pazienti con neoplasie mieloproliferative (MPN). Questa scoperta ha generato un grande interesse nel trattamento MPNs di mira JAK2 con piccole molecole inibitori della chinasi. Ora, quasi una dozzina di inibitori della JAK2 sono in studi clinici e uno di loro è stato recentemente approvato per il trattamento della mielofibrosi. Mentre mirata delle chinasi oncogeniche da piccole molecole inibitrici nei tumori portare risultati promettenti, ma soffre anche di sviluppare resistenza al trattamento. In realtà, fino ad ora, i pazienti trattati con inibitori delle chinasi, come l&#39;Imatinib, Gefitinib, Erlotinib e Dasatinib sviluppate mutazioni di resistenza per lo più con l&#39;acquisizione di mutazioni nel dominio chinasi a cui bersagli farmacologici 8-10. Resistenza a seguito di mutazione genica evidenzia i limiti of mirate monoterapia contro le chinasi oncogeniche, e rappresenta la prossima sfida per lo sviluppo di sempre più successo chemioterapia. Conseguenze meccanicistici e funzionali di resistenza ai farmaci dovrebbero fornire un razionale per la selezione e la progettazione di composti gratuiti per lo sviluppo di farmaci. Le mutazioni identificate tramite schermi in vitro, hanno mostrato un alto grado di correlazione con quelli riscontrati in pazienti. Pertanto, lo screening in vitro per le mutazioni che conferiscono resistenza ai farmaci per un dato farmaco coppie bersaglio assist di sviluppo clinico o preclinico per identificare i modelli di resistenza che possono causare una ricaduta clinica. L&#39;identificazione di queste forme mutanti non solo è utile nel monitoraggio dei pazienti per risposta ai farmaci e la recidiva ma anche essenziale per la progettazione di inibitori più robusti prossima generazione. Ad esempio, lo sviluppo della prossima generazione di inibitori BCR / ABL, Nilotinib e Ponatinib, sono stati resi possibili a causa di una maggiore mecintese hanistic acquisita mutagenesi, strutturale, e studi funzionali. In precedenza, abbiamo riportato i risultati del nostro schermo utilizzando mutagenesi casuale di BCR / ABL a rivelare lo spettro di mutazioni che conferiscono resistenza agli inibitori come Imatinib 11,12, PD166326 12, e AP24163 13. I risultati non solo identificare le mutazioni che conferiscono resistenza e malattia clinica ricaduta, ma anche fornito la comprensione meccanicistica della resistenza e dei principi che regolano la funzione chinasi 11,14 droga. Qui forniamo ulteriori dettagli metodologico, utilizzando Ruxolitinib e JAK2 come una coppia di destinazione della droga, per consentire una più ampia applicazione di questa strategia di screening.

<table border="0" cellpadding="0" cellspacing="0" width="1074">
	<tbody>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell and Tissue culture&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BaF3 Cells</td>
			<td>ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEK293T cells</td>
			<td>ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pMSCV-JAK2-V617F-puro.GW</td>
			<td>A gift from Ross Levine</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pMSCV-JAK2-V617F/Y931C.GW</td>
			<td>Made in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pMSCV-JAK2-V617F/L983F.GW</td>
			<td>Made in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pMSCV-JAK2-V617F/P58A.GW</td>
			<td>Made in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pMSCV-V617F-Cherry.GW</td>
			<td>Made in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pMSCV-JAK2-V617F/Y931C-cherry.GW</td>
			<td>Made in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pMSCV-JAK2-V617F/L983F-cherry.GW</td>
			<td>Made in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pMSCV-Luciferase-puro.GW</td>
			<td>Made in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI</td>
			<td>Cellgro (corning)</td>
			<td>15-040-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td>Cellgro (corning)</td>
			<td>15-013-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penn/Strep</td>
			<td>Cellgro (corning)</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td>Atlanta biological</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin EDTA 1X</td>
			<td>Cellgro (corning)</td>
			<td>25-052-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1XPBS</td>
			<td>Cellgro (corning)</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Glutamine</td>
			<td>Cellgro (corning)</td>
			<td>25-005-CL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Puromycin</td>
			<td>Gibco (life technologies)</td>
			<td>A11138-03</td>
			<td></td>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trapan Blue solution (0.4%)</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td>Cellgro (corning)</td>
			<td>25-950-CQC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">INCB018424 (Ruxolitinib)</td>
			<td>ChemieTeK</td>
			<td>941678-49-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">WST-1</td>
			<td>Roche</td>
			<td>11644807001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.45uM acro disc filter</td>
			<td>PALL</td>
			<td>2016-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70um nylon cell stariner</td>
			<td>Becton Dickinson</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacterial Culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SOC</td>
			<td>New England Biolabs</td>
			<td>B90920s</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ampicillin</td>
			<td>Sigma</td>
			<td>A0166</td>
			<td>100mg/ml stock solution&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto agar</td>
			<td>Difco</td>
			<td>214050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Terrific broth</td>
			<td>Becton Dickinson</td>
			<td>243820</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose</td>
			<td>Genemate</td>
			<td>E-3119-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kits</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dneasy Blood&#38; tissue kit</td>
			<td>Qiagen</td>
			<td>69506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Expand long template PCR system</td>
			<td>Roche</td>
			<td>1168134001</td>
			<td></td>
		</tr>
		<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>
		</tr>
		<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

La scoperta di BCR / ABL come oncogene conducente leucemia mieloide cronica (CML) ha portato allo sviluppo di Imatinib, che, di fatto, ha dimostrato il potenziale di targeting chinasi in tumori trattando efficacemente i pazienti CML. Questa osservazione ha rivoluzionato lo sviluppo di farmaci a bersaglio le chinasi oncogeni implicati in varie altre neoplasie, come, EGFR, B-RAF, KIT e PDGFRs. Tuttavia, uno svantaggio principale di terapie anti-chinasi è l&#39;emergere di mutazioni di resistenza ai farmaci che rendono l&#39;obiettivo di aver ridotto o perso affinità per la droga. La comprensione dei meccanismi impiegati da varianti resistenti non solo aiuta a sviluppare gli inibitori di nuova generazione, ma dà anche impulso alla gestione clinica utilizzando la medicina personalizzata. Abbiamo riportato una strategia di vettore retrovirale basato di screening per identificare lo spettro di resistenza conferire mutazioni BCR / ABL, che ha contribuito a sviluppare la prossima generazione di BCR / ABL inibitori. Utilizzando Ruxolitinib e JAK2 come una coppia di destinazione della droga, qui si descrivono in metodi di screening in vitro che utilizza le cellule del mouse BAF3 esprimono la biblioteca mutazione casuale del JAK2 chinasi.

L&#39;emergere di mutazioni genetiche pone grande sfida per la terapia chinasi contro mirata. Studi mutazionali, oltre a fornire approfondimenti meccanicistici e funzionali che sono strumentali nella selezione e progettazione di sviluppo di farmaci di nuova generazione, permette anche una migliore gestione clinica e possano in futuro essere più utile per il trattamento personalizzato. In questo esperimento, mostriamo lo screening per le mutazioni di resistenza ruxolitinib in JAK2-V617F chinasi (Figura 1).</stro...

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

Emersione di resistenza genetica contro terapia con inibitori della chinasi pone sfida importante per la terapia del cancro efficace. Identificazione e caratterizzazione di mutazioni resistenti contro un farmaco di nuova concezione contribuisce a una migliore gestione clinica e di progettazione di farmaci di nuova generazione. Qui, descriviamo il nostro protocollo per lo screening e la validazione di mutazioni resistenti in vitro.

Un metodo per lo screening e validazione di mutazioni resistenti contro Kinase Inhibitors

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 Views.  Cincinnati Children's Hospital Medical Center.  Emersione di resistenza genetica contro terapia con inibitori della chinasi pone sfida importante per la terapia del cancro efficace. Identificazione e caratterizzazione di mutazioni resistenti contro un farmaco di nuova concezione contribuisce a una migliore gestione clinica e di progettazione di farmaci di nuova generazione. Qui, descriviamo il nostro protocollo per lo screening e la validazione di mutazioni resistenti in vitro. 

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.

Iniezione di An. Gli embrioni stephensi per generare la malaria resistenti zanzare

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

Ricerca

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.

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.

L&#39;applicazione di una degenerazione C. elegans Dopamina Saggio Neuron per la convalida dei potenziali morbo di Parkinson geni

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.

Il metodo MODS per la diagnosi di tubercolosi e di tubercolosi multiresistente

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

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

Metodo per la Cultura di Chick primi embrioni ex vivo (Nuova Cultura)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

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.

Saggiare l&#39;attività chinasi di 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 ...

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue1,2. GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (Gi and Go) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K+ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate.
GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders3. However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations3.
Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K+ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K+ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry4 or radiotracer analysis5, the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.

A real-time screening procedure for identifying drugs that interact with G protein-gated inward rectifier K+ (GIRK) channels is described. The assay utilizes membrane potential-sensitive fluorescent dyes to measure GIRK channel activity. This technique is adaptable for use on a number of cell lines.

Un test di screening a fluorescenza per l&#39;identificazione di modulatori Canali GIRK

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...

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.

Conversione di un ELISA Capture ad un saggio Luminex xMAP utilizzando un anticorpo Multiplex metodo di screening

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 Catalyst come reporter per lo screening dei farmaci contro l&#39;RNA Editing in tripanosomi

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.

Generazione efficiente delle cellule iPS da Blood Utilizzando episomi e HDAC inibitori

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.

Screening ad alto rendimento per i modulatori chimici di Genes post-trascrizionale regolamentati

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

Un metodo per lo screening e validazione di mutazioni resistenti contro Kinase Inhibitors

Riepilogo

Esplora Altri Video

Capitoli in questo video

Iniezione di An. Gli embrioni stephensi per generare la malaria resistenti zanzare

L'applicazione di una degenerazione C. elegans Dopamina Saggio Neuron per la convalida dei potenziali morbo di Parkinson geni

Il metodo MODS per la diagnosi di tubercolosi e di tubercolosi multiresistente

Metodo per la Cultura di Chick primi embrioni ex vivo (Nuova Cultura)

Saggiare l'attività chinasi di LRRK2 In vitro

Un test di screening a fluorescenza per l'identificazione di modulatori Canali GIRK

Conversione di un ELISA Capture ad un saggio Luminex xMAP utilizzando un anticorpo Multiplex metodo di screening

RNA Catalyst come reporter per lo screening dei farmaci contro l'RNA Editing in tripanosomi

Generazione efficiente delle cellule iPS da Blood Utilizzando episomi e HDAC inibitori

Screening ad alto rendimento per i modulatori chimici di Genes post-trascrizionale regolamentati