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.

NOTE: All procedures in this protocol were conducted according to the National Institute of Health guidelines for the ethical treatment and care of animals, and according to an approved IACUC animal use protocol.
1. Cell Line Maintenance
<ol>
	<li>Culture BAF3 cells in RPMI-1640 medium supplemented with 10% fetal bovine serum and penicillin/streptomycin (100 units/ml and 100 &#181;g/ml) and spent culture medium of Wehi Cells. Grow HEK293T cells in DMEM supplemented with 10% fetal bovine serum and penicillin/streptomycin (100 units/ml and 100 &#956;g/ml). Maintain cells at 37 &#176;C in a humidified atmosphere containing 5% CO2.</li>
</ol>
2. Plasmid Construction
<ol>
	<li>Clone the mouse JAK2 and its oncogenic isoform JAK2-V617F in pMSCV-puro-GW by LR clonase using standard recombination cloning procedure.</li>
	<li>For the construction of luciferase expression vector (pMSCV-Luc-Cherry-GW) used in in-vivo imaging, amplify luciferase from pLVX-Tet-ON vector by PCR using primers (LUC-SD/RP TTACAATTTGGACTTTCCG and LUC-SD/FP-CACCATGGAAGACGCCAAAAAC) followed with cloning in pENTR-SD-TOPO to generate pENTR-Luc, which is used to transfer the Luciferase gene in retroviral vector, pMSCV-Cherry-GW by recombination mediated cloning using LR clonase.</li>
</ol>
3. Preparation of Random Mutation Library, Screening and Identifying the Mutations
3.1) Random Mutagenesis
<ol>
	<li>Clone full length of JAK2 wild type and the V617F mutation into pMSCV-puro-GW retroviral vector using a commercial recombination cloning technology.</li>
	<li>Use 1 &#181;g of JAK2-V617F plasmid DNA to transform, XL-1 Red, E. coli cells, which is defective in DNA repair mechanisms thus allowing them to incorporate random mutations during replication. More specifically, mix 50 - 100 ng of DNA (more than 100 ng of DNA decreases transformation efficiency) with 100 &#181;l of competent cells in a pre-chilled polypropylene tube and incubated on ice for 30 min, gently swirling every 5 min. For a good library coverage, use four to six tubes of competent cells. 
	NOTE: More than 100 ng of DNA decreases transformation efficiency</li>
	<li>Immerse the tube in a water bath at 42 &#176;C for a heat shock of 45 sec and incubate on ice for 2 min. Next, add 1 ml of SOC medium (2.0% tryptone, 0.5% yeast extract, 0.05% NaCl, 10 mM MgCl2, 10 mM MgSO4, and 0.4% D-glucose). Incubate the tube at 37 &#176;C while shaking at 225-250 rpm for 90 min.</li>
	<li>Plate cells from each tube on four 10 cm LB-agar plates containing 100 &#181;g/ml ampicillin. Incubate the plates for 16 - 24 hr at 37 &#176;C.</li>
	<li>Following visible colonies, collect them by scraping the plates with a sterile plate scraper. Pool cells from each plate and isolate plasmid DNA using a commercial plasmid extraction kit. 
	NOTE: Usually colonies are smaller and take 18-24 hr to be visible because these are slow growing bacterial strains. At this stage, assess the heterogeneity of mutations in the library by restriction digestion with a frequent cutter such as Sau3A1 or Taq1 or sequencing.</li>
</ol>
3.2) Production of Retroviral Supernatants and Transduction
<ol>
	<li>One day before transfection, plate 4 x 106 HEK 293T cells onto 100 cm dishes in DMEM containing 10% FCS, penicillin/streptomycin and 2 mM L-glutamine.</li>
	<li>Next day, replace the medium and transfect the cells with mutagenized pMSCV-JAK2-V617F library. For each 10 cm plate, mix 10 &#181;g of DNA (5 &#181;g of the plasmid library and 5 &#181;g of retroviral packaging plasmid, pCLEco) with 40 &#181;l of FuGENE to a total volume of 400 &#181;l using serum free DMEM media. Incubate the DNA and lipid mix for 20 min at room temperature. After 20 min add the DNA-lipid complex drop wise on top of HEK293T cells.</li>
	<li>After six hr, change the transfection media with fresh media and incubate the plates for 48 hr at 37 &#176;C for virus production. After 48 hr of incubation, collect viral supernatants filtered through a 0.45 &#181;m acrodisc filter.</li>
</ol>
3.3) Selection of Resistant Clones In Vitro
<ol>
	<li>For drug resistant JAK2-V617F mutants, transduce 100 million BAF3 cell with 100 ml of virus supernatants having a viral titer of 0.5-1 x106. More specifically, mix 108 cells with 100 ml of virus supernatant to a total volume of 300 ml using RPMI media, containing 10% serum and 24 &#181;g/ml of polyberene (final concentration of ployberene is 8 &#181;g/ml). Distribute these cell mixers in multiple six well plates (4-5 ml per well).</li>
	<li>Centrifuge the plates at 1,250 x g for 90 min at 25 &#176;C. After centrifugation, incubate the plates at 37 &#176;C for 24 hr.</li>
	<li>Next day, pool the cells together and mix with Ruxolitinib and medium containing soft agar and plated in six well plates. For each drug concentration, mix 10 ml of transduced BAF3 cells (10-20 million cells) with the appropriate amount of Ruxolitinib in a 50 ml tube. Make up the volume to 40 ml using RPMI containing 20% serum. Add 10 ml of 1.2 % agar to the cells, mixcarefully and plate in six well plates.</li>
	<li>Incubate the plates at 37 &#176;C for two weeks. Pick the resistant colonies using 0.2 ml pipette tips and sub-culture them individually in 24 well plates for 3-4 days.</li>
	<li>Harvest the resistant BAF3 cells by centrifugation at 450 x g for five min at room temperature. Isolate the genomic DNA using a commercial genomic DNA isolation kit. PCR amplify full length JAK2 cDNA from 100 ng of genomic DNA using primers (mJAK2FP-ATGGCCTGCCTTACAATGACAG and mJAK2RP-GGTTCTGGAAGTCTGCTTGG) and long-template expand high fidelity PCR systems.</li>
	<li>Separate the PCR products by agarose gel electrophoresis. Excise the 3.4 kb of DNA band representing JAK2 coding frame and isolate the DNA using a commercial gel extraction kit. Sequence the full length of cDNA using 8 internal primers (P1 - P8) spanning the coding sequence and analyze sequences using commercial software.</li>
</ol>
4. In Vitro Validation of Resistant Mutations
Many cell clones carry more than one mutation, To test the contribution of each mutation in resistance phenotype, generate selected variants by site-directed mutagenesis using pMSCV-JAK2V-617F plasmid as template.
<ol>
	<li>Perform site directed mutagenesis on pMSCV-JAK2-V617F plasmid using a commercial mutagenesis kit and oligonucleotides designed to create point mutations. Confirm the identities of mutant clones by sequencing.</li>
	<li>Transduce BAF3 cells with retroviruses made using mutant plasmids followed with selection using puromycin at 1&#181;g/ml.</li>
	<li>Plate 104 BAF3-JAK2-V617F cells (50 &#181;l of RPMI with 10% FCS) into each well of a 96 well plate. Separately add 50 &#181;l of the ruxolitinib containing media to the wells across the plate (final concentration: 0, 1, 3, 5, 10, and 20 &#181;M), and incubate the plates for 60 hr at 37 &#176;C.</li>
	<li>Assess cell viability by adding 10 &#181;l of WST-1 reagent to each well followed with incubation at 37&#176;C for 2-4 hr. Record absorbance at A450 using a plate reader. Perform all assays in triplicate and plot averaged absorbance against INCB018424 concentrations. Perform a best-fit sigmoidal curve using a nonlinear curve fitting algorithm. Score the drug concentration resulting in 50% cell viability as the cellular IC50.</li>
	<li>Next, plate six million BAF3 cells expressing JAK2 variants in six well plates in RPMI medium containing 10% serum with increasing concentrations of ruxolitinib and incubated for 4&#8211;6 hr at 37 &#176;C.</li>
	<li>Collect the cells by centrifugation at 450 x g for five min at 4 &#176;C. Wash the cells one time with PBS, and lyse in 20 mM Tris-Cl (pH 7.5), 50 mM NaCl, 1% NP-40, 0.1% SDS, 5 mM EDTA, 1mM EGTA, 1 mM sodium fluoride, 2 mM Sodium Vanadate, and 5% Glycerol supplemented with protease inhibitor cocktail and phosphatase inhibitors. Sonicate the cell suspension for 1 min followed by addition of 5x gel loading buffer (350 mM Tris-HCl [pH 6.8], 500 mM DTT, 15% SDS, 10 mM Benzamidine, 5 mM EDTA, 5 mM EGTA, 5 mM Sodium Vanadate, Protease cocktail, 50% Glycerol, and 0.001% Bromophenol blue) and denaturation at 70 &#176;C for 5&#8211;10 min.</li>
	<li>Resolve the proteins on a 8% SDS polyacrylamide gel under denaturing conditions. Perform immunoblotting with mouse monoclonal anti-phopsho STAT5. Strip the blots and reprobed with a rabbit polyclonal anti-SATA5 antibody. Visualize the bands using ECL reagents according to the supplier&#8217;s recommendation.</li>
</ol>
5. In Vivo Validation
<ol>
	<li>Transduce BAF3 cells expressing luciferase and cherry (transduced with retroviruses made from pMSCV-Luc-cherry GW) with viruses expressing JAK2-V617F and resistant variants. Select cells that survive puromycin selection for the expression of JAK2 and its resistance variants.</li>
	<li>Inject two million actively growing BAF3-Luc/cherry cells carrying JAK2-V617F and its resistant variants in 200 &#181;l of PBS to 6 Balb/C mice via tail-vein injection.</li>
	<li>After three days of cell transplantations, inject the mice with Ruxolitinib (100 mg/kg) twice daily for two weeks.</li>
	<li>Perform luciferase-based bioluminescence imaging with an imaging system. .
	<ol>
		<li>In brief, prepare luciferin solution by dissolving 300 mg to 25 ml of sterile deionized water, aliquot in smaller volumes and store. 
		NOTE: Store luciferin at -80 &#176;C and protect from light until use.</li>
		<li>Inject 250 &#181;l to each mouse by IP to achieve 125 mg/kg in each mouse. Anesthetize the mice from same group together using inhaled isoflurane (1.5%-2.0%) in a sealed box. Apply vet ointment on the eye to prevent dryness. 
		NOTE: The time taken for mice to sleep (10-15 min) allowed luciferin activity to peak. Keep the time consistent between the groups to avoid variation.</li>
	</ol>
	</li>
	<li>Transfer the mice immediately to imaging chamber stage and maintain anesthesia at 1.5-2% isofluorane during imaging procedures. Image mice with sequential 0.1, 0.5, 1, 5, 30, 60, and 300 sec exposure. Capture images and quantitate bioluminescence intensity using image acquisition and analysis software. Following imaging return the mice back to its cage.</li>
	<li>For, in vivo chimerism harvest total bonemarrow cells. Euthanize the mice with CO2 for 5 min followed with cervical dislocation. Harvest the bones and crush in 4 ml cold PBS to collect the bone marrow. Analyze the percent cherry positive cells under fluorescence microscope and quantify using FACS. Collect twenty thousand events from each sample.</li>
</ol>

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No conflicts of interest declared.

The clinical success of Imatinib in treating CML demonstrated not only the potential of targeting the rouge kinases by small molecule inhibitors, but also uncovered limitations of targeted therapy: clinical relapse and emergence of drug resistance mutations in the target gene. Identification of resistance mutations helps in better clinical management and development of next-generation inhibitors. This protocol describes a methodology to identify drug-resistant mutations in the targeted gene. This method uses a randomly m...

Protein kinases are key regulatory enzymes of intracellular signal transduction pathways that seemingly modulate every cellular function. A proper control of kinase mediated signaling is crucial to homeostasis and development, which mostly relies on proper regulation of kinases, phosphatases and its degradation by UPS (ubiquitin proteasome system). Deregulated kinases are at the center stage of many cancers and implicated in host of human diseases 1. Human genome encodes more than 500 protein kinases that have been linked, directly or indirectly, to ~400 human diseases 2. These observations supported the concept for therapeutic targeting of kinases by small molecule inhibitors 3-5.
The demonstration of ABL kinase inhibitors, such as Imatinib, in the treatment of chronic myeloid leukemia (CML) provided the proof of concept for this approach6,7. This observation not only revolutionized the anti-kinase therapy but also enforced the idea to identify the genetic lesions in other neoplastic diseases for therapeutic targeting, which lead to discovery of oncogenic mutations in the JAK2 from polycythemia vera (PV) and patients with myeloproliferative neoplasms (MPN). This discovery generated great interest in treating MPNs by targeting JAK2 with small molecule kinase inhibitors. Now, almost a dozen of JAK2 inhibitors are in clinical trials and one of them has been approved recently for the treatment of myelofibrosis. While specific targeting of oncogenic kinases by small molecule inhibitors in cancers bring promising outcome, it also suffers from developing resistance to the treatment. In fact, so far, patients treated with kinase inhibitors, such as Imatinib, Gefitinib, Erlotinib and Dasatinib developed resistance mutations mostly by acquiring mutations in the kinase domain to which drug targets 8-10. Resistance as a result of gene mutation highlights the limitations of targeted monotherapy against the oncogenic kinases, and represents the next challenge in the development of ever more successful cancer chemotherapy. Mechanistic and functional consequences of drug resistance should provide a rationale for selection and design of complimentary compounds for drug development. Mutations identified via in vitro screens, have shown a high degree of correlation with those found in patients. Therefore, in vitro screening for mutations that confer drug-resistance for a given drug target pairs in clinical or preclinical development assists in identifying the resistance patterns that are likely to cause clinical relapse. The identification of these mutant forms is not only helpful in monitoring the patients for drug response and relapse but also essential for the design of more robust next generation inhibitors. For instance, development of next generation BCR/ABL inhibitors, Nilotinib and Ponatinib, were made possible because of greater mechanistic understandings gained from mutagenesis, structural, and functional studies.
Earlier, we have reported the results of our screen using random mutagenesis of BCR/ABL to reveal the spectrum of mutations conferring resistance to inhibitors such as Imatinib11,12, PD16632612, and AP2416313. The results not only identified the mutations conferring clinical resistance and disease relapse, but also provided the mechanistic understanding of drug resistance and principles governing the kinase function11,14. Here we provide additional methodological detail, using Ruxolitinib and JAK2 as a drug target pair, to enable a broader application of this screening strategy.

<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

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 potential of targeting the kinase in cancers by effectively treating the CML patients. This observation revolutionized drug development to target the oncogenic kinases implicated in various other malignancies, such as, EGFR, B-RAF, KIT and PDGFRs. However, one major drawback of anti-kinase therapies is the emergence of drug resistance mutations rendering the target to have reduced or lost affinity for the drug. Understanding the mechanisms employed by resistant variants not only helps in developing the next generation inhibitors but also gives impetus to clinical management using personalized medicine. We reported a retroviral vector based screening strategy to identify the spectrum of resistance conferring mutations in BCR/ABL, which has helped in developing the next generation BCR/ABL inhibitors. Using Ruxolitinib and JAK2 as a drug target pair, here we describe in vitro screening methods that utilizes the mouse BAF3 cells expressing the random mutation library of JAK2 kinase.

Emergence of genetic mutations poses great challenge for the targeted anti kinase therapy. Mutational studies, besides providing mechanistic and functional insights that are instrumental in selection and design of next-generation drug development, also allows better clinical management and may in future be more helpful for personalized treatment. In this experiment, we show screening for ruxolitinib resistance mutations in JAK2-V617F kinase (Figure 1). We constructed pMSCV-JAK2-V617F-cherry.gateway vecto...

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

Emergence of genetic resistance against kinase inhibitor therapy poses significant challenge for effective cancer therapy. Identification and characterization of resistant mutations against a newly developed drug helps in better clinical management and next generation drug design. Here, we describe our protocol for in vitro screening and validation of resistant mutations.

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 ...

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JoVE Journal

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14.8K Views.  Cincinnati Children's Hospital Medical Center. Emergence of genetic resistance against kinase inhibitor therapy poses significant challenge for effective cancer therapy. Identification and characterization of resistant mutations against a newly developed drug helps in better clinical management and next generation drug design. Here, we describe our protocol for in vitro screening and validation of resistant mutations.

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.

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

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.

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.

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.

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.

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.

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

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.

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.


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

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that involves the construction, cloning and expression of mutant libraries, coupled to high frequency homologous DNA recombination in vivo. Here, we present a protocol to create and screen mutant libraries in yeast based on the example of a fungal aryl-alcohol oxidase (AAO) to enhance its total activity. Two protein segments were subjected to focused-directed evolution by random mutagenesis and in vivo DNA recombination. Overhangs of ~50 bp flanking each segment allowed the correct reassembly of the AAO-fusion gene in a linearized vector giving rise to a full autonomously replicating plasmid. Mutant libraries enriched with functional AAO variants were screened in S. cerevisiae supernatants with a sensitive high-throughput assay based on the Fenton reaction. The general process of library construction in S. cerevisiae described here can be readily applied to evolve many other eukaryotic genes, avoiding extra PCR reactions, in vitro DNA recombination and ligation steps.

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

We present a detailed protocol to construct and screen mutant libraries for directed evolution campaigns in Saccharomyces cerevisiae.

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that ...

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.

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