Name: a method for screening and validation of resistant mutations against kinase inhibitors
Uploaded: 2014-12-07T15:00:00
Description: Watch this Scientific Journal Video about A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors at JoVE.com

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.

注意：在这个协议中的所有程序都根据对动物伦理治疗和护理健康准则国家研究所进行的，并且根据批准的IA​​CUC动物使用的协议。 1.细胞的维护<ol><li>培养BaF3细胞在RPMI-1640培养基补充有10％胎牛血清和青霉素/链霉素（100单位/ ml和100μg/ ml）和WEHI细胞的废培养基。生长HEK293T细胞在DMEM补充有10％胎牛血清和青霉素/链霉素（100单位/毫升和100微克/毫升）。保持在含有5％CO 2的?...

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伊马替尼的治疗慢性粒细胞白血病的临床成功证明了小分子抑制剂靶向的胭脂激酶不仅潜力，但靶向治疗还发现限制：临床复发和耐药性的突变的靶基因中的出现。耐药突变鉴定有助于更好的临床管理和下一代抑制剂的发展。这个协议描述了一种方法，以确定在目标基因的耐药突变。此方法使用内置在大肠杆菌中随机诱变的质粒文库大肠杆菌 ，表达的突变体蛋白。库，然后在BaF3细?...

蛋白激酶是细胞内的信号转导通路的调节似乎每个细胞功能调控的关键酶。激酶介导的信号的适当控制是至关重要的稳态和发展，这主要是依赖于激酶，磷酸酶及其降解适当调控UPS（泛素蛋白酶体系统）。放松管制的激酶是许多癌症的中心舞台，并牵连到主机人类疾病1。人类基因组编码超过500蛋白激酶已被链接，直接或间接地，以400〜人类疾病2。这些观察结果支持这一概念用于激酶的小分子抑制剂3-5治疗靶向。 ABL的激酶抑制剂，如伊马替尼的论证，慢性粒细胞白血病（CML）的治疗提供了概念证明这种方法6,7。这一观察，不仅彻底改变了蚂蚁的i-激酶疗法而且执行的想法，以确定遗传病变的其他肿瘤疾病的治疗为目标，这导致发现的致癌基因突变的从真性红细胞增多症（PV）和患者的骨髓增生性肿瘤（MPN）的JAK2。这一发现产生的靶向JAK2小分子激酶抑制剂治疗MPNS极大的兴趣。现在，JAK2抑制剂几乎一打的临床试验，其中一人已被批准用于骨髓纤维化的治疗。而致癌激酶在癌症的小分子抑制剂特异性靶向携带有为结果如何，还患有显影抵抗的治疗。实际上，到目前为止，患者大多通过获得突变的激酶域的药物靶向8-10治疗激酶抑制剂，如伊马替尼，吉非替尼，埃罗替尼和达沙替尼抗药性的突变。阻力基因突变的结果凸显了限制Ø˚F靶向单一疗法对抗致癌激酶，并且表示在以往更成功的癌症化学疗法的发展下一个挑战。耐药机理和功能的后果应提供选择和药物开发的免费化合物的设计理念。通过在体外筛选鉴定的突变，都表现出与在患者中发现的相关程度高。因此， 在体外筛选赋予识别的电阻图案，有可能导致临床复发耐药在临床或临床前开发协助一个给定的药物靶标对突变。这些突变体形式的识别是不仅在监测患者对药物的反应和复发，还必需为更健壮的下一代抑制剂的设计有帮助的。例如，下一代BCR / ABL抑制剂，尼罗替尼和Ponatinib的发展，分别因为更大MEC成为可能hanistic理解的突变，结构和功能的研究中获得。 此前，我们曾报道我们的屏幕采用随机突变BCR / ABL的揭示突变赋予抗抑制剂如伊马替尼11,12，PD166326 12和AP24163 13的频 ​​谱的结果。结果不但确定授予临床耐药和疾病复发的基因突变，但也提供了耐药性和管理的激酶功能11,14原理的机械理解。在这里，我们提供额外的方法的细节，使用Ruxolitinib和JAK2作为药物靶标对，使这一筛选策略的更广泛的应用。

<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>
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		<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>
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		<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

BCR / ABL的发现作为驱动基因在慢性骨髓性白血病（CML）导致了伊马替尼，其中，实际上，证明有效地治疗慢性粒细胞白血病患者靶向激酶在癌症的潜力的发展。这一观察革命性的药物开发目标牵连其他各种恶性肿瘤，如，表皮生长因子受体，B-RAF，KIT和PDGFRs致癌激酶。然而，抗激酶疗法的一个主要的缺点是耐药突变渲染目标的出现，已经减弱或消失亲和性的药物。了解了耐药变异不仅有助于开发下一代抑制剂也采用个性化医疗带动了临床管理使用的机制。我们报道了逆转录病毒载体基于筛选策略来识别抗性赋予在BCR / ABL，突变这在开发下一代BCR / ABL抑制剂帮助的频谱。使用Ruxoli替尼和JAK2作为药物靶标对，在这里我们将介绍在利用表达JAK2激酶的随机突变库鼠标BAF3细胞的体外筛选方法。

基因突变的出现带来了针对性的抗激酶治疗的巨大挑战。突变的研究，除了提供那些有助于选择和下一代药物开发的机械设计和功能的见解，也可以更好地临床管理，可能在未来成为个性化的治疗更有帮助。在这个实验中，我们显示筛选ruxolitinib抗性突变JAK2-V617F激酶（ 图1）。我们构建了通过引入全长小鼠JAK2-V617FcDNA成的pMSCV樱桃-GW的pMSCV-JAK2-V617F-cherry.gateway载体。它建议使用的细菌宿?...

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

针对激酶抑制剂治疗遗传性的出现带来了有效的癌症治疗显著的挑战。对新开发的药物鉴定和耐药突变特性有助于更好的临床管理和下一代药物设计。在这里，我们描述了体外筛选及耐药突变的验证协议。

对于耐药性突变对激酶抑制剂的筛选和验证方法

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 ...

Research

JoVE Journal

Biology

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

注射的。 stephensi胚胎生成抗药性疟疾的蚊子

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

科研

生物学

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

一个线虫多巴胺神经元退化含量的应用验证潜在的帕金森氏症基因

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

MODS的方法和诊断结核病耐药结核病

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

Assaying the Kinase Activity of LRRK2 in vitro

含量测定的LRRK2的激酶活性在体外</em

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

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

转换到一个点阵仪XMAP含量，使用多重抗体筛查方法的捕获ELISA

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

RNA催化剂作为记者为筛选药物对RNA编辑在锥虫

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

高通量筛选转录后调控基因的化学调节剂

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

激酶底物对鉴定使用高通量筛选


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

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

小鼠模型的验证扰乱LINC配合物在细胞特异性的方式

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

Interactome-Seq: A Protocol for Domainome Library Construction, Validation and Selection by Phage Display and Next Generation Sequencing

Interactome: Domainome 图书馆建设、噬菌体展示和下一代测序的验证与选择协议

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to ...