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的潮湿气氛中的细胞在37℃。 </li></ol> 2.质粒构建<ol><li>克隆小鼠JAK2及其致癌亚型JAK2-V617F中的pMSCV-PURO-GW通过LR使用标准重组克隆过程克隆酶。 </li><li>对于荧光素酶表达载体（的pMSCV -吕克-樱桃-GW）用于建造在-VI沃成像，使用引物（LUC-SD / RP TTACAATTTGGACTTTCCG和LUC-SD / FP-CACCATGGAAGACGCCAAAAAC），随后用克隆在载体pENTR-SD-TOPO生成载体pENTR-吕克，其用于通过PCR扩增来自pLVX-TET-ON载体的萤光素酶通过使用LR克隆酶介导的重组克隆转移荧光素酶基因的逆转录病毒载体，的pMSCV樱桃-GW。 </li></ol> 3.准备随机突变库，筛选和鉴定的突变 3.1）随机突变<ol><li>克隆全长的JAK2野生型和V617F突变成的pMSCV-PURO-GW逆转录病毒载体使用市售的重组克隆技术。 </li><li>使用1 JAK2-V617F质粒DNA微克改造，XL-1红，E.大肠杆菌细胞，这是有缺陷的DNA修复机制从而使他们能够在复制期间掺入随机突变。更具体地说，混合50 - 100纳克的DNA（超过100毫微克的DNA降低转化效率）和100微升感受态细胞中预冷的聚丙烯管中，并在冰上孵育30分钟，轻轻摇动，每5分钟。一个良好的库覆盖，使用感受态细胞的四到六管中。 注：超过100毫微克DNA的降低转化效率</li><li>浸入管在水浴中于42℃45秒的热冲击，并在冰上孵育2分钟。接着，加入1 ml的SOC培养基（2.0％胰蛋白胨，0.5％酵母提取物，0.05％的NaCl，10mM的氯化镁，10mM的硫酸镁和0.4％D-葡萄糖）。孵育所述管在37℃下以225-250转摇动90分钟。 </li><li>板细胞上410厘米的LB琼脂平板上含有100微克/毫升氨苄青霉素的每个管中。孵育板16 - 24小时，在37℃下。 </li><li>下面可见菌落，通过刮板用无菌板刮板收集它们。池单元从用市售的质粒提取试剂盒各板及分离质粒DNA。 注：通常菌落较小，并采取18-24小时是可见的，因为这些都是生长缓慢的细菌菌株。在此阶段，评估在库突变通过限制性消化的异质用频繁切割器如Sau3A1或Taq1或测序。 </li></ol>逆转录病毒上清转导和3.2）生产<ol><li>前一天的转染，板4×10 6的HEK 293T细胞在含有10％FCS，青霉素/链霉素和2mM L-谷氨酰胺100厘米菜肴的DMEM。 </li><li>第二天，更换培养基和转染细胞诱变的pMSCV-JAK2-V617F库。对于每个10cm平板，混合10微克的DNA（5微克质粒文库和5微克逆转录病毒包装质粒，pCLEco）与40微升的FuGENE对使用无血清DMEM培养基加入400μl的总体积。孵育DNA和脂质混合20分钟，在室温下进行。 20分钟后，添加明智的DNA-脂质复合物滴在HEK293T细胞的顶部。 </li><li> 6小时后，改转染媒体瓦特第i个新鲜培养基并孵育板48小时，在37℃下进行病毒生产。 48小时后培养，收集通过0.45μm的acrodisc的过滤器过滤的病毒上清。 </li></ol> 3.3）选择抗性克隆的体外 <ol><li>对于耐药JAK2-V617F突变体，转导亿BAF3用100ml具有0.5-1×10 6病毒滴度的病毒上清的细胞。更具体地说，混合10 8个细胞用100ml的病毒上清以使用RPMI培养基300毫升的总体积，含有10％血清和polyberene的24微克/毫升（ployberene的终浓度为8微克/毫升）。发布这些细胞搅拌器在多个6孔板（4-5毫升，每孔）。 </li><li>离心板在1250×g离心90分钟，在25℃。离心后，孵育所述板在37℃下24小时。 </li><li>第二天，凝聚细胞一起，并与Ruxolitinib和介质含有软琼脂混合并铺在SIX孔板中。对于每一个药物浓度，混合10毫升转导的BaF3细胞（10-20个细胞）与Ruxolitinib在50ml管适量的。补足体积至用含有20％血清的RPMI 40毫升在6孔板中加入10 mL 1.2％琼脂的细胞，mixcarefully和板。 </li><li>孵育所述板在37℃下两周。挑用0.2毫升吸管尖端和亚培养它们分别在24孔板3-4天的抗性菌落。 </li><li>通过离心收获抗性的BaF3细胞在450×g离心，在室温下5分钟。隔离使用市售的基因组DNA分离试剂盒的基因组DNA。 PCR扩增全长的JAK2基因从100ng的基因组DNA使用引物（mJAK2FP-ATGGCCTGCCTTACAATGACAG和mJAK2RP-GGTTCTGGAAGTCTGCTTGG）和长模板扩大高保真PCR系统。 </li><li>分离的PCR产物通过琼脂糖凝胶电泳。切除的DNA条带的3.4 kb的代表JAK2编码框架和隔离DNA使用市售的凝胶提取试剂盒。序列的cDNA的使用8个内部引物（P1 - P8）全长跨越的编码序列，并分析序列使用商业软件。 </li></ol>耐药突变的4 体外验证许多细胞克隆携带一个​​以上的突变，为了测试在抗性表型的每个突变的贡献，产生所选的变体通过使用的pMSCV-JAK2V-617F质粒为模板位点定向诱变。 <ol><li>执行使用商业突变试剂盒和设计创造的点突变寡核苷酸的pMSCV-JAK2-V617F质粒定点突变。通过测序证实突变克隆的身份。 </li><li>转导的BaF3细胞与逆转录病毒用突变质粒，随后用选择使用嘌呤霉素在1微克/毫升制成。 </li><li>板10 4 BAF3-JAK2-V617F细胞（50微升的RPMI 10％FCS）中加入96孔板的每个孔中。另LY添加50微升含ruxolitinib媒体的横跨板的孔中（最终浓度：0，1，3，5，10，和20微米），并孵育板60小时，在37℃。 </li><li>通过加入10微升的WST-1试剂向每个井与在37℃下进行2-4小时，评估细胞生存力。在A450用酶标仪记录吸光度。完成所有实验一式三份和情节均对吸收INCB018424浓度。执行使用非线性曲线拟合算法的最佳拟合S形曲线。得分导致50％细胞生存力的细胞的IC 50中的药物浓度。 </li><li>接着，板6000000 BaF3细胞表达JAK2变种在6孔板中含有10％血清的增加ruxolitinib的浓度和在37℃下温育4-6小时的RPMI培养基。 </li><li>收集细胞，离心，在450×g离心在4℃下5分钟。在20mM Tris-CL一次用PBS，并裂解洗涤细胞（pH7.5）中，50mM氯化钠，1％NP-40，0.1％SDS，5mM的EDTA，1mM的EGTA，1mM的氟化钠，2mM的钒酸钠，和5％的甘油补充有蛋白酶抑制剂混合物和磷酸酶抑制剂。超声处理的细胞悬浮液1分钟，随后加入5倍的凝胶上样缓冲液（350毫摩尔Tris盐酸[pH为6.8]，500mM的DTT，15％SDS的10mM苯甲脒，5mM的EDTA，5mM的EGTA，5mM的钒酸钠，蛋白酶鸡尾酒，50％甘油，和0.001％溴酚蓝）和变性在70℃下5-10分钟。 </li><li>解决在8％SDS聚丙烯酰胺凝胶中的蛋白质变性条件下。执行与鼠单克隆抗phopsho STAT5免疫印迹。剥离印迹，并再次探测与兔多克隆抗SATA5抗体。使用可视化ECL试剂根据供应商的建议波段。 </li></ol> 5. 在体内验证<ol><li>转导BAF3细胞表达荧光素酶和樱桃（转导自体pMSCV - 吕克 - 雪儿做逆转录病毒RY GW）病毒表达JAK2-V617F和抗药性的变种。选择细胞存活嘌呤选择用于JAK2和其电阻变体的表达。 </li><li>注入经尾静脉注射携带JAK2-V617F及其耐药变异体在200μl的PBS中，以6只Balb / C小鼠2000000活跃生长BAF3吕克/樱桃细胞。 </li><li>后细胞移植的三天，每天两次两周注射小鼠Ruxolitinib（100毫克/千克）。 </li><li>与成像系统执行的荧光素酶为基础的生物发光成像。 。 <ol><li>简言之，通过将300毫克至25ml无菌去离子水，分装在较小体积和存储制备荧光素溶液。 注：储存荧光素在-80℃下并避光保护直到使用。 </li><li>注入250μl到每只小鼠通过IP来实现在每只小鼠125毫克/公斤。麻醉从一起使用，在密封箱吸入异氟烷（1.5％-2.0％），同一组中的小鼠。适用于日兽医药膏Ë眼睛，防止干涩。 注：采取小鼠的睡眠时间（10-15分钟）使荧光素活性峰值。保持时间的组之间是一致的，以避免变形。 </li></ol></li><li>马上转移的小鼠成像室阶段，在成像过程维持麻醉在1.5-2％异氟醚。图像小鼠顺序0.1，0.5，1，5，30，60和300秒的曝光。捕捉图像，并使用图像采集和分析软件定量生物发光强度。继成像返回老鼠回到笼子里。 </li><li>为， 在体内嵌合收获总骨髓细胞。安乐死的小鼠用CO 2进行5分钟，随后用颈椎脱位。收获的骨头和4毫升粉碎冷PBS以收集骨髓。分析荧光显微镜下的百分樱桃阳性细胞和量化使用FACS。收集从每个样品两万事件。 </li></ol>

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No conflicts of interest declared.

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>
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		<tr height="21">
			<td height="21" style="height:21px;">HEK293T cells</td>
			<td>ATCC</td>
			<td></td>
			<td></td>
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		<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>
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		<tr height="21">
			<td height="21" style="height:21px;">pMSCV-JAK2-V617F/L983F.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-JAK2-V617F/P58A.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-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

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 ...

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

Research

JoVE Journal

Biology

14.8K 次观看.  Cincinnati Children's Hospital Medical Center. 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。 

视频: 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.

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

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.

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

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.

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

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.

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

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.

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

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 ...

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.

从血液高效的iPS细胞生成使用附加体和HDAC抑制剂

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.

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

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.

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 ...

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.

使用基于比色细胞的测定法检测针对 AAV 的中和抗体的分步方法

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