Name: methods for evaluating the role of c-fos and dusp1 in oncogene dependence
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence at JoVE.com

作者感谢 g. q. dley 为 mscv-bcr-abres-yefp 结构提供了 baf3 和 wehi 细胞和 t. reya。作者感谢 m. carroll 提供了 cml 爆炸危机的病人样本。这项研究得到了 nci (1ro1ca155091)、白血病研究基金会和 v 基金会以及 nhlbi (R21HL114074-01) 向硕士提供的赠款的支持。

所有动物实验都是根据辛辛那提儿童医院医疗中心 (cchmc) 动物护理和使用机构委员会 (iacuc) 的指导方针进行的。人类标本 (正常 bm 和 cml (p210-bcr-abl +) 白血病) 的样本是通过机构审查委员会批准的协议 (机构审查委员会: 全联邦保证 #00002988 辛辛那提儿童医院医疗中心) 获得的;得到 cchmc 和辛辛那提大学的捐助方知情同意。
1. 实时 qpcr 分析
<ol><li>从 rpmi 中生长的 bf3 细胞中分离总...

<ol>
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Qiagen-miRNAeasy kit Available from: <a target="_blank" href="https://www.qiagen.com/us/shop/sample-technologies/rna/mirna/mirneasy-mini-kit/#resources">https://www.qiagen.com/us/shop/sample-technologies/rna/mirna/mirneasy-mini-kit/#resources</a> (2018)</li><li>. DNA-free DNA removal kit Available from: <a target="_blank" href="https://www.thermofisher.com/order/catalog/product/AM1906?gclid=EAIaIQobChMIg4D-_ZvC2wIVx5-zCh00Cg8fEAAYASAAEgIv6vD_BwE&amp;s_kwcid=AL!3652!3!264318446624!b!!g!!&amp;ef_id=V7SO5AAAAck3ba89:20180607185004:s">https://www.thermofisher.com/order/catalog/product/AM1906?gclid=EAIaIQobChMIg4D-_ZvC2wIVx5-zCh00Cg8fEAAYASAAEgIv6vD_BwE&amp;s_kwcid=AL!3652!3!264318446624!b!!g!!&amp;ef_id=V7SO5AAAAck3ba89:20180607185004:s</a> (2018)</li><li>. SuperScript&#8482; III First-Strand Synthesis System Available from: <a target="_blank" href="https://www.thermofisher.com/order/catalog/product/18080051">https://www.thermofisher.com/order/catalog/product/18080051</a> (2018)</li><li>. 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对于大部分癌细胞, 对 tki 的治疗反应是通过阻断肿瘤所依赖的酪氨酸激酶-onco蛋白信号来介导的。然而, 对少数促成 mrd 的癌细胞如何摆脱癌基因依赖和治疗的了解相对较少4。最近的研究表明, 生长因子信号介导白血病和实体器官肿瘤的耐药性。这表明, 各种分子机制可能是内在阻力10、11、12 的基础。为了了?...

bcr-abl1 融合癌基因的本构酪氨酸激酶活性引起 cml, 为小分子抑制剂靶向激酶活性提供了依据。tki 在治疗 cml 患者方面的成功彻底改变了靶向治疗的概念1,2。随后, 抗激酶治疗作为精确的医学被开发了为其他几个恶性肿瘤, 包括实体肿瘤。到目前为止, 已有30多种激酶抑制剂被美国 fda 批准用于治疗各种恶性肿瘤。虽然 tki 治疗对抑制疾病非常有效, 但并不治疗。此外, 在治疗过程中, 少量癌细胞持续存在: mrd3、4、5。即使是表现出完全缓解的患者, 也会留下 mrd, 如果不持续抑制, 最终会导致复发。因此, 需要根除 mrd 细胞, 以实现持久或治疗性的反应。cml 代表了一个有价值的范式, 用于定义精确医学的概念、肿瘤发生的机制、合理的靶向治疗、疾病进展和耐药性。然而, 即使在今天, 导致 tki 诱导的癌细胞死亡的机制还没有完全了解, 也没有完全了解为什么 mrd 细胞 (由白血病干细胞组成) 对 tki4,6具有内在的耐药性。然而, "癌基因依赖" 突变激酶蛋白的现象与 tki 的有效性有关, tki 对靶向癌基因的急性抑制会导致致癌休克, 导致细胞出现大规模的原凋亡反应或静止上下文相关的方式6,7,8,9。然而, 癌基因依赖的机械基础是缺乏的。最近的研究表明, 生长因子信号减少了癌基因的依赖性, 从而导致抵抗 tki 治疗10,11,12。因此, 为了深入了解癌基因依赖的机制, 我们从 bcr-abl1 成瘾和非成瘾细胞 (随生长因子生长) 进行了全基因组表达谱分析, 揭示了 c-fos 和 dusp1 是犯罪基因依赖的关键介质。癌基因成瘾13。c-fos 和 dusp1 的遗传缺失对 bcr-abl1 表达细胞是合成致死的, 实验中使用的小鼠没有发生白血病。此外, 小分子抑制剂对 c-fos 和 dusp1 的抑制作用, 治愈了 bcr-abl1 诱导的小鼠 cml。结果表明, c-fos 和 dusp1 的表达水平定义了癌细胞的凋亡阈值, 因此较低的水平会产生药物敏感性,而较高的水平会导致对治疗的抵抗力13。
为了确定导致癌基因依赖性的基因, 我们在生长因子存在的情况下进行了几次全基因组表达分析实验, 并使用了鼠肉和 cml 患者衍生细胞 (k562)。这些数据与 cml 患者数据集在伊马替尼治疗前后从 cd34+造血干细胞中获得的数据进行了并行分析。这一分析揭示了三个基因 (转录因子 [c-fos]、双特异性磷酸酶 1 [dusp1] 和 rna 结合蛋白 [zfp36]) 通常在 tki 耐药细胞中被凝聚。为了验证这些基因在赋予耐药方面的意义, 我们进行了体外和体内的逐步分析。通过实时 qpcr (rt-qpcr) 和耐药细胞中的西方印迹证实了这些基因的表达水平。此外, cdna 过度表达和击倒的 shrna 发夹的 c-fos, dusp1 和 zfp36 揭示了升高的 c-fos 和 dusp1 表达是足够和必要的, 以赋予 tki 的抵抗力。因此, 我们仅使用 c-fos 和 dusp1 的小鼠模型进行了体内验证。为了对 c-fos 和 dusp1 进行基因验证, 我们创造了 rosacreert 诱导的 c-fosf函(条件淘汰赛)14 , 并与 dusp1-p/(直接淘汰赛)15交叉, 使 rosacreert2-c-fosfl双转基因 小鼠。在一个菌落形成单元 (cfu)中, 对表达 bcr-abl1的骨髓衍生的 c-kit + 细胞 (来自 c-fos fl/fl-/---和 c-fos fusp1-\-) 进行了体外分析, 活体骨髓移植在小鼠体内, 单独或共同检测白血病发展中对 c-fos 和 dusp1 的要求。同样, 用 bcr-abl1 表示, 用 bcr-abl1表达, 用 dfc (二氟姜黄素) 16 和 dusp1 对 c-fos 的化学抑制, 用 bci (苯并二己胺)-2 , 3-二氢-1h-1) 17 在体外和体内进行了测试。来自野生类型 (wt) 鼠标的骨髓衍生的 c-kit+细胞。为了确认白血病干细胞中对 c-fos 和 dusp1 的要求, 我们利用 cml 小鼠模型, 通过多西环素 (tet-转动剂在小鼠干细胞白血病 (scl) 基因 3 ' 基因下表达) 在其干细胞中特异性诱导 bcr-abl1章程)18,19。我们在体内移植试验中使用了这些小鼠的骨髓林-sca+c-kit+ (lsk) 细胞。此外, 我们还建立了 phopsho-p38 水平和 il-6 的表达, 分别作为药物动态生物标志物,分别用于 dusp1 和 c-fos 的抑制。最后, 为了扩大这项研究对人类的相关性, 患者衍生的 c-Kit+细胞 (相当于小鼠的 c-kit+细胞) 接受了长期的体外培养细胞检测 (ltcic) 和体内人性化的小鼠模型。cml20,21。免疫缺陷小鼠移植 cml cd34 + 细胞, 其次进行药物治疗和人白血病细胞存活分析。
在这个项目中, 我们开发了使用遗传和化学工具进行目标识别和验证的方法, 使用不同的临床前模型。这些方法可以成功地应用于验证其他目标开发化学模式的治疗发展。

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			<td height="21" style="height:21px;width:424px;">Biological Materials</td>
			<td style="width:247px;"></td>
			<td style="width:147px;"></td>
			<td style="width:131px;"></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;">IMDM</td>
			<td>Cellgro (corning)</td>
			<td>15-016-CVR</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RetroNectin Recombinant Human Fibronectin Fragment</td>
			<td>Takara</td>
			<td>T100B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MethoCult GF M3434 (Methylcellulose for Mouse CFU)</td>
			<td>Stem Cell</td>
			<td>3434</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MethoCult H4434 Classic (Methylcellulose for Human CFU)</td>
			<td>Stem Cell</td>
			<td>4434</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-Hydroxytamoxifen</td>
			<td>Sigma</td>
			<td>H6278</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recombinant Murine SCF</td>
			<td>Prospec</td>
			<td>CYT-275</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recombinant Murine Flt3-Ligand</td>
			<td>Prospec</td>
			<td>CYT-340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recombinant Murine IL-6</td>
			<td>Prospec</td>
			<td>CYT-350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recombinant Murine IL-7</td>
			<td>Peprotech</td>
			<td>217-17</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DFC</td>
			<td>LKT Laboratories Inc.</td>
			<td>D3420</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BCI</td>
			<td>Chemzon Scientific</td>
			<td>NZ-06-195</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Imatinib</td>
			<td>LC Laboratory</td>
			<td>I-5508</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Curcumin</td>
			<td>Sigma</td>
			<td>458-37-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NDGA</td>
			<td>Sigma</td>
			<td>500-38-9</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="42">
			<td height="42" style="height:42px;">L-Glutamine</td>
			<td>Cellgro (corning)</td>
			<td>25-005-CL</td>
			<td style="width:131px;">5mg/ml stock in water</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;">HEPES</td>
			<td>Sigma</td>
			<td>H7006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4.7H2O</td>
			<td>Sigma</td>
			<td>S9390</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Protamine sulfate</td>
			<td>Sigma</td>
			<td>P3369</td>
			<td style="width:131px;">5mg/ml stock in water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan 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;">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;">FICOL (Histopaque 1083) (polysucrose)</td>
			<td>Simga</td>
			<td>1083</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Corning</td>
			<td>21040CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LS Columns</td>
			<td>Miltenyi</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease Inhibitor Cocktail</td>
			<td>Roche</td>
			<td>CO-RO</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphatase Inhibitor Cocktail 2</td>
			<td>Sigma</td>
			<td>P5762</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nitrocullulose Membrane</td>
			<td>Bio-Rad</td>
			<td>1620115</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:424px;">SuperSignal West Dura Extended Duration Substrate ( chemiluminiscence substrate)</td>
			<td>Thermo Scientific</td>
			<td>34075</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD5</td>
			<td>eBioscience</td>
			<td>13-0051-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD11b</td>
			<td>eBioscience</td>
			<td>13-0112-75</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD45R (B220)</td>
			<td>BD biosciences</td>
			<td>553092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD45.1-FITC</td>
			<td>eBioscience</td>
			<td>11-0453-85</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD45.2-PE</td>
			<td>eBioscience</td>
			<td>12-0454-83</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">hCD45-FITC</td>
			<td>BD Biosciences</td>
			<td>555482</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Biotin-FITC</td>
			<td>Miltenyi</td>
			<td>130-090-857&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-7-4</td>
			<td>eBioscience</td>
			<td>MA5-16539</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Gr-1 (Ly-6G/c)</td>
			<td>eBioscience</td>
			<td>13-5931-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Ter-119</td>
			<td>eBioscience</td>
			<td>13-5921-75</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ly-6 A/E (Sca1) PE Cy7</td>
			<td>BD&#160;</td>
			<td>558612</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD117 APC&#160;</td>
			<td>BD&#160;</td>
			<td>553356</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD Pharm Lyse</td>
			<td>BD&#160;</td>
			<td>555899</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD Cytofix/Cytoperm (Fixing and permeabilization solution)</td>
			<td>BD&#160;</td>
			<td>554714</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:424px;">BD Perm/Wash&#160; (permeabilization and wash solution for phospho flow)</td>
			<td>BD&#160;</td>
			<td>554723</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">phospho p38</td>
			<td>Cell Signaling Technologies</td>
			<td>4511S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">total p38</td>
			<td>Cell Signaling Technologies</td>
			<td>9212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse IgG control</td>
			<td>BD&#160;</td>
			<td>554121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Flour 488 conjugated&#160;</td>
			<td>Invitrogen</td>
			<td>A-11034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium Chloride</td>
			<td>Invitrogen</td>
			<td>K278001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2X HBS</td>
			<td>Invitrogen</td>
			<td>K278002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA</td>
			<td>Ambion</td>
			<td>AM9261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blood Capillary Tubes</td>
			<td>Fisher</td>
			<td>22-260-950</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blood Collection Tube</td>
			<td>Giene Bio-One</td>
			<td>450480</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Newborn Calf Serum</td>
			<td>Atlanta biological</td>
			<td>S11295</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Erythropoiein</td>
			<td>Amgen</td>
			<td>5513-267-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">human SCF</td>
			<td>Prospec</td>
			<td>CYT-255</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human IL-3</td>
			<td>Prospec</td>
			<td>CYT-210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">G-SCF</td>
			<td>Prospec</td>
			<td>CYT-220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GM-CSF</td>
			<td>Prospec</td>
			<td>CYT-221</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MyeloCult (media for LTCIC assay)</td>
			<td>Stem Cell Technologies</td>
			<td>5100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrocortisone Sodium Hemisuccinate</td>
			<td>Stem Cell Technologies</td>
			<td>7904</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM alpha</td>
			<td>Gibco</td>
			<td>12561-056</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>60316 and 60317</td>
			<td></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;">SOC</td>
			<td>New England Biolabs</td>
			<td>B90920s</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Ampicillin</td>
			<td>Sigma</td>
			<td>A0166</td>
			<td style="width:131px;">100mg/ml stock in water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto agar (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="105">
			<td height="105" style="height:105px;">Doxycycline chow</td>
			<td>TestDiet.com</td>
			<td>52662</td>
			<td style="width:131px;">modified RMH1500, Autoclavable 5LK8&#160; with 0.0625% Doxycycline&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tamoxifen</td>
			<td>Sigma</td>
			<td>T5648</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iodonitrotetrazolium chloride&#160;</td>
			<td>Sigma</td>
			<td>I10406</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;">GoTaq Green (taq polymerase with Green loadign dye)</td>
			<td>Promega</td>
			<td>M1722</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">miRNeasy Mini Kit&#160; (RNA isolation kit)</td>
			<td>Qiagen</td>
			<td>217084</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNA Free Dnase Kit (DNAse treatment for RT PCR)</td>
			<td>Ambion, Life Technologies</td>
			<td>AM1906</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:424px;">Superscript III First Strand Synthesis (reverse transcriptase for cDNA synthesis)</td>
			<td>Invitrogen</td>
			<td>18080051</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:424px;">SYBR Green (taq polymerase mix with green interchalating dye for qPCR)</td>
			<td>Bio-Rad</td>
			<td>1725270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD117 MicroBead Kit</td>
			<td>Miltenyi</td>
			<td>130-091-224</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human Long-Term Culture Initiating Cell Assay</td>
			<td>Stemp Cell Technologies</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 cetrifuge 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></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C-1000 Thermal cycler</td>
			<td>Bio-RAD</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mastercycler Real Plex 2</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:424px;">ChemiDoc Imaging System (imaging system for gels and western blots)</td>
			<td>Bio-RAD</td>
			<td>17001401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hemavet ( boold counter)</td>
			<td>Drew-Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LSR II ( FACS analyzer)</td>
			<td>BD&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fortessa I ( FACS analyzer)</td>
			<td>BD&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACSAriaII ( FACS Sorter)</td>
			<td>BD&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnet Stand</td>
			<td>Miltenyi</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Irradiator</td>
			<td style="width:247px;">&#160;J.L. Shepherd and Associates, San Fernando CA</td>
			<td>Mark I Model 68A</td>
			<td>source Cs 137</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ROSACreERT2</td>
			<td>Jackson Laboratory</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scl-tTA&#160;</td>
			<td>Dr. Claudia Huettner&#8217;s lab</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BoyJ&#160;</td>
			<td>mouse core facility at CCHMC</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C57Bl/6&#160;</td>
			<td>Jackson Laboratory</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NSGS</td>
			<td>mouse core facility at CCHMC</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">ROSACreERT2/c-Fosfl/fl Dusp1-/-&#160;</td>
			<td>Made in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ROSACreERT2/c-Fosfl/fl</td>
			<td>Made in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">BaF3</td>
			<td style="width:247px;">Gift from George Daley, Harvard Medical School, Boston</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">WEHI</td>
			<td style="width:247px;">Gift from George Daley, Harvard Medical School, Boston</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">CML-CD34+ and Normal CD34+ cells</td>
			<td style="width:247px;">University Hospital, University of Cincinnati</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

methods for evaluating the role of c-fos and dusp1 in oncogene dependence

酪氨酸激酶抑制剂 (tki) 在治疗慢性髓系白血病 (cml) 方面的示范预示着癌症治疗的一个新时代。然而, 少数细胞对 tki 治疗没有反应, 导致残留疾病最小;即使是最强大的 tki 也无法消灭这些细胞。这些 mrd 细胞作为对治疗产生抵抗力的水库。为什么 tki 治疗对 mrd 细胞无效尚不清楚。生长因子信号转导与 tki 治疗过程中支持 mrd 细胞存活有关, 但缺乏机械理解。最近的研究表明, 由于 mrd 细胞中收敛的致癌和生长因子信号, c-fos 和 dusp1 表达升高, 可以介导 tki 的抗性。c-fos 和 dusp1 的遗传和化学抑制使 cml 对 tki 非常敏感, 并在遗传和人性化的小鼠模型中治愈 cml。我们使用来自 tki 敏感和耐药细胞的多个微阵列确定了这些目标基因。在这里, 我们提供了在体外和体内使用小鼠模型进行目标验证的方法。这些方法可以很容易地应用于任何目标的遗传验证和治疗发展。

癌基因成瘾与 tki 的治疗效果有关。然而, 驱动癌基因依赖的机制并不清楚。我们进行了多次无偏见的基因表达分析, 以确定参与策划成瘾的基因成分。这些分析揭示了 c-fos、dusp1 和 zfp36 这三个基因在癌细胞中的上升, 这些基因不依赖于肿瘤信号的生存, 因此对 tki 治疗不敏感。由 shrna 介导的击倒降低 c-fos 和 dusp1 的调节, 足以恢复其他 tki 耐药细胞的药物敏感性。
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Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence

c-fos 和 dusp1 在癌基因依赖性中的作用评价方法

在这里, 我们描述了 c-fos 和 dusp1 作为白血病药物靶标的遗传和化学验证协议, 在体外和体内使用遗传和人性化的小鼠模型。该方法可应用于遗传验证和治疗发展的任何目标。

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, ...

这种方法可以帮助回答癌症治疗领域的关键问题，例如识别与基因依赖有关的关键基因。该技术的主要优点是，它利用许多激酶驱动的癌症常见的肿瘤依赖性，因此它适用于多种肿瘤类型。虽然这种方法可以提供白血病的洞察力，它也可以应用于其他系统，如实体肿瘤，如肺癌，脑肿瘤和胰腺肿瘤。一般来说，由于在分子生物学和细胞生理学方面缺乏经验，对这种方法的新鲜人将难以为之奋斗。首先，从安乐死小鼠中收获腿骨。用手术刀清洁骨头上的组织。然后把骨头放在冰冷的PBS上，然后用刺的刺压碎骨头。在此之后，通过 70 微米细胞滤网将电池悬浮液过滤到 50 毫升螺帽管中，并配有 10 毫升移液器。用冷 PBS 多次清洗砂浆和刺，并将细胞悬浮液添加到 50 毫升管中。接下来，用真空和玻璃移液器将骨髓离心并取出上流液。将细胞颗粒重新在5毫升PBS中。使用移液器，慢慢将悬浮骨髓添加到室温多蔗糖的顶部。在此之后，在关闭制动器时，以 400 G 旋转聚蔗糖 20 分钟。离心后，用移液器收集多云的完全单核细胞接口，并转移到新的15毫升管。使用 PBS 将体积达到 10 毫升，并取出 20 微升进行计数。离心后，丢弃上经剂，将颗粒放在冰上。接下来，用 EDTA 和 BSA 重新在 PBS 中重新暂停细胞颗粒。将 CD117 微珠添加到细胞悬浮液中，并加入涡流混合。在此之后，在4摄氏度下孵育10分钟的暂停。然后将体积高达 10 毫升，使用 EDTA 和 BSA，在 1200 G 下以 4 摄氏度的离心机使用更多 PBS，使用 5 分钟。使用真空去除上一级，并使用 EDTA 和 BSA 将 PBS 中的细胞颗粒悬浮。然后将 3 毫升 PBS 与 EDTA 和 BSA 添加到带磁柱的磁支架中，并允许其流入收集管。缓冲区完成流过列后，添加单元格悬浮液，并允许它流经柱进入收集管。接下来，在柱上再加入 5 毫升 PBS 和 EDTA 和 BSA，然后立即用柱塞冲洗。取出柱塞，在计数细胞之前重复冲洗。离心细胞后，取出上经剂，将细胞颗粒悬浮在完整的 IMDM 中，以进行培养。在37摄氏度下一夜之间用5%的二氧化碳培养细胞。首先，将逆转录病毒质粒DNA与包装质粒、氯化钙和水混合在1.5毫升管中。然后将500微升的HBS加入另一个1.5毫升的管中。将转染混合物滴入含有HBS的管子中，同时与涡流剂混合设置为最低设置。在室温下孵育转染混合物20至30分钟。在孵化期间，将氯奎因加入 HEK 细胞，并将其留在孵化器中。添加转染混合物后，在37摄氏度5%CO2培养箱中孵育细胞8小时，通过缓慢地加入14毫升新鲜DMEM10培养基来改变细胞介质。缓慢地对着盘子的壁移液有助于防止 HEK 细胞的任何剥离。然后在37摄氏度的5%CO2培养箱中孵育细胞过夜。使用10毫升注射器收集病毒上一道液。然后将 0.45 微米注射器过滤器连接到注射器上。将病毒上一种药物放入新的收集管中。大约16小时后收集病毒上一道液。在此之后，在PBS中加入两毫升重组的人类纤维素片段，加入未经处理的6个井培养板。第二天，使用移液器从盘子中去除纤维素。在 PBS 中用 2%BSA 将板块住，并在室温下孵育板 30 分钟。使用真空去除 BSA 并使用 PBS 清洗板，然后用真空清除板。在此之后，立即添加过滤的病毒上清液，并离心板两个小时。使用真空去除上一液，然后再次旋转板。接下来用真空去除上一代，用PBS清洗病毒涂层孔。用真空去除PBS，将先前培养的c-Kip加小鼠细胞添加到病毒涂层孔中。离心后，在37摄氏度和5%的二氧化碳下孵育细胞。转导后48小时，通过离心对细胞进行颗粒化，并在FACS缓冲液1中重新填充。首先在室温下用细胞因子解冻甲基纤维素。然后在FACS管中加入3毫升甲基纤维素。将100微升的细胞悬浮液与适当的抑制剂添加到3毫升甲基纤维素。然后将悬浮液高至高 10 到 30 秒。大多数气泡逸出后，使用 1 毫升注射器将 1 毫升介质板在三个复制板中。将盘子放在一个大的培养皿中，以及一个含有无菌水的开放式盘子。然后覆盖大培养皿，在37摄氏度下孵育细胞。通过溶解10毫升水中的10毫克氯化碘三极氯化物，准备碘三氯化物的污渍。使用配备 0.2 微米过滤器的 Luer 锁注射器对染色溶液进行消毒。在组织培养罩中，在CFU板周围添加100微升的染色溶液。然后在37摄氏度的细胞培养箱中孵育板。最后，在菌落变成深红色棕色后，使用无菌组织培养罩中的白色背景拍摄染色的CFU板的照片。在该协议中，使用了多种公正的基因表达分析来识别协调基因成瘾的遗传成分。为了验证c-Fos和Dusp1作为白血病治疗靶点的作用，用表达BCR-ABL1肿瘤基因的细胞确认了它们过度表达。通过RT-qPCR和西部印迹的分析证实，两者都是由BCR-ABL1引起的，并且它们的表情被生长因子IL-3增强。YFP 加表达细胞通过 FACS 排序，以进一步进行体外和体内测定。结果表明，仅删除 c-Fos 和 Dusp1 就抑制了 CFU 编号。缺乏 c-Fos 和 Dusp1 的细胞在形成菌落的能力方面受到显著损害，Imatinib 治疗消灭了所有 CDU，表明 c-Fos 和 Dusp1 的丧失对 BCR-ABL1 表达具有合成致命性。经过这一开发，这项技术为癌症领域的研究人员探索基因网络和基因驱动基因依赖，以及这些基因如何影响他们在小鼠模型和人类患者中的特殊反应铺平了道路。在执行此过程时，不要忘记使用逆转录病毒和患者样本可能极其危险和安全的实验室做法。

Research

JoVE Journal

Cancer Research

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

共培养化验，以研究胶质母细胞瘤入侵的巨噬细胞和小胶质细胞刺激

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

科研

癌症研究

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

该<em&gt;果蝇</em&gt;成虫盘瘤模型：基因的表达与肿瘤侵袭性的可视化和定量遗传马赛克

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

Evaluating In Vitro DNA Damage Using Comet Assay

Evaluating In Vitro DNA Damage Using Comet Assay

利用彗星试验评价体外DNA 损伤

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer ...

Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome

二维凝胶电泳结合质谱法分析人垂体腺瘤组织蛋白质组

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may ...

Evaluating the Role of Mitochondrial Function in Cancer-related Fatigue

线粒体功能在癌症相关疲劳中的作用评价

Fatigue is a common and debilitating condition that affects most cancer patients. To date, fatigue remains poorly characterized with no diagnostic test to ...

3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer

3维细胞培养系统研究膀胱癌侵袭和评价治疗方法

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. ...

Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods

使用一套实用可行的方法,描述英国皇家空军家族激酶与疾病相关的突变体

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental ...

Evaluating the Differentiation Capacity of Mouse Prostate Epithelial Cells Using Organoid Culture

利用有机体培养评价小鼠前列腺皮细胞的分化能力

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation ...

Integration of Bioinformatics Approaches and Experimental Validations to Understand the Role of Notch Signaling in Ovarian Cancer

生物信息学方法与实验验证的集成，了解诺奇信号在卵巢癌中的作用

Notch signaling is a highly conserved regulatory pathway involved in many cellular processes. Dysregulation of this signaling pathway often leads to ...

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

在结肠直肠癌细胞的三维球体培养物中使用无细胞益生菌超常剂评估细胞死亡

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using ...