Name: identification of egfr and ras inhibitors using caenorhabditis elegans
Uploaded: 2020-10-05T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans at JoVE.com

我们感谢斯瓦蒂·阿鲁尔博士（MD安德森癌症中心）提供了 让60（n1046）。我们还要感谢大卫·雷纳博士（休斯顿得克萨斯A&M健康科学中心生物科学与技术研究所）的 林-1 菌株。最后，我们感谢丹妮尔·加辛博士和她的实验室（德克萨斯大学麦戈文医学院）提供了一些试剂。一些蠕虫菌株由国家卫生研究院基础设施项目办公室（P40 OD010440）资助的CGC提供。这项研究得到了德克萨斯州癌症预防和研究所（CPRIT）向JF汉考克提供RP200047的支持。

1. 线虫生长介质 （NGM） 板准备
<ol><li>在 2 L 埃伦迈尔烧瓶中加入 2.5 克辣椒和 3 克 NaCl 到 970 mL 的去离子水。使用磁搅拌棒搅拌内容。此后，在烧瓶中加入20克醋。在 121 °C 和 15 磅/在2 的压力下将烧瓶的含量高压 30 分钟。消毒后，将烧瓶放在搅拌盘上，让中等温度冷却至 50 °C。</li>
	<li>要准备 NGM 板，请在冷却介质中添加以下试剂:25 mL 的 1 M 磷酸钾缓冲器 （pH = 6.0），1 mL 的 1 M MgSO4，1mL 的 1 M CaCl2，1 mL （5 毫克/毫升在 95% 乙醇） 胆固醇， 1 mL（乙醇中10%v/w）尼他汀，1 mL 25毫克/mL链霉素。</li>
	<li>在层压流下，将冷却的介质倒入 60 mm x 15 mm 无菌培养皿中。让板凝固 2 小时。这些板可以在4°C下保存1个月。</li>
</ol>2. 传播 C.埃莱根斯
<ol><li>将 100 μL 隔夜生长的 大肠杆菌 OP50 点到每个 NGM 板的中心，让板在层压罩中干燥 24 小时。随后，这些板可以储存在聚苯乙烯容器中。 注: 大肠杆菌 OP50培养在板播种前生长在卢里亚-贝尔塔尼 （LB）介质的37°C介质中。培养植物可以在4°C下储存1~2个月，用于板的定期播种。</li>
	<li>使用无菌蠕虫采摘，在解剖显微镜下从以前生长的盘子中收集 10/12 重力成年蠕虫。将这些蠕虫转移到新鲜的 NGM 板中，用 大肠杆菌 OP50 播种，并在 20 °C 下孵育板 24 小时。</li>
	<li>24小时后，使用无菌蠕虫采摘从板中去除成年蠕虫。在 20 °C 下孵育板 3 天。胚胎会发育成重力成年蠕虫。</li>
</ol>3. 准备同步 的 C. 埃莱根斯 文化
<ol><li>通过用 M9W 清洗 2 到 4 个盘子，将重力成年蠕虫收集到 15 mL 圆锥管中。<ol>
<li>M9W 的准备:溶解 5 克 NaCl，6 克 Na2HPO4，3克 KH2PO4在去离子水中溶解到最终体积为 1 L. 自动清洁溶液在 121 °C 在 15 磅/在2的压力 30 分钟。在冷却溶液中加入 1 mL 的 1 M MgSO4。</li>
	</ol>
</li>
	<li>通过在 450 x g 下离心 1 分钟来对蠕虫进行离心。在不干扰蠕虫颗粒的情况下对超强的超强体进行排泄。</li>
	<li>将 400 μL 的 8.25% 次氯酸钠（家用漂白剂）和 5 N NaOH 的 100 μL 组合在一起，准备蠕虫裂解溶液。将此溶液添加到蠕虫颗粒中，然后轻拂管子以混合管子中的内容。通过在解剖显微镜下观察蠕虫的裂解，防止管内胚胎过度出血。</li>
	<li>在锥形管中加入 10 mL 的 M9W，当 70% 的成年蠕虫裂解时稀释裂解混合物。</li>
	<li>以 450 x g 离心管 1 分钟。用 10 mL 的 M9W 替换超高纳特。</li>
	<li>重复步骤 3.5 两次。</li>
	<li>完成洗涤步骤后，加入 3/5 mL 的 M9W 以重新悬念蛋丸。在室温下（RT）以18转速在管旋转器上旋转管子过夜。</li>
	<li>过夜孵化后，从旋转器中取出管子，以 450 x g 离心 1 分钟离心将 L1 幼虫颗粒化。将 M9W 吸气至 250 μL 留在管中。</li>
	<li>摇动管子以重新使用幼虫。将三滴含有幼虫的 5 微升滴加入培养皿盖上。使用解剖显微镜计算每滴蠕虫的数量，并确定 1 μL 中的蠕虫数量。</li>
</ol>4. 药物检测的准备
注:此检测中的步骤显示在 图 1中。
<ol><li>在 50 mL 圆锥形管中生长 30 mL 大肠杆 菌 OP50，在 150 rpm 的轨道摇床中在 37 °C 的夜间生长。</li>
	<li>旋转一夜之间生长 大肠杆菌 OP50培养在4000 x g 10分钟颗粒细胞。取出超高颗粒，在 M9W 的 3 mL 中重新使用颗粒以集中培养物。</li>
	<li>在为药物检测准备工作解决方案之前，在 100 mL 的 M9W 中加入 0.1 mL（95% 乙醇中的 5 毫克/毫升）胆固醇。</li>
	<li>通过稀释每种药物加车辆（二甲基硫氧化物）来准备每个实验药物的工作解决方案:DMSO）在4.8ml M9W补充胆固醇，以获得适当的浓度。溶解 DMSO 在 4.8 mL 的 M9W 补充胆固醇，以准备车辆控制。</li>
	<li>在每个含有车辆控制或药物和漩涡管的管中加入 200 μL 浓缩 大肠杆菌 OP50 培养物，以确保它们混合。</li>
	<li>要执行实验，在 12 井组织培养板中将每个工作药物溶液或车辆控制中的 2 mL 添加到每个井中。重复测试每个药物浓度和车辆控制。</li>
	<li>使用无菌微管添加每口井 100 L1 幼虫（参见同步 C. elegans 培养步骤）。将蠕虫体积限制在 10 μL 以内。</li>
	<li>在 20 °C 下孵育板。</li>
	<li>如果需要，在检测的第 3 天用 50 微升 10 倍浓缩 大肠杆菌 OP50 补充井。</li>
</ol>5. 为显微镜准备的阿加罗斯垫
<ol><li>在 5 mL 的去离子水中溶解 0.1 克藻醇，准备 2% 的葡萄糖 w/v 溶液。加热微波炉中的内容以溶解藻子。20 张幻灯片可以从 5 mL 的阿加罗斯溶液中准备。</li>
	<li>将实验室胶带条沿两个玻璃幻灯片放置。胶带将充当隔板，限制玫瑰垫的厚度。之后，在带胶带的幻灯片之间放置第三个干净的玻璃滑梯。</li>
	<li>要制作一个玫瑰垫，将 100 μL 的熔融玫瑰点到干净的滑梯中心。将另一个干净的玻璃滑梯穿过，放在玫瑰的顶部，轻轻向下按压以形成垫子。当垫子凝固时，移除顶部滑动。</li>
</ol>6. 观察 让-60、让-23和林-1 菌株中的Muv表型
注:只有候选药物，抑制Muv表型在 let-23 和 让-60 菌株将使用 林-1 菌株进行检测，以确定抑制是否发生在RAS或EGFR的水平。
<ol><li>当达到生命周期的适当阶段（让-60和让-23菌株3天，林-1菌株5天），从孵化器中取出板。并使用移液器在 15 mL 圆锥管中收集蠕虫。</li>
	<li>以 450 x g 离心管 1 分钟。移除 M9W 而不干扰蠕虫颗粒，并添加 5 mL 的新鲜 M9W。</li>
	<li>重复步骤 6.2 两次。</li>
	<li>在不干扰蠕虫颗粒的情况下，通过吸入去除所有剩余的 M9W。此后，加入 500 μL 的 2 mM 钠阿齐德或 2 mM 四米盐酸四米索。允许管子在RT孵育15分钟2mM钠阿齐德或2mM四米盐酸四米索将麻醉蠕虫成像。 注意:在处理钠阿齐德时，一定要佩戴个人防护装备 （PPE）。解决方案必须在化学罩下准备。</li>
	<li>将麻醉蠕虫悬架的 10 μL 添加到玫瑰垫的中心。将#1.5 盖片轻轻放在蠕虫悬架上。如果需要，用指甲油固定盖唇，以防止干燥。</li>
	<li>使用DIC显微镜观察让-60（n1046），让-23（sa62）和林-1（sy254）菌株。在 10 倍和 20 倍放大倍数下对蠕虫进行成像。</li>
	<li>对于 让 60 （n1046） 和 让 23 （sa62）， 根据 Muv 表型的存在或不存在给成年蠕虫打分。而对于 lin-1 菌株，使用高分辨率DIC显微镜计算L4幼虫腹侧采用1°或2°细胞命运的VPC数量。</li>
	<li>在为每个菌株和药物治疗评分后，执行学生 t 测试，以确定治疗之间的统计差异。</li>
</ol>

<ol>
	<li>Marshall, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+Ras+and+Raf:+key+regulatory+proteins+in+cellular+transformation.">Interactions between Ras and Raf: key regulatory proteins in cellular transformation.</a> Molecular Reproduction and Development. 42 (4), 493-499 (1995).</li><li>Whelan, J. T., Hollis, S. E., Cha, D. S., Asch, A. S., Lee, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-transcriptional+regulation+of+the+Ras-ERK/MAPK+signaling+pathway.">Post-transcriptional regulation of the Ras-ERK/MAPK signaling pathway.</a> Journal of Cellular Physiology. 227 (3), 1235-1241 (2012).</li><li>Grandis, J. R., Tweardy, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevated+levels+of+transforming+growth+factor+alpha+and+epidermal+growth+factor+receptor+messenger+RNA+are+early+markers+of+carcinogenesis+in+head+and+neck+cancer.">Elevated levels of transforming growth factor alpha and epidermal growth factor receptor messenger RNA are early markers of carcinogenesis in head and neck cancer.</a> Cancer Research. 53 (15), 3579-3584 (1993).</li><li>Sasahira, T., Kirita, T., Kuniyasu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+of+molecular+pathobiology+in+oral+cancer:+a+review.">Update of molecular pathobiology in oral cancer: a review.</a> International Journal of Clinical Oncology. 19 (3), 431-436 (2014).</li><li>Stransky, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mutational+landscape+of+head+and+neck+squamous+cell+carcinoma.">The mutational landscape of head and neck squamous cell carcinoma.</a> Science. 333 (6046), 1157-1160 (2011).</li><li>Bos, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ras+oncogenes+in+human+cancer:+a+review.">ras oncogenes in human cancer: a review.</a> Cancer Research. 49 (17), 4682-4689 (1989).</li><li>Downward, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+RAS+signalling+pathways+in+cancer+therapy.">Targeting RAS signalling pathways in cancer therapy.</a> Nature Reviews Cancer. 3 (1), 11-22 (2003).</li><li>Prior, I. A., Lewis, P. D., Mattos, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+survey+of+Ras+mutations+in+cancer.">A comprehensive survey of Ras mutations in cancer.</a> Cancer Research. 72 (10), 2457-2467 (2012).</li><li>Hancock, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ras+proteins:+different+signals+from+different+locations.">Ras proteins: different signals from different locations.</a> Nature Reviews Molecular Cell Biology. 4 (5), 373-384 (2003).</li><li>Hancock, J. F., Parton, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ras+plasma+membrane+signalling+platforms.">Ras plasma membrane signalling platforms.</a> Biochemical Journal. 389, 1-11 (2005).</li><li>van der Hoeven, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fendiline+inhibits+K-Ras+plasma+membrane+localization+and+blocks+K-Ras+signal+transmission.">Fendiline inhibits K-Ras plasma membrane localization and blocks K-Ras signal transmission.</a> Molecular and Cellular Biology. 33 (2), 237-251 (2013).</li><li>Moghal, N., Sternberg, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epidermal+growth+factor+system+in+Caenorhabditis+elegans.">The epidermal growth factor system in Caenorhabditis elegans.</a> Experimental Cell Research. 284 (1), 150-159 (2003).</li><li>Sundaram, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RTK/Ras/MAPK+signaling.">RTK/Ras/MAPK signaling.</a> WormBook. , 1-19 (2006).</li><li>Ferguson, E. L., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+22+genes+that+affect+the+vulval+cell+lineages+of+the+nematode+Caenorhabditis+elegans.">Identification and characterization of 22 genes that affect the vulval cell lineages of the nematode Caenorhabditis elegans.</a> Genetics. 110 (1), 17-72 (1985).</li><li>Katz, W. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+point+mutation+in+the+extracellular+domain+activates+LET-23,+the+Caenorhabditis+elegans+epidermal+growth+factor+receptor+homolog.">A point mutation in the extracellular domain activates LET-23, the Caenorhabditis elegans epidermal growth factor receptor homolog.</a> Molecular and Cellular Biology. 16 (2), 529-537 (1996).</li><li>Hara, M., Han, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ras+farnesyltransferase+inhibitors+suppress+the+phenotype+resulting+from+an+activated+ras+mutation+in+Caenorhabditis+elegans.">Ras farnesyltransferase inhibitors suppress the phenotype resulting from an activated ras mutation in Caenorhabditis elegans.</a> Proceedings of the National Academy of Sciences of the United States of America. 92 (8), 3333-3337 (1995).</li><li>Reiner, D. J., Gonzalez-Perez, V., Der, C. J., Cox, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Caenorhabditis+elegans+to+evaluate+inhibitors+of+Ras+function+in+vivo.">Use of Caenorhabditis elegans to evaluate inhibitors of Ras function in vivo.</a> Methods in Enzymology. 439, 425-449 (2008).</li><li>van der Hoeven, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sphingomyelin+Metabolism+Is+a+Regulator+of+K-Ras+Function.">Sphingomyelin Metabolism Is a Regulator of K-Ras Function.</a> Molecular and Cellular Biology. 38 (3), (2018).</li><li>Beitel, G. J., Tuck, S., Greenwald, I., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Caenorhabditis+elegans+gene+lin-1+encodes+an+ETS-domain+protein+and+defines+a+branch+of+the+vulval+induction+pathway.">The Caenorhabditis elegans gene lin-1 encodes an ETS-domain protein and defines a branch of the vulval induction pathway.</a> Genes &amp; Development. 9 (24), 3149-3162 (1995).</li><li>Porta-de-la-Riva, M., Fontrodona, L., Villanueva, A., Ceron, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Caenorhabditis+elegans+methods:+synchronization+and+observation.">Basic Caenorhabditis elegans methods: synchronization and observation.</a> Journal Visualized Experiments. (64), e4019 (2012).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> WormBook. , 1-11 (2006).</li><li>Revtovich, A. V., Lee, R., Kirienko, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+between+mitochondria+and+diet+mediates+pathogen+and+stress+resistance+in+Caenorhabditis+elegans.">Interplay between mitochondria and diet mediates pathogen and stress resistance in Caenorhabditis elegans.</a> PLoS Genetics. 15 (3), 1008011 (2019).</li><li>Zimmermann, M., Zimmermann-Kogadeeva, M., Wegmann, R., Goodman, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+human+microbiome+drug+metabolism+by+gut+bacteria+and+their+genes.">Mapping human microbiome drug metabolism by gut bacteria and their genes.</a> Nature. 570 (7762), 462-467 (2019).</li><li>Moghal, N., Garcia, L. R., Khan, L. A., Iwasaki, K., Sternberg, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+EGF+receptor-mediated+vulva+development+by+the+heterotrimeric+G-protein+G-alpha+q+and+excitable+cells+in+C.+elegans.">Modulation of EGF receptor-mediated vulva development by the heterotrimeric G-protein G-alpha q and excitable cells in C. elegans.</a> Development. 130 (19), 4553-4566 (2003).</li></ol>

作者声明没有相互竞争的经济利益。

我们描述使用蠕虫的检测是简单和廉价的，以确定EGFR和RAS功能的抑制剂。 C. elegans 是药物发现有吸引力的模型，因为由于生命周期短（3 天在 20 °C）和产生大量幼虫的能力，在实验室中很容易生长。更重要的是，EGFR-RAS-ERK MAPK通路在进化和功能上得到保护，哺乳动物提供了一个基因可传导系统来分析EGFR和RAS抑制剂的影响。此外，蠕虫的透明性使研究者能够可视化不同的结构，以及绿色荧光...

调节生物体内发育事件的细胞通路在所有元子中都得到了高度保护。其中一个途径是EGFR-RAS-ERK线粒体活性蛋白激酶（MAPK）信号级联，这是控制细胞增殖、分化、迁移和生存的关键途径1，2。此信号通路中的缺陷可能导致病理或疾病状态，如癌症。表皮生长因子受体（EGFR）在人类肿瘤中表现出高度表达，包括50%的口腔鳞状细胞癌，并有助于恶性肿瘤3、4、5的发展。而三个RAS同位素H-，K-和N-RAS的突变是多种人类癌症恶性转化的主要驱动因素。在这三种RAS同位素中，K-RAS的致癌突变最为普遍，为6、7、8。要使EGFR和RAS发挥作用，它们必须定位到等离子膜（PM）。防止这些分子的本地化到PM可以完全废除这个信号通路9，10的生物活性。因此，抑制这些蛋白质的本地化到PM是一种治疗策略，以阻止下游信号和由此产生的不良后果。使用高含量筛选检测，芬迪林，L型钙通道阻滞剂，被确定为K-RAS活性的抑制剂11。在芬迪林的存在下，K-RAS 到 PM 内部传单的纳米集群显著减少。此外，K-RAS从等离子体膜重新分配到内质视网膜（ER）、高尔吉仪器、内分泌体和细胞溶胶。更重要的是，胰腺癌、结肠癌、肺癌和子宫内膜癌细胞系的增殖被芬迪林11抑制下游信号所阻断。这些数据表明，芬迪林作为一种特定的K-RAS抗癌疗法，导致RAS蛋白对PM的误定位。
线虫 卡诺哈布德炎埃莱甘斯 已在发展的背景下进行了广泛的研究。许多控制蠕虫发展的信号通路都是进化的，在功能上是保守的。例如，EGFR 介解 RAS 激活和随后激活的 ERK MAPK 信号级联在蠕虫12中保存。级联由以下蛋白质表示:LET-23 > LET-60 > LIN-45 > MEK-2 >MPK-1。LET-60 与 RAS 同源，而 LET-23 与 EGFR 同源。在蠕虫中，这种途径调节外阴13的发育。外阴是蠕虫腹壁上的上皮孔径，允许产卵。蠕虫外阴的形成取决于外阴前体细胞 （VPC） 暴露在 EGFR-RAS-MAPK 信号级联激活梯度下。在正常发育过程中，近亲VC从淋瑙锚细胞接收强信号，以分化成1°和2°细胞的命运，从而产生功能性外阴12。而离散性VC区分成3°细胞的命运，融合到皮下同步，不会形成外阴由于耗尽的信号。在没有信号的情况下，所有 VPC 都区分为 3° 细胞命运，从而形成无外阴。然而，构成信号导致形成一个或多个非功能性外阴，由于所有VPC的诱导，以承担1°和2°细胞的命运。
导致外阴诱导缺陷或过度的突变已经为代表该通路的蛋白质编码的许多基因被识别出来。有缺陷的外阴诱导导致无外阴（Vul）表型，而过度的外阴诱导导致多外阴（Muv）表型，其表现是在整个心室壁上出现大量非功能性异位伪外阴。让-60（n1046）菌株表达的Muv表型是由于RAS功能突变的增加，而在让-23（sa62）菌株中，它是由于EGFR14，15的激活突变。这些突变菌株中的强Muv表型已被药理干预所困扰，通过MEK-1抑制剂U0126 16、17治疗让-60（n1046）蠕虫就证明了这一点。有趣的是，我们已经表明，R-芬迪林和抑制剂，影响苯丙胺代谢抑制在蠕虫18的Muv表型。为了证明这些抑制剂块让60信号在RAS的水平，林-1空菌株已被使用17。Lin-1是一种类似Ets的抑制转录因子，在外阴19的发育中起到抑制作用。在让-60（n1046）蠕虫中，Muv表型的强烈回归，对lin-1空蠕虫没有影响，这表明这些抑制发生在RAS的水平。
在此协议中，我们演示了使用C. elegans作为模型来识别 RAS 和 EGFR 蛋白的抑制剂。使用液体基检测，我们通过抑制让-60（n1046）和让-23（sa62）突变菌株的C.elegans的Muv表型来证明R-芬迪林的抑制作用。这种检测验证了在抗癌治疗药物发现初始阶段使用C.elegans作为工具。

<table><tbody><tr><td>&lt;strong&gt;Media and chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Agarose&amp;nbsp;</td><td>Millipore Sigma&amp;nbsp;</td><td>A9539-50G</td><td></td></tr><tr><td>Bacto Peptone&amp;nbsp;</td><td>Fisher Scientific</td><td>DF0118-17-0</td><td></td></tr><tr><td>BD Difco Agar&amp;nbsp;</td><td>Fisher Scientific</td><td>DF0145-17-0</td><td></td></tr><tr><td>BD Difco LB Broth</td><td>Fisher Scientific</td><td>DF0446-17-3</td><td></td></tr><tr><td>Calcium Chloride</td><td>Fisher Scientific</td><td>BP510-500</td><td></td></tr><tr><td>Cholesterol</td><td>Fisher Scientific</td><td>ICN10138201</td><td></td></tr><tr><td>Magnesium Sulfate</td><td>Fisher Scientific</td><td>BP213-1</td><td></td></tr><tr><td>Nystatin</td><td>Acros organics</td><td>AC455500050</td><td></td></tr><tr><td>Potassium Phosphate Dibasic</td><td>Fisher Scientific</td><td>BP363-500</td><td></td></tr><tr><td>Potassium pPhosphate Monobasic</td><td>Fisher Scientific</td><td>BP362-500</td><td></td></tr><tr><td>R-Fendiline</td><td></td><td></td><td>Commercially Synthesized (Pharmaceutical grade)</td></tr><tr><td>Sodium Azide</td><td>Millipore Sigma&amp;nbsp;</td><td>S2002-25G</td><td></td></tr><tr><td>Sodium chloride&amp;nbsp;</td><td>Fisher Scientific</td><td>BP358-1</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>Fisher Scientific</td><td>SS266-1</td><td></td></tr><tr><td>8.25% Sodium Hypochlorite&amp;nbsp;</td><td></td><td></td><td>Bleach</td></tr><tr><td>Sodium Phosphate Dibasic&amp;nbsp;</td><td>Fisher Scientific</td><td>BP332-500</td><td></td></tr><tr><td>Streptomycin Sulfate&amp;nbsp;</td><td>Fisher Scientific</td><td>BP910-50</td><td></td></tr><tr><td>(&amp;minus;)-Tetramisole Hydrochloride</td><td>Millipore Sigma&amp;nbsp;</td><td>L9756</td><td></td></tr><tr><td>UO126 (MEK inhibitor)</td><td>Millipore Sigma&amp;nbsp;</td><td>19-147</td><td></td></tr><tr><td>&lt;strong&gt;Consumables&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>15mL Conical Sterile Polypropylene Centrifuge Tubes&amp;nbsp;</td><td>Fisher Scientific</td><td>12-565-269</td><td></td></tr><tr><td>50mL Conical Sterile Polypropylene Centrifuge Tubes</td><td>Fisher Scientific</td><td>12-565-271</td><td></td></tr><tr><td>Disposable Polystyrene Serological Pipettes 10mL</td><td>Fisher Scientific</td><td>07-200-574</td><td></td></tr><tr><td>Disposable Polystyrene Serological Pipettes 25mL</td><td>Fisher Scientific</td><td>07-200-575</td><td></td></tr><tr><td>No. 1.5&amp;nbsp; 18 mm X 18 mm Cover Slips</td><td>Fisher Scientific</td><td>12-541A</td><td></td></tr><tr><td>Petri Dish with Clear Lid (60 x 15 mm)</td><td>Fisher Scientific</td><td>FB0875713A</td><td></td></tr><tr><td>Petri Dishes with Clear Lid (100X15mm)</td><td>Fisher Scientific</td><td>FB0875712</td><td></td></tr><tr><td>Plain Glass Microscope Slides (75 x 25 mm)</td><td>Fisher Scientific</td><td>12-544-4</td><td></td></tr><tr><td>12- Well Tissue Culture Plates</td><td>Fisher Scientific</td><td>50-197-4804</td><td></td></tr><tr><td>&lt;strong&gt;Software&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Prism</td><td>Graphpad</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Bacterial Strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; OP50</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Worm Strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Strain&lt;/strong&gt;</td><td>&lt;strong&gt;Genotype&lt;/strong&gt;</td><td>&lt;strong&gt;Transgene&lt;/strong&gt;</td><td>&lt;strong&gt;Source&lt;/strong&gt;</td></tr><tr><td>MT2124 &amp;nbsp;</td><td>&lt;em&gt;let-60(n1046) IV.&lt;/em&gt;</td><td></td><td>CGC</td></tr><tr><td>MT7567</td><td>&lt;em&gt;lin-1(sy254) IV.&lt;/em&gt;</td><td></td><td>CGC</td></tr><tr><td>PS1839</td><td>&lt;em&gt;let-23(sa62) II.&lt;/em&gt;</td><td></td><td>CGC</td></tr></tbody></table>

identification of egfr and ras inhibitors using caenorhabditis elegans

表皮生长因子受体（EGFR）及其下游效应器RAS的血浆膜定位变化与包括癌症在内的多种疾病有关。自由活的线虫 C.elegans 具有进化和功能保存的EGFR-RAS-ERK MAP信号级联，是外阴发育的核心。RAS同源LET-60和EGFR同源LET-23功能突变的增益诱导这些蠕虫的腹壁产生可见的非功能性异位伪外阴。以前，这些蠕虫中的多外阴（Muv）表型已被小化学分子抑制。在这里，我们描述了在液体基检测中使用蠕虫来识别消除EGFR和RAS蛋白活动的抑制剂的协议。通过此检测，我们显示 K-RAS 的间接抑制剂 R-fendiline 抑制 了在 let-60 （n1046） 和 让-23 （sa62） 突变蠕虫中表达的 Muv 表型。检测简单、价格低廉，设置不费时，可作为发现抗癌治疗的初始平台。

我们首先证明，与DMSO治疗的蠕虫相比，R-fendiline能够抑制 让-60（n1046） 突变菌株中的Muv表型。我们的数据显示，R-芬迪林能够以依赖剂量的方式阻断 让-60（n1046） 中的Muv表型（图2A，B）。然而，在 林-1 空突变菌株中观察到Muv表型的未逆转，以响应R-芬迪林浓度的增加（图2B）。数据表明，R-芬迪林块激活 让60 信?...

Watch this Scientific Journal Video about Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans at JoVE.com

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

基因可处理的线虫 卡诺哈布迪炎的叶原体 可以用作药物发现一个简单和廉价的模型。这里描述的是一个协议，以确定抗癌治疗，抑制RAS和EGFR蛋白的下游信号。

使用卡诺哈布迪炎埃莱甘斯识别 EGFR 和 RAS 抑制剂

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in ...

identification-of-egfr-and-ras-inhibitors-using-caenorhabditis-elegans

Research

JoVE Journal

Cancer Research

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

3.0K 次观看.  University of Texas Health Science Center. 基因可处理的线虫 卡诺哈布迪炎的叶原体 可以用作药物发现一个简单和廉价的模型。这里描述的是一个协议，以确定抗癌治疗，抑制RAS和EGFR蛋白的下游信号。

视频: Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the lumen and basolateral membranes contacting each other and/or the extracellular matrix. How this distinctive membrane asymmetry is established and maintained during organ morphogenesis is still an unresolved question of cell biology. This protocol describes the C. elegans intestine as a&#160;model for the analysis of polarized membrane biogenesis during tube morphogenesis, with emphasis on apical membrane and lumen biogenesis. The C. elegans twenty-cell single-layered intestinal epithelium is arranged into a simple bilaterally symmetrical tube, permitting analysis on a single-cell level. Membrane polarization occurs concomitantly with polarized cell division and migration during early embryogenesis, but de novo polarized membrane biogenesis continues throughout larval growth, when cells no longer proliferate and move. The latter setting allows one to separate subcellular changes that simultaneously mediate these different polarizing processes, difficult to distinguish in most polarity models. Apical-, basolateral membrane-, junctional-, cytoskeletal- and endomembrane components can be labeled and tracked throughout development by GFP fusion proteins, or assessed by in situ antibody staining. Together with the organism&#39;s genetic versatility, the C. elegans intestine thus provides a unique in vivo model for the visual, developmental, and molecular genetic analysis of polarized membrane and tube biogenesis. The specific methods (all standard) described here include how to: label intestinal subcellular components by antibody staining; analyze genes involved in polarized membrane biogenesis by loss-of-function studies adapted to the typically essential tubulogenesis genes; assess polarity defects during different developmental stages; interpret phenotypes by epifluorescence, differential interference contrast (DIC) and confocal microscopy; quantify visual defects. This protocol can be adapted to analyze any of the often highly conserved molecules involved in epithelial polarity, membrane biogenesis, tube and lumen morphogenesis.

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analy

The transparent C. elegans intestine can serve as an "in vivo tissue chamber" for studying apicobasal membrane and lumen biogenesis at the single-cell and subcellular level during multicellular tubulogenesis. This protocol describes how to combine standard labeling, loss-of-function genetic/RNAi and microscopic approaches to dissect these processes on a molecular level.

C. 线虫小肠作为细胞间腔形态发生和体内极化膜生物单细胞水平的模型: 抗体染色、rna 干扰功能分析和成像标记

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the ...

科研

发育生物学

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many historical and contemporary studies have identified thousands of lifespan-altering paradigms, including genetic mutations, transgenic gene expression, and hormesis, a beneficial, low-grade exposure to stress. With its many advantages, including a short lifespan, easy and low-cost maintenance, and fully sequenced genome with homology to almost two-thirds of all human genes, C. elegans has quickly been adopted as an outstanding model for stress and aging biology. Here, several standardized methods are surveyed for measuring lifespan and healthspan that can be easily adapted into almost any research environment, especially those with limited equipment and funds. The incredible utility of C. elegans is featured, highlighting the capacity to perform powerful genetic analyses in aging biology without the necessity of extensive infrastructure. Finally, the limitations of each analysis and alternative approaches are discussed for consideration.

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

Caenorhabditis elegans serve as an excellent model system with robust and low-cost methods for surveying healthspan, lifespan, and resilience to stress.

调查低成本方法，以测量秀丽隐杆线虫的寿命和健康寿命

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many ...

遗传学

Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism

Neurexins and neuroligins are cell adhesion molecules present in excitatory and inhibitory synapses, and they are required for correct neuron network function1. These proteins are found at the presynaptic and postsynaptic membranes 2. Studies in mice indicate that neurexins and neurologins have an essential role in synaptic transmission 1. Recent reports have shown that altered neuronal connections during the development of the human nervous system could constitute the basis of the etiology of numerous cases of autism spectrum disorders 3.
Caenorhabditis elegans could be used as an experimental tool to facilitate the study of the functioning of synaptic components, because of its simplicity for laboratory experimentation, and given that its nervous system and synaptic wiring has been fully characterized. In C. elegans nrx-1 and nlg-1 genes are orthologous to human NRXN1 and NLGN1 genes which encode alpha-neurexin-1 and neuroligin-1 proteins, respectively. In humans and nematodes, the organization of neurexins and neuroligins is similar in respect to functional domains.
The head of the nematode contains the amphid, a sensory organ of the nematode, which mediates responses to different stimuli, including osmotic strength. The amphid is made of 12 sensory bipolar neurons with ciliated dendrites and one presynaptic terminal axon 4. Two of these neurons, named ASHR and ASHL are particularly important in osmotic sensory function, detecting water-soluble repellents with high osmotic strength 5. The dendrites of these two neurons lengthen to the tip of the mouth and the axons extend to the nerve ring, where they make synaptic connections with other neurons determining the behavioral response 6.
To evaluate the implications of neurexin and neuroligin in high osmotic strength avoidance, we show the different response of C. elegans mutants defective in nrx-1 and nlg-1 genes, using a method based on a 4M fructose ring 7. The behavioral phenotypes were confirmed using specific RNAi clones 8. In C. elegans, the dsRNA required to trigger RNAi can be administered by feeding 9. The delivery of dsRNA through food induces the RNAi interference of the gene of interest thus allowing the identification of genetic components and network pathways.

Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism

Neurexins and neuroligins are membrane-neuron adhesion proteins which perform essential roles in synaptic differentiation and transmission. Neuroligin deficient mutants of C. elegans are defective in detecting osmotic strength, but when they also contain a mutation in the gene coding neurexin, they recover the wild type phenotype.

渗透避免IN线虫：突触功能的两个基因，人类同源 NRXN1 NLGN1，作为候选人自闭症

Neurexins and neuroligins are cell adhesion molecules present in excitatory and inhibitory synapses, and they are required for correct neuron network ...

生物学

Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans

Co-immunoprecipitation methods are frequently used to study protein-protein interactions. Confirmation of hypothesized protein-protein interactions or identification of new ones can provide invaluable information about the function of a protein of interest. Some of the traditional methods for extract preparation frequently require labor-intensive and time-consuming techniques. Here, a modified extract preparation protocol using a bead mill homogenizer and metal beads is described as a rapid alternative to traditional protein preparation methods. This extract preparation method is compatible with downstream co-immunoprecipitation studies. As an example, the method was used to successfully co-immunoprecipitate&#160;C. elegans microRNA Argonaute ALG-1 and two known ALG-1 interactors: AIN-1, and HRPK-1. This protocol includes descriptions of animal sample collection, extract preparation, extract clarification, and protein immunoprecipitation. The described protocol can be adapted to test for interactions between any two or more endogenous, endogenously tagged, or overexpressed C. elegans proteins in a variety of genetic backgrounds.

Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans

This method describes a protocol for high-throughput protein extract preparation from Caenorhabditis elegans samples and subsequent co-immunoprecipitation.

来自卡诺哈布迪炎精英的蛋白质提取制剂制备和共免疫沉淀

Co-immunoprecipitation methods are frequently used to study protein-protein interactions. Confirmation of hypothesized protein-protein interactions or ...

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

高通量筛选和生物传感与荧光 C。线虫菌株

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi筛选识别胚后的表型 C。线虫</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

定量和自动化高通量的全基因组RNAi屏幕 C。线虫</em

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems. For example, blood transfusions in mammalian systems and grafting techniques in plants enable asking questions of circulatory system composition and signaling. The circulatory system of the worm, the pseudocoelom, has until recently been impossible to assay directly. To answer questions of intercellular signaling and circulatory system composition C. elegans researchers have traditionally turned to genetic analysis, cell/tissue specific rescue, and mosaic analysis. These techniques provide a means to infer what is happening between cells, but are not universally applicable in identification and characterization of extracellular molecules. Here we present a newly developed technique to directly assay the pseudocoelomic fluid of C. elegans. The technique begins with either genetic or physical manipulation to increase the volume of extracellular fluid. Afterward the animals are subjected to a vampiric reverse microinjection technique using a microinjection rig that allows fine balance pressure control. After isolation of extracellular fluid, the collected fluid can be assayed by transfer into other animals or by molecular means. To demonstrate the effectiveness of this technique we present a detailed approach to assay a specific example of extracellular signaling molecules, long dsRNA during a systemic RNAi response. Although characterization of systemic RNAi is a proof of principle example, we see this technique as being adaptable to answer a variety of questions of circulatory system composition and signaling.

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The model organism C. elegans uses pseudocoelomic fluid as a passive circulatory system. Direct assay of this fluid has not been previously possible. Here we present a novel technique to directly assay the extracellular space, and use systemic silencing signals during an RNAi response as a proof of principle example.

吸血鬼从细胞外液分离<em&gt;秀丽隐杆线虫</em

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ...

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans lab.

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

While dsRNA feeding in C. elegans is a powerful tool to assess gene function, current protocols for large scale feeding screens result in variable knockdown efficiencies. We describe an improved protocol for performing large scale RNAi feeding screens that results in highly efficacious and reproducible knockdown of gene expression.

在大规模的基因敲除<em&gt; C。线虫</em&gt;使用双链RNA料库产生失去功能的鲁棒表型

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well ...

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein subcellular localization changes; in particular, a nuclear translocation under a heat stress condition. Proteins react correspondingly to external and internal signals. A common mechanism is to change its subcellular localization. This article describes a protocol to track protein localization that does not require an antibody, radioactive labeling, or a confocal microscope. In this article, GFP is used to tag the target protein EXL-1 in C. elegans, a member of the chloride intracellular channel proteins (CLICs) family, including mammalian CLIC4. An integrated translational exl-1::gfp transgenic line (with a promoter and a full gene sequence) was created by transformation and &#947;-radiation, and stably expresses the gene and gfp. Recent research showed that upon heat stress, not oxidative stress, EXL-1::GFP accumulates in the nucleus. Overlapping the GFP signal with both the nuclei structure and the DAPI signals confirms the EXL-1 subcellular localization changes under stress. This protocol presents two different fixation methods for DAPI staining: ethanol fixation and acetone fixation. The DAPI staining protocol presented in this article is fast and efficient and preserves both the GFP signal and the protein subcellular localization changes. This method only requires a fluorescence microscope with Nomarski, a FITC filter, and a DAPI filter. It is suitable for a small laboratory setting, undergraduate student research, high school student research, and biotechnology classrooms.

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

This protocol demonstrates how to track a protein nuclear translocation under heat stress by using a green fluorescence protein (GFP) fusion protein as a marker and 4',6-diamidino-2-phenylindole (DAPI) staining. The DAPI staining protocol is fast and preserves the GFP and protein subcellular localization signals.

使用卡诺哈布迪炎埃莱甘斯识别 EGFR 和 RAS 抑制剂

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吸血鬼从细胞外液分离

在大规模的基因敲除

秀丽线虫绿色荧光蛋白标记和 4 '、6-Diamidino-2-苯基吲哚染色检测蛋白质亚细胞定位