Name: high-throughput screening and biosensing with fluorescent c. elegans strains
Uploaded: 2011-05-19T13:00:00
Description: Watch this Scientific Journal Video about High-throughput Screening and Biosensing with Fluorescent C. elegans Strains at JoVE.com

线虫转基因线虫遗传学中心（美国明尼苏达大学，明尼阿波利斯，明尼苏达州）。这项工作是支持由美国国立卫生研究院R21授予NS0667678 - 01到金水。参加所有作者在收集，分析和解释数据。 CKL，KS和KPC参加了编写和修改的手稿。

1。细菌的蠕虫食品的制备第1天<ol><li>加入5毫升饱和大肠杆菌大肠杆菌 OP50细菌培养到500毫升了不起的肉汤补充50微克/ ml链霉素和成长在一个摇床（225转）隔夜在37 ° C。 </li></ol>第2天<ol start="2"><li> 2500 RCF为20分钟，分割成10个50毫升管和一个冷冻离心机离心机细菌过夜的细菌培养。 </li><li>倒出关闭LB培养基，并在每10毫升的液体线虫的生长介质（NGM）重?...

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	<li>Simeonov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+high-throughput+screen+identifies+inhibitors+of+the+Schistosoma+mansoni+redox+cascade.">Quantitative high-throughput screen identifies inhibitors of the Schistosoma mansoni redox cascade.</a> PLoS Negl Trop Dis. 2, e127-e127 (2008).</li><li>Giacomotto, J., Segalat, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+and+small+animal+models,+where+are+we.">High-throughput screening and small animal models, where are we.</a> Br J Pharmacol. 160, 204-216 (2010).</li><li>Artal-Sanz, M., de Jong, L., Tavernarakis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans:+a+versatile+platform+for+drug+discovery.">Caenorhabditis elegans: a versatile platform for drug discovery.</a> Biotechnol J. 1, 1405-1418 (2006).</li><li>An, J. H., Blackwell, T. 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Sci. 101, 215-225 (2008).</li><li>Tullet, J. M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+inhibition+of+the+longevity-promoting+factor+SKN-1+by+insulin-like+signaling+in+C.+elegans.">Direct inhibition of the longevity-promoting factor SKN-1 by insulin-like signaling in C. elegans.</a> Cell. 132, 1025-1038 (2008).</li><li>Oliveira, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Condition-adapted+stress+and+longevity+gene+regulation+by+Caenorhabditis+elegans+SKN-1/Nrf.">Condition-adapted stress and longevity gene regulation by Caenorhabditis elegans SKN-1/Nrf.</a> Aging Cell. , (2009).</li><li>Park, S. -. K., Tedesco, P. M., Johnson, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+and+longevity+in+Caenorhabditis+elegans+as+mediated+by+SKN-1.">Oxidative stress and longevity in Caenorhabditis elegans as mediated by SKN-1.</a> Aging Cell. 8, 258-269 (2009).</li><li>Hayes, J. D., McMahon, M., Chowdhry, S., Dinkova-Kostova, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+chemoprevention+mechanisms+mediated+through+the+Keap1-Nrf2+pathway.">Cancer chemoprevention mechanisms mediated through the Keap1-Nrf2 pathway.</a> Antioxid Redox Signal. , (2010).</li><li>Kwak, M. K., Kensler, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+NRF2+signaling+for+cancer+chemoprevention.">Targeting NRF2 signaling for cancer chemoprevention.</a> Toxicol Appl Pharmacol. 244, 66-76 (2010).</li><li>Leiser, S. F., Miller, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nrf2+signaling,+a+mechanism+for+cellular+stress+resistance+in+long-lived+mice.">Nrf2 signaling, a mechanism for cellular stress resistance in long-lived mice.</a> Mol Cell Biol. 30, 871-884 (2010).</li><li>Link, C. D., Johnson, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reporter+transgenes+for+study+of+oxidant+stress+in+Caenorhabditis+elegans.">Reporter transgenes for study of oxidant stress in Caenorhabditis elegans.</a> Methods Enzymol. 353, 497-505 (2002).</li><li>Vanduyn, N., Settivari, R., Wong, G., Nass, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SKN-1/Nrf2+inhibits+dopamine+neuron+degeneration+in+a+Caenorhabditis+elegans+model+of+methylmercury+toxicity.">SKN-1/Nrf2 inhibits dopamine neuron degeneration in a Caenorhabditis elegans model of methylmercury toxicity.</a> Toxicol Sci. , (2010).</li><li>Hasegawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+inexpensive+method+to+screen+for+common+foods+that+reduce+the+action+of+acrylamide,+a+harmful+substance+in+food.">A rapid and inexpensive method to screen for common foods that reduce the action of acrylamide, a harmful substance in food.</a> Toxicol Lett. 175, 82-88 (2007).</li><li>Chase, D. 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我们提出了培养方法，并配发大量的转基因线虫。培养蠕虫使用的设备是标准的实验室进行分子克隆和液体处理和设备的荧光微孔板处理大量的实验室标准。配药的活C.大量的其他方法线虫需要昂贵的粒子分拣设备19。 Pgst - 4：绿色荧光蛋白检测可用于屏幕上的第“N”白领转录因子的小分子调制器和检测异生和氧化剂污染物在环境和食品样品14,16。乐百氏诱导?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td> LB培养基</td><td>研究产品国际公司</td><td> L24066 </td><td></td></tr><tr><td>太棒了肉汤</td><td>研究产品国际公司</td><td> T15100 </td><td></td></tr><tr><td>协同™HT多模酶标仪</td><td>百特</td><td></td><td>过滤器：GFP 485/20ex 528/20em; RFP的540/25ex 590/35em </td></tr><tr><td> MicroFlo选择饮水机</td><td>百特</td><td></td><td></td></tr><tr><td>蠕虫配药烧瓶</td><td>南部科学公司，佛罗里达州，Micanopy </td><td></td><td>自定义组装（352）284-2531 </td></tr><tr><td> 384微孔板</td><td>格雷纳生物的一</td><td> 5678-1209 </td><td></td></tr><tr><td>透气封箱胶带</td><td> NUNC </td><td> 241205 </td><td></td></tr><tr><td> （5 - 羟基- P - naphthoqinone）胡桃</td><td>阿库罗斯福冈</td><td> 121640010 </td><td>胡桃是溶于DMSO </td></tr><tr><td>二甲基亚砜</td><td> Sigma - Aldrich公司</td><td> D - 5879 </td><td></td></tr><tr><td> 线虫转基因株</td><td>作者实验室</td><td> VP596 </td><td> Pgst - 4：：GFP和PDOP - 3：招标书 </td></tr></tbody></table>

high-throughput screening and biosensing with fluorescent c. elegans strains

高通量筛选（HTS）是一个强大的方法确定的生物过程的化学调制。然而，在使用细胞培养模型的屏幕中发现了许多化合物， 经常 发现 在 1-2体内有毒或药物活动。在整体动物模型的筛选，可以帮助避免这些缺陷，并简化药物开发的路径。 线虫是一个多细胞生物模型，非常适合用于高通量筛选。这是小（&lt;1毫米），并可以在经济上的培养，在液体中的处方。 C。 线虫也是最听话的实验动物模型允许快速和详细的鉴定药物模式的行动之一。 我们描述了培养协议，并配发荧光的 C株线虫为高通量筛选化学库或改变特定基因的表达的环境污染物的检测。发展同步蠕虫的大量液体培养基中生长，收获，洗涤，并悬浮在一个定义的密度。蠕虫，然后添加到黑，平底的384孔板使用蠕动的液体分配器。从一种化学品库或测试样本（例如，水，食物，或土壤）的小分子可以被添加到井与蠕虫体内，实时荧光强度与荧光酶标仪测定。这种方法可以适应任何C.诱导基因一个合适的记者提供的线虫。许多诱导应力和发展的转录途径是定义在 C 线虫和GFP的转基因记者菌株已经存在许多其中4人。当与适当的转基因记者结合，我们的方法可用于屏幕通路调制器，或发展为强大的生物传感器检测环境污染物。 我们证明我们的C 。 线虫文化和配药协议，高温超导实验，我们发展到显示器的 C. 线虫第“N”白领转录因子SKN - 1。 SKN - 1和哺乳动物同源Nrf2的激活过程中的氧化和异生强调5-10细胞保护基因。 Nrf2的保护哺乳动物和许多与年龄有关的，如癌症，神经退行性疾病，和慢性炎症疾病已成为一个主要的化疗目标11-13。我们的实验是基于GFP的转基因SKN - 1靶基因 GST -4 14记者，编码谷胱甘肽- S转移6。 商品及服务税-4记者也是一个传感器异生和氧化的化学物质，激活SKN - 1，可用于检测水平低的污染物，如丙烯酰胺和甲基水银15-16。

Watch this Scientific Journal Video about High-throughput Screening and Biosensing with Fluorescent C. elegans Strains at JoVE.com

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

液为基础的培养和分配过程<em&gt; C。线虫</em&gt;菌株表达荧光记者蛋白质的描述，不需要昂贵的分拣设备。这种方法可以适用于众多诱导<em&gt; C。线虫</em&gt;基因药物发现或污染物的生物传感。

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 ...

Research

JoVE Journal

Biology

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

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

生产 C。线虫</em通过重组工程与转基因 galK选择标记

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and ...

科研

生物学

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

真菌内切葡聚糖酶的活性高通量筛选大肠埃希氏菌</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

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

Quantitative and Automated High-throughput Genome-wide RNAi Screens 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. ...

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

内向整流钾通道的小分子调节剂的高通量筛选

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

C. elegans Chemotaxis Assay

C. elegans Chemotaxis Assay

线虫趋含量的

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

生化和高通量的微观评估的脂肪含量<em&gt;秀丽隐杆线虫</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

使用自制双辉光萤光素酶检测的高通量筛选功能

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

激酶底物对鉴定使用高通量筛选


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