Name: quantitative and automated high-throughput genome-wide rnai screens in c. elegans
Uploaded: 2012-02-27T12:00:00
Duration: 10 min 58 s
Description: Watch this Scientific Journal Video about Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans at JoVE.com

我们感谢他们的贡献D. Braendle，CL库尔兹和F.Montañana-桑奇斯。这个项目是由ANR和行政法院区域PACA的，（INSERM）和国家科学研究中心的机构资金补助支持。

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K., Miteva, Y., Hannenhalli, S., Lamitina, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+physiological+activation+of+osmosensitive+gene+expression+mimics+transcriptional+signatures+of+pathogen+infection+in+C.+elegans.">Genetic and physiological activation of osmosensitive gene expression mimics transcriptional signatures of pathogen infection in C. elegans.</a> PLoS One. 5, e9010-e9010 (2010).</li><li>Lee, K. Z., Kniazeva, M., Han, M., Pujol, N., Ewbank, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fatty+acid+synthase+fasn-1+acts+upstream+of+WNK+and+Ste20&#47;GCK-VI+kinases+to+modulate+antimicrobial+peptide+expression+in+C.+elegans+epidermis.">The fatty acid synthase fasn-1 acts upstream of WNK and Ste20&#47;GCK-VI kinases to modulate antimicrobial peptide expression in C. elegans epidermis.</a> Virulence. 1, 113-122 (2010).</li><li>Duverger, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+semi-automated+high-throughput+approach+to+the+generation+of+transposon+insertion+mutants+in+the+nematode+Caenorhabditis+elegans.">A semi-automated high-throughput approach to the generation of transposon insertion mutants in the nematode Caenorhabditis elegans.</a> Nucleic Acids Res. 35, e11-e11 (2007).</li><li>Pierce-Shimomura, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+crawling+and+swimming+locomotory+patterns+in+C.+elegans.">Genetic analysis of crawling and swimming locomotory patterns in C. elegans.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 20982-20987 (2008).</li><li>Ruzanov, P., Riddle, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+SAGE+analysis+of+the+Caenorhabditis+elegans+transcriptome.">Deep SAGE analysis of the Caenorhabditis elegans transcriptome.</a> Nucleic Acids Res. 38, 3252-3262 (2010).</li><li>Kurz, C. L., Ewbank, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection+in+a+dish:+high-throughput+analyses+of+bacterial+pathogenesis.">Infection in a dish: high-throughput analyses of bacterial pathogenesis.</a> Curr. Opin. Microbiol. 10, 10-16 (2007).</li><li>Okoli, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+antifungal+compounds+active+against+Candida+albicans+using+an+improved+high-throughput+Caenorhabditis+elegans+assay.">Identification of antifungal compounds active against Candida albicans using an improved high-throughput Caenorhabditis elegans assay.</a> PLoS One. 4, e7025-e7025 (2009).</li><li>Moy, T. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+novel+antimicrobials+using+a+live-animal+infection+model.">Identification of novel antimicrobials using a live-animal infection model.</a> Proc. Natl. Acad. Sci. U. S. A. 103, 10414-10419 (2006).</li><li>Ballestriero, F., Thomas, T., Burke, C., Egan, S., Kjelleberg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+compounds+with+bioactivity+against+the+nematode+Caenorhabditis+elegans+by+a+screen+based+on+the+functional+genomics+of+the+marine+bacterium+Pseudoalteromonas+tunicata+D2.">Identification of compounds with bioactivity against the nematode Caenorhabditis elegans by a screen based on the functional genomics of the marine bacterium Pseudoalteromonas tunicata D2.</a> Appl. Environ. Microbiol. 76, 5710-5717 (2010).</li></ol>

的Ewbank实验室合作联盟Biometrica和MODUL生物。

<p class="jove_content"目前的协议允许大规模RNAi筛选<em&gt; C。线虫</em&gt;固体介质，采用荧光报告基因的表达水平作为一个读出。它已被优化与自动化工具，以方便高通量检测，可以轻松快速地处理大量的样品，在一个高效，可靠和可重复性的方式。其他方法已不依靠自动化液体样品处理如此严重<sup&gt; 8</sup&gt;。</p&gt;<p class="jove_content"一个关键的组织方面的协议已经发展强大的样品跟踪方法的RNAi屏幕程序与公司ModulBio合作适应商业MBioLIMS的软件。在每一个阶段的过程中，板内LIMS生成条形码标识。作为LIMS也记录在每口井的克隆的身份，每块板良好公认的基因也很简单。和超越，LIMS的链接社区数据库WormBase，允许直接访问到任何特定基因的分子和功能数据。 LIMS系统也有利于板甚至染色体和全基因组水平上进行分析。对于这些分析，数据存储的一种方式，是完全兼容先前所描述的工具如COPAmulti<sup&gt; 16</sup&gt;。</p&gt;<p class="jove_content"&gt;实验方法的一个关键点是调整食品的数量相对于蠕虫的数量。对于许多的分析，它是必不可少的，蠕虫不饿死在实验结束，但不会有开始了过多的食物也很重要。我们观察到高密度的食物，减少在使用蠕虫携带的P协议荧光诱导<em&gt; NLP-29</em&gt; :: GFP的记者。这可能反映了当蠕虫在太多的细菌培养（学士，结果未显示）的感染效率下降。同时，RNA干扰细菌的培养条件也很重要，因为这决定是否在最佳水平产生双链RNA。一些大型的RNAi协议需要文化琼脂平板上的RNAi克隆之前播种液体培养<sup&gt; 8</sup&gt;。我们获得强大的基因沉默，省略这一步。我们选择成长超过每一种文化，以确保均匀的结果。其实描述的协议提供了足够的材料，同时进行2个或3个屏幕。在利益获得最​​强大的结果，我们赞成，但是，进行屏幕重复使用的蠕虫病毒，RNA干扰细菌和真菌孢子的独立文化。</p&gt;<p class="jove_content"另一个关键步骤与使用介质既不是太潮湿也不过于干燥。如果还没干板，RNA干扰细菌不干燥正确和蠕虫将在检测游泳。这可能导致问题。例如，蠕虫都不能有效地感染<em&gt; D coniospora</em在液体（C. Couillault，未发表结果）。另一方面，在干板，培养基的渗透压增加。这有蠕虫生理上的直接影响<sup&gt; 17</sup&gt;，其中包括一些抗菌肽基因，如感应，<em&gt; NLP 29</em&gt;<sup&gt; 18</sup&gt;。用于免除蠕虫和孢子的体积是至关重要的，以便有效和快速干燥。如果超过10μL的液体添加到新鲜的板，井可以采取多小时，晾干。这可能意味着它是很难用机器人的方法来添加蠕虫板。例如，每个蠕虫Biosort约1μL液体配药。目前，我们手动分发的蠕虫，也使我们能够克服解决泥沙蠕虫的趋势。</p&gt;<p class="jove_content"我们以前报道的Biosort的调整，允许一个96孔板，在36分钟内分析<sup&gt; 19</sup&gt;。充氧机的制造商现在提供了修改Biosort，进一步减少分析时间。样品与“FastReFlex”，是直接经由流细胞进行分析，过滤绕过泡沫陷阱。这样的结果，在数据采集的时间显着减少;整个板块可以在22分钟允许各地每天要处理20板块分析。然而，如果不冻结板，将之间的第一个和最后的时间间隔8小时。由于实验的时间相对较短，历时仅18小时的感染，这将推出跨板不可接受的变量。不仅冻结板块分析前解决这个问题，但它也是在高通量分析中是必不可少的，与当前的协议和标准的液体处理机器人，2人可以很容易地处理多达50板块一周。目前，我们经常一个星期屏幕30板，这使得整个基因组屏幕在不到2个月进行一次。</p&gt;<p class="jove_content"这个协议的一个有趣的特点是，它采用固体琼脂，允许不能在培养液中进行的实验事实。由于蠕虫生理上的文化条件不同而异<sup&gt; 20,21</sup&gt;，能够以固体培养基上进行检测，还可以补充平时液基屏幕。对于某些表型（如运动），分析，可以直接与96孔固体培养基板进行。但是，对于当前的屏幕，作为Biosort可以不品尝从固体培养基平板，蠕虫首先转移到液体冻结前。另一方面，COPAS的Biosort允许兼容许多不同表型的测量多参数定量分析。很可能，对于任何给定的屏幕并不是所有的测量参数，将主读出。然而，这些测量结果有以下几个原因可能是有用的。他们允许之一，以确定是否检测正确运行。例如，可以检查每口井的蠕虫病毒的数量和规模。唯一的RNAi克隆应该阻止生长或繁殖应该有一个预期值的偏差。井不少于20蠕虫和/或动物减少30％（TOF）的一个标准相比dard控制生长没有影响（<em&gt;如绿色荧光蛋白（RNA干扰</em&gt;）），可以排除后续分析。这些克隆的功能，可以使用替代协议，其中蠕虫接触到的RNAi细菌后，他们的发展是先进<sup&gt; 15</sup&gt;。不同的参数，也可以用来提炼的候选基因（HIT）的选择，例如，限制只有那些影响绿色荧光，但不红的命中。精确的方法，选择点击选择将取决于屏幕类型和它的准确读出。鉴于从定量的全基因组的屏幕产生的数据的复杂性，可能需要与统计学家合作。</p&gt;<p class="jove_content"我们所描述的协议，可以很容易地适应其他类型的检测。例如，在感染步骤<em&gt; D coniospora</em&gt;可取代任何其他的病原体能够感染固体培养基上。这可能是有用的，因为我们已经找到了许多重要的致病因素，在培养液中粘质沙雷氏菌感染不感染过程中发挥作用固体培养基上（E. Pradel，个人通讯）。该协议也是完全兼容测试化学品化合物<sup&gt; 22-24</sup&gt;，或以确定使用不同的细菌库的生物活性化合物<sup&gt; 25</sup&gt;，所以应该找一个在一般工具<em&gt; C。线虫</em&gt;研究界。</p

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> BactoAgar </td><td>屋宇署诊断系统</td><td> 214010 </td><td></td></tr><tr><td>氨苄青霉素</td><td> Sigma-Aldrich公司</td><td> A9518 </td><td></td></tr><tr><td> BactoPeptone </td><td>屋宇署诊断系统</td><td> 211677 </td><td></td></tr><tr><td> 氯化钙 </td><td>任何供应商</td><td></td><td></td></tr><tr><td> AeraSeal胶薄膜</td><td> dutscher </td><td> 760214 </td><td></td></tr><tr><td>异丙基β-D-半乳糖苷（知识产权TG）， </td><td> Thermo Scientific的</td><td> AB-0481 </td><td></td></tr><tr><td> K 2 HPO 4 </td><td>任何供应商</td><td></td><td></td></tr><tr><td> KH 2 PO 4 </td><td>任何供应商</td><td></td><td></td></tr><tr><td> 硫酸镁 </td><td>任何供应商</td><td></td><td></td></tr><tr><td>氯化钠</td><td>任何供应商</td><td></td><td></td></tr><tr><td>的Na 2 HPO 4 </td><td>任何供应商</td><td></td><td></td></tr><tr><td>氢氧化钠</td><td>任何供应商</td><td></td><td></td></tr><tr><td> 96孔盘</td><td> Thermo Scientific的</td><td> AB-0932 </td><td></td></tr><tr><td> 96孔平板</td><td>鹰</td><td> 353072 </td><td></td></tr><tr><td> 96轮板</td><td>鹰</td><td> 353077 </td><td></td></tr><tr><td>四环素</td><td> Sigma-Aldrich公司</td><td> T8032 </td><td></td></tr><tr><td>的Triton X-100 </td><td>任何供应商</td><td></td><td></td></tr><tr><td> TECAN机器人</td><td> TECAN </td><td></td><td></td></tr><tr><td> liquidator96 商标 </td><td> RAININ </td><td></td><td></td></tr><tr><td> COPAS Biosort </td><td>联盟Biometrica </td><td></td><td></td></tr><tr><td> LIMS系统</td><td> MODUL-生物</td><td></td><td></td></tr></tbody></table>

quantitative and automated high-throughput genome-wide rnai screens in c. elegans

RNA干扰是一个功能强大的方法来了解基因功能，尤其是当在整个基因组的规模和在定量的情况下进行。 在 C 线虫基因功能可以被撞倒，简单而有效地喂养与对应一个特定的基因1表达双链RNA的细菌的蠕虫。而RNAi技术克隆库的建立覆盖大部分的C.线虫基因组2,3开真正的功能基因组研究（例如4-7）的方式，最成熟的方法是费力。莫伊和他的同事们已经开发出了半自动化的协议，促进全基因组的屏幕8。该方法依赖于显微成像和图像分析。 在这里，我们描述为一种高通量的基因组全屏幕的替代协议，根据机器人细菌RNA干扰克隆，定量分析使用的COPAS的Biosort（联盟Biometrica（充氧））的处理，以及集成软件：MBioLIMS（MODUL生物实验室信息管理系统）技术，提供数据管理和样品跟踪吞吐量增加。该方法允许屏幕固体培养基平板上进行。这是特别重要的一些研究，如处理宿主-病原体相互作用在 C， 线虫 ，因为某些微生物不有效地感染蠕虫液体培养。 我们展示如何使用该方法可用于量化C.先天免疫在抗真菌基因的重要性线虫 。在这种情况下，该方法依赖于利用携带GFP的抗菌肽基因NLP 29的启动和红色荧光记者的控制下表皮感染诱导荧光报告基因，转基因的菌株组成，表示在表皮。后者提供了一个功能完整的表皮的内部控制和非特异性基因沉默9。当控制蠕虫是由真菌感染荧光绿色。 NLP 29日在感染后减弱荧光表达结果需要一个基因的RNAi击倒。目前，该协议允许每周进行测试和分析了3000多RNAi的克隆，开放在不到2个月的全基因组筛选的可能性。

Watch this Scientific Journal Video about Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans at JoVE.com

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

我们描述了协议使用<em&gt; C。线虫</em&gt;和RNAi喂养库，可以自动测量多个参数，如荧光，规模和不透明的人口中的个别蠕虫。我们给一个屏幕上的一个例子，以确定在抗真菌先天免疫有关的基因<em&gt; C。线虫</em&gt;。

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. ...

Research

JoVE Journal

Biology

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

Development of automated imaging and analysis for zebrafish chemical screens.

斑马鱼化工屏幕自动成像和分析的发展。

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK ...

科研

生物学

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

采用芯片识别亚型特定的靶基因的全基因组分析

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the ...

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

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

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

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 ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

高通量使用自动染色体的物理图谱<em&gt;原位</em&gt;杂交

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

全基因组基因缺失<em&gt;链球菌血统</em通过高通量PCR

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

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 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 ...

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin

药物与DNA相互作用的细胞全基因组映射与COSMIC（小分子交联，染色质隔离）

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to ...