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。

<ol>
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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. ...

quantitative-automated-high-throughput-genome-wide-rnai-screens-c

Research

JoVE Journal

Biology

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

17.5K 次观看.  Université de la Méditerranée. 我们描述了协议使用 C。线虫和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 C。线虫。

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

Development of automated imaging and analysis for zebrafish chemical screens.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens 1. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates 2. In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling 3. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos.

We report the development of a system for automated imaging and analysis of zebrafish transgenic embryos in multiwell plates. This demonstrates the ability to measure dose dependent effects of a small molecule, BCI, on Fibroblast Growth Factor reporter gene expression and provide technology for establishing high-throughput 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

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3)1. The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes2. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me33. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform4. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner.
To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.

Here we are presenting a chromatin immunoprecipitation (ChIP) procedure for genome-wide location analysis of protein isoforms that differ in a histone-binding domain. We are applying it to ChIP-Seq analysis to identify the targets of the KDM5A/JARID1A/RBP2 histone demethylase.

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 (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 ...

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

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 over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

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

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

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

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

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

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

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

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

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

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 obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

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

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

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

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

高通量筛选转录后调控基因的化学调节剂

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

DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is an alternative approach to chromatin immunoprecipitation (ChIP). An expressed fusion protein consisting of the protein of interest and the E. coli DNA adenine methyltransferase can methylate the adenine base in GATC motifs near the sites of protein-DNA interactions. Adenine-methylated DNA fragments can then be specifically amplified and detected. The original DamID assay detects the genomic locations of methylated DNA fragments by hybridization to DNA microarrays, which is limited by the availability of microarrays and the density of predetermined probes. In this paper, we report the detailed protocol of integrating high throughput DNA sequencing into DamID (DamID-seq). The large number of short reads generated from DamID-seq enables detecting and localizing protein-DNA interactions genome-wide with high precision and sensitivity. We have used the DamID-seq assay to study genome-nuclear lamina (NL) interactions in mammalian cells, and have noticed that DamID-seq provides a high resolution and a wide dynamic range in detecting genome-NL interactions. The DamID-seq approach enables probing NL associations within gene structures and allows comparing genome-NL interaction maps with other functional genomic data, such as ChIP-seq and RNA-seq.

We describe herein an assay by coupling DNA adenine methyltransferase identification (DamID) to high throughput sequencing (DamID-seq). This improved method provides a higher resolution and a wider dynamic range, and allows analyzing DamID-seq data in conjunction with other high throughput sequencing data such as ChIP-seq, RNA-seq, etc.

DamID-SEQ：蛋白质-DNA相互作用的全基因组映射腺嘌呤甲基化的DNA片段通过高通量测序

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is ...