我们感谢罗恩戴维斯，亚当Deutschbauer，整个臀部在多伦多大学的讨论和意见的跳实验室。 CN是由国家人类基因组研究所（批准号HG000205），RO1 HG003317，CIHR澳门币84305，加拿大癌症协会（＃020380）的赠款支持。联办是由斯坦福大学的基因组培训计划（批准数的T32 HG00044从国家人类基因组研究所）和国立卫生研究院（批准号P01 GH000205）支持。 GG是在NHGRI RO1 HG003317和加拿大癌症协会，格兰特＃020380，运输署和唐纳利测序中心的支持，部分是从加拿大创新到DRS基金会的赠款支持。布兰达安德鲁斯和杰克布拉特。 AMS是由多伦多大学打开奖学金支持。

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没有利益冲突的声明。

在这里，我们提出了一个协议，适度的修改，可以很容易地适应广泛的现有馆藏的创建标签突变馆藏的条形码不同的微生物突变集合。我们强调，虽然我们已经报道了标签的转座子突变致病性酵母菌 C.一个协议白色念珠菌 ，一个非常类似的协议，可以适应各种各样的单细胞真菌。修改，该协议运作良好13中的细菌，和目前正在建设中一些额外的真菌和细菌的基因组的集合。目前，这个实验提供了唯一的综合性，全基因组基因的小分子的相互作用的公正屏幕。其中特别引人注目的检测功能是没有的基因或小分子的先验知识是必需的。尽管这些检测的范围和权力，其可转让给其他实验室已经有点阻碍，由最初的资本投资和信息工具，分析的结果。通过结合分析的强大工具的下一代序列读数，我们期望通过增加。

<table border="1"><tbody><tr><td> 抗生素 </td><td> 供应商和产品目录号</td></tr><tr><td>羧</td><td> Sigma公司的一部分＃C1613 </td></tr><tr><td>卡那霉素</td><td> Sigma公司的一部分＃K1876 </td></tr><tr><td>大观</td><td> Sigma公司的一部分＃S0692 </td></tr><tr><td>氯霉素</td><td> Sigma公司的一部分＃C0378 </td></tr><tr><td> 清理和浓度试剂盒的DNA </td><td></td></tr><tr><td> QIAprep自旋小量制备试剂盒</td><td> Qiagen公司，部分＃27106 </td></tr><tr><td> HiSpeed​​质粒马克西套件</td><td> Qiagen公司，部分＃12663 </td></tr><tr><td> QIAquick PCR纯化试剂盒</td><td> Qiagen公司，部分＃28106 </td></tr><tr><td> QIAquick凝胶提取试剂盒</td><td> Qiagen公司，部分＃28704 </td></tr><tr><td> PCR和电泳试剂 </td><td></td></tr><tr><td> Taq DNA聚合酶（MG -免费）缓冲区</td><td> New England Biolabs公司的一部分，＃M0320L </td></tr><tr><td>脱氧核苷酸的解决方案组合</td><td> New England Biolabs公司的一部分，＃N0447L </td></tr><tr><td> 25毫米氯化镁2 </td><td>西格玛，部分＃63036 </td></tr><tr><td>琼脂糖，装载染料，核酸染色适用于凝胶电泳</td><td>各种</td></tr><tr><td> 10X TAE缓冲液</td><td> Sigma公司的一部分＃T8280 </td></tr><tr><td> 1 KB PLUS DNA梯状</td><td> Invitrogen公司的一部分＃10787026 </td></tr><tr><td> YPD肉汤 </td><td></td></tr><tr><td> 10克酵母提取物</td><td> Sigma公司的一部分＃Y1625 </td></tr><tr><td>细菌用胰蛋白胨20克</td><td> BD公司的一部分，＃211677 </td></tr><tr><td> 20克的葡萄糖</td><td> Sigma公司的一部分＃D9434 </td></tr><tr><td> 医学实验 </td><td></td></tr><tr><td>多通道移液器（1000，200，和20μL） </td><td>各种</td></tr><tr><td>一次性移液器水库</td><td>各种</td></tr><tr><td> 15日和50 mL离心管</td><td>各种</td></tr><tr><td> 96 - 384孔深井板</td><td>爱思进科学，一部分的P - 2ML - SQ - CS＆P - 384240SQCS </td></tr><tr><td> 96孔和384孔PCR板和密封膜</td><td>各种</td></tr><tr><td>版辊密封多孔板</td><td> Sigma公司的一部分＃R1275 </td></tr><tr><td> 30 ° C和37 ° C摇晃增长的平板上的细菌和酵母和孵化器在管</td><td>各种</td></tr><tr><td> 在体外转座子诱变 </td><td></td></tr><tr><td> EZ - Tn5的转座子</td><td>震中生物技术的一部分，＃TNP92110 </td></tr><tr><td> 高通量转型 </td><td></td></tr><tr><td> Seqprep 96 HT质粒准备工具包</td><td>边缘生物系统公司的一部分，＃84359 </td></tr><tr><td>聚乙二醇，分子量3350 </td><td>各种</td></tr><tr><td>醋酸锂</td><td> Sigma公司的一部分＃517992 </td></tr><tr><td> 6孔板，无菌</td><td>康宁，部分＃3335 </td></tr><tr><td> 50毫克/毫升尿苷</td><td> Sigma公司的一部分＃U3750 </td></tr><tr><td> 100X的Tris - EDTA缓冲液</td><td> Sigma公司的一部分＃T9285 </td></tr><tr><td> 1X TE/0.1M LiOAc </td><td>各种</td></tr><tr><td>鲑鱼睾丸DNA </td><td> Sigma公司的一部分＃1626 </td></tr><tr><td> 增长的条形码集合 </td><td></td></tr><tr><td> 48孔板;如果增长板中的文化</td><td>格雷纳，部分＃M9437 </td></tr><tr><td>胶粘板密封</td><td> ABgene，部分＃AB - 0580 </td></tr><tr><td> 200毫升的培养瓶中</td><td>各种</td></tr><tr><td>能够分光光度计吸光度</td><td>各种</td></tr><tr><td>控温摇床，250毫升烧瓶或晃动分光光度计</td><td>各种</td></tr><tr><td>安全锁的离心管中，2毫升</td><td> Eppendorf公司的一部分，0030 120.094 </td></tr><tr><td> 杂交设备 </td><td></td></tr><tr><td>杂交炉640 </td><td> Affymetrix公司的一部分＃800138 </td></tr><tr><td>基因芯片流体站450 </td><td> Affymetrix公司，部分＃00-0079 </td></tr><tr><td> GeneArray扫描仪3000 </td><td> Affymetrix公司，部分＃00-0212 </td></tr><tr><td>沸水浴浮动机架</td><td>各种</td></tr><tr><td> 杂交耗材 </td><td></td></tr><tr><td> Genflex标签16K阵列V2 </td><td> Affymetrix，一部分＃511331 </td></tr><tr><td>登哈特的解决方案，集中50X </td><td> Sigma公司的一部分＃D2532 </td></tr><tr><td>链霉亲和，R -藻红蛋白共轭（萨佩） </td><td> Invitrogen公司的一部分＃S866 </td></tr><tr><td>安全锁的离心管，0.5毫升</td><td> Eppendorf公司的一部分，0030 123.301 </td></tr><tr><td>蝇头强硬点</td><td>多样化的生物技术，部分＃LTTM - 1000 </td></tr><tr><td> 0.5 M EDTA </td><td> BioRad公司，部分＃161-0729 </td></tr><tr><td> 10％吐温</td><td> Sigma公司的一部分＃T2700 </td></tr><tr><td> MES的自由酸水合物</td><td> Sigma公司的一部分＃M5287 </td></tr><tr><td> MES钠盐</td><td> Sigma公司的一部分＃M5057 </td></tr><tr><td> 5 M氯化钠</td><td>西格玛，部分＃71386 </td></tr><tr><td> 20X SSPE </td><td> Sigma公司的一部分＃S2015 </td></tr><tr><td>分子生物学级水</td><td> Sigma公司的一部分＃W4502 </td></tr><tr><td> 杂交引物 </td><td> 各供应商（标准除盐） </td></tr><tr><td> Uptag </td><td> 5&#39;GATGTCCACGAGGTCTCT 3&#39; </td></tr><tr><td> Buptagkanmx4 </td><td> 5&#39;生物素GTCGACCTGCAGCGTACG 3 </td></tr><tr><td> Dntag </td><td> 5&#39;CGGTGTCGGTCTCGTAG 3&#39; </td></tr><tr><td> Bdntagkanmx4 </td><td> 5&#39;生物素GAAAACGAGCTCGAATTCATCG 3 </td></tr><tr><td> B213 </td><td> 5&#39;生物素CTGAACGGTAGCATCTTGAC 3 </td></tr><tr><td> Uptagkanmx </td><td> 5&#39;GTCGACCTGCAGCGTACG 3“ </td></tr><tr><td> Dntagkanmx </td><td> 5&#39;GAAAACGAGCTCGAATTCATCG 3“ </td></tr><tr><td> Uptagcomp </td><td> 5&#39;AGAGACCTCGTGGACATC 3“ </td></tr><tr><td> Dntagcomp </td><td> 5&#39;CTACGAGACCGACACCG 3“ </td></tr><tr><td> Upkancomp </td><td> 5&#39;CGTACGCTGCAGGTCGAC 3“ </td></tr><tr><td> Dnkancomp </td><td> 5&#39;CGATGAATTCGAGCTCGTTTTC 3“ </td></tr><tr><td> 测序引物：Illumina公司平台 </td><td> 各供应商 </td></tr><tr><td> UpTag远期（100um的） </td><td> GGC GAC的阿拉木图5“民航局GCA GAA CGA GCT CTT CCG ATC T GAT GTC CAC GAG GTC的大老山隧道3” </td></tr><tr><td> UpTag反向（100um的） </td><td> GCG ACG的行政协调会5“学能测验的GAT ACC GAC中的ACT铁通台泥税务师CAC GAC GCT CTT CCG ATC T NNNNN GTC GAC CTG CAG总工会ACG的3&#39; </td></tr><tr><td> DownTag远期（100um的） </td><td> GGC GAC的阿拉木图 5“ 民航局GCA GAA CGA GCT CTT CCG ATC T GAA AAC聚糖反恐委员会GAA TTC ATC地下3” </td></tr><tr><td> DownTag反向（100um的） </td><td> GCG ACG的行政协调会 5“ 学能测验的GAT ACC GAC中的ACT铁通台泥税务师CAC GAC GCT CTT CCG ATC T NNNNN CGG公司的TGT CGG大老山隧道总工会公司3` </td></tr><tr><td>阅读1标签SEQ引物（100um的） </td><td> 5&#39;GTC GAC CTG C​​AG总工会ACG的3&#39; </td></tr><tr><td>阅读1下标签SEQ引物（100um的） </td><td> CGG公司的TGT CGG大老山隧道CGT公司5&#39;3&#39; </td></tr><tr><td>阅读指数测序引物（标准Illumina的R1底漆）（100um的） </td><td> 5“交流使铁通台泥税务师CAC GAC GCT CTT CCG ATC T 3” </td></tr><tr><td> 更多的测序试剂/设备</td><td></td></tr><tr><td> Qiagen公司MinElute 96 UF PCR纯化试剂盒</td><td> Qiagen公司，部分＃29051 </td></tr><tr><td>真空泵</td><td>任何供应商</td></tr><tr><td>马歇雷-纳格尔真空歧管</td><td>马歇雷纳格尔，部分＃740 681 </td></tr><tr><td> Invitrogen公司QUANT它双链BR检测试剂盒</td><td> Invitrogen公司的一部分＃Q32853 </td></tr><tr><td> Invitrogen公司的量子位的检测管</td><td> Invitrogen公司的一部分＃Q32856 </td></tr><tr><td>丙烯酰胺40％加1％N，N&#39; -亚甲基双丙烯酰胺，37.5:1 </td><td>生物RAD，部分＃161-0148 </td></tr><tr><td>三基地</td><td> Sigma公司的一部分＃T1503 - 1KG </td></tr><tr><td>硼酸</td><td> Sigma公司的一部分＃B6768 - 500G </td></tr><tr><td> 0.5M EDTA，pH值8.0 </td><td> Teknova，部分＃E0306 </td></tr><tr><td>过硫酸铵</td><td> Sigma公司的一部分＃A3678 - 25G </td></tr><tr><td> TEMED </td><td> Bioshop，部分＃TEM001.25 </td></tr><tr><td>溴化乙锭溶液</td><td> Bioshop，部分＃ETB444.10 </td></tr><tr><td> 0.5M铵酯</td><td> Teknova，部分＃A2000 </td></tr><tr><td> 10MM镁醋酸水合物</td><td> Sigma公司的一部分＃M0631 - 100G </td></tr><tr><td>为1mM EDTA，pH值8.0 </td><td> 0.5M EDTA，pH值8.0 </td></tr><tr><td>乙醇</td><td>各种</td></tr><tr><td>醋酸钠，pH值5.2 </td><td> Teknova，部分＃S0297 </td></tr><tr><td>高速真空</td><td>各种</td></tr><tr><td>单读集群生成工具包</td><td> Illumina公司的一部分，GD - 300 - 1001 </td></tr><tr><td> 36C测序试剂盒V4 </td><td>Illumina公司，部分FC - 104 - 4002 </td></tr><tr><td></td><td></td></tr></tbody></table> 10X TBE缓冲液配方<table border="1"><tbody><tr><td>应收</td><td>试剂</td></tr><tr><td> 108克</td><td>三基地</td></tr><tr><td> 55克</td><td>硼酸</td></tr><tr><td> 40ML </td><td> 0.5M EDTA（pH值8.0） </td></tr><tr><td colspan="2"> DH 2 O至1L标志</td></tr></tbody></table> 12％的聚丙烯酰胺凝胶配方<table border="1"><tbody><tr><td>卷</td><td>试剂</td></tr><tr><td> 5.8毫升</td><td>丙烯酰胺40％加1％N，N&#39; -亚甲基双丙烯酰胺，37.5:1 </td></tr><tr><td> 12毫升</td><td> DH 2 O </td></tr><tr><td> 2毫升</td><td> 10X TBE </td></tr><tr><td> 140μL </td><td> 10％过硫酸铵</td></tr><tr><td>总成交量：20毫升</td><td></td></tr></tbody></table> Uptag引物组合： 悬浮Uptag Buptagkanmx4在DDH 2 O的每100微米，然后以1:1的比例混合，终浓度为50微米。储存在-20 ° C。 Downtag引物组合： 悬浮Dntag Bdntagkanmx4 DDH 2 O在100微米，然后以1:1的比例混合，终浓度为50微米。储存在-20 ° C。 混合寡核苷酸： 重悬在DDH 2 O在100微米以下八个寡核苷酸（标准脱盐）： Uptag，Dntag，Uptagkanmx，Dntagkanmx，Uptagcomp，Dntagcomp，Upkancomp，Dnkancomp。 混合同等体积的八个寡核苷酸终浓度为12.5μM。 12X MES的股票： 10毫升，0.7克的MES游离酸水合物和1.93克MES钠盐溶于8毫升分子生物学级水。搅拌好后，检查pH值和调整，如果需要的话pH值6.5-6.7。加水到10毫升的总量。过滤消毒，并保存在4 ° C的光（例如，包装箔管）保护。替换，如果溶液变成明显的黄色或6个月后。 2X杂交缓冲液： 对于50毫升，混合12X MES的股票（从14年9月2日）8.3毫升，17.7毫升5 M氯化钠，4.0毫升的0.5 M EDTA，0.1毫升10％的吐温20（体积/体积），19.9毫升，过滤DDH 2 O。过滤消毒。 洗了：混合300毫升20X SSPE，1 mL 10％的吐温（体积/体积），699毫升DDH 2 O。过滤消毒。 洗净乙：混合150毫升20X SSPE，1 mL 10％的吐温（体积/体积），849毫升DDH 2 O过滤消毒。 条码测序引物 -标记引物序列的5&#39;尾巴（ 粗体 ）Illumina的特定适配器纳入F和R引物序列。变量序列（ 斜体字 ）5 MER索引标签，用于在复用/索引读出。 3&#39;尾巴（下划线）代表的共同引物侧翼uptag条码，需要放大酵母条码。 下调标记的引物序列5&#39;尾巴是相同的5&#39;UP标记的引物（Illumina的具体序列）的尾巴，然而，3&#39;尾巴（下划线）所使用的共同引物扩增取代向下标签条形码。

competitive genomic screens of barcoded yeast libraries

凭借在新一代测序技术的进步，我们几乎每天都获得新的基因组序列。这些进步的步伐正在加快，前途更大的深度和广度。鉴于这些非凡的进步，需要快速，并行的方法，以确定基因功能变得更加重要。集合的全基因组的缺失突变体， 酵母菌和大肠杆菌大肠杆菌曾担任基因功能的功能特性的工作母机，但这种方法是不可伸缩的，目前的基因缺失方法要求每个成千上万的基因组成基因组中被删除，并核实的。只有这项工作完成后，我们可以追求高通量的表型。在过去的十年，我们的实验室已提炼组合的竞争力，小型化，高通量的基因组范围内，可并行执行检测。这是可能的并行，因为DNA“标签”，或“条码”，到每个突变列入，条形码突变的代理服务和一个可以衡量条码丰评估突变体健身。在这项研究中，我们设法填补DNA序列和条形码突变集合之间的差距。而要做到这一点，我们引入一个结合转座子中断条码的方法，开辟了并行条码检测新测序，但较差的特点微生物。为了说明这种方法，我们提出一个新的白色念珠菌条形码中断收集和描述如何将两个芯片为基础的下一代测序为基础的平台，可以用来收集万- 100万在一次实验中基因-基因和基因药物的相互作用。

Watch this Scientific Journal Video about Competitive Genomic Screens of Barcoded Yeast Libraries at JoVE.com

Competitive Genomic Screens of Barcoded Yeast Libraries

我们已经开发出全面的，无偏见的全基因组的屏幕，了解基因药物和基因与环境的相互作用。筛选这些突变集合的方法。

条形码酵母图书馆竞争力的基因屏幕

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

competitive-genomic-screens-of-barcoded-yeast-libraries

Research

JoVE Journal

Biology

18.1K 次观看.  University of Toronto. 我们已经开发出全面的，无偏见的全基因组的屏幕，了解基因药物和基因与环境的相互作用。筛选这些突变集合的方法。

视频: Competitive Genomic Screens of Barcoded Yeast Libraries

Large-Scale Screens of Metagenomic Libraries

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation. Generating a streamlined procedure for creating and screening metagenomic libraries is therefore useful for efficient high-throughput investigations into the genetic and metabolic properties of uncultured microbial assemblages. Here, key protocols are presented on video, which we propose is the most useful format for accurately describing a long process that alternately depends on robotic instrumentation and (human) manual interventions. First, we employed robotics to spot library clones onto high-density macroarray membranes, each of which can contain duplicate colonies from twenty-four 384-well library plates. Automation is essential for this procedure not only for accuracy and speed, but also due to the miniaturization of scale required to fit the large number of library clones into highly dense spatial arrangements. Once generated, we next demonstrated how the macroarray membranes can be screened for genes of interest using modified versions of standard protocols for probe labeling, membrane hybridization, and signal detection. We complemented the visual demonstration of these procedures with detailed written descriptions of the steps involved and the materials required, all of which are available online alongside the video.

宏基因组库的大型屏幕

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation.  Generating a ...

科研

生物学

Isolation of Genomic DNA from Mouse Tails

从鼠尾基因组DNA提取

Large Insert Environmental Genomic Library Production

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approaches have emerged, enabling researchers to bridge this cultivation gap by capturing the genetic content of indigenous microbial communities directly from the environment. To this end, genomic DNA libraries are constructed using standard albeit artful laboratory cloning techniques. Here we describe the construction of a large insert environmental genomic fosmid library with DNA derived from the vertical depth continuum of a seasonally hypoxic fjord. This protocol is directly linked to a series of connected protocols including coastal marine water sampling [1], large volume filtration of microbial biomass [2] and a DNA extraction and purification protocol [3]. At the outset, high quality genomic DNA is end-repaired with the creation of 5 -phosphorylated blunt ends. End-repaired DNA is subjected to pulsed-field gel electrophoresis (PFGE) for size selection and gel extraction is performed to recover DNA fragments between 30 and 60 thousand base pairs (Kb) in length. Size selected DNA is purified away from the PFGE gel matrix and ligated to the phosphatase-treated blunt-end fosmid CopyControl vector pCC1 (EPICENTRE <a href="http://www.epibio.com/item.asp?ID=385">http://www.epibio.com/item.asp?ID=385</a>). Linear concatemers of pCC1 and insert DNA are subsequently headfull packaged into phage particles by lambda terminase, with subsequent infection of phage-resistant E. coli cells. Successfully transduced clones are recovered on LB agar plates under antibiotic selection and archived in 384-well plate format using an automated colony picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the CopyControl Fosmid Library Production Kit from EPICENTRE and the published works of multiple research groups [4-7]. Each step is presented with best practice in mind. Whenever possible we highlight subtleties in execution to improve overall quality and efficiency of library production. The whole process of fosmid library production and automated colony picking takes at least 7-10 days as there are many incubation steps included. However, there are several stopping points possible which are mentioned within the protocol.

Construction of a fosmid library with environmental genomic DNA isolated from the vertical depth continuum of a seasonally hypoxic fjord is described. The resulting clone library is picked into 384-well plates and archived for downstream sequencing and functional screening by the application of an automated colony picking system.

大型插入环境基因组文库生产

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent ...

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

从酵母细胞线粒体的纯化

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

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 ...

Yeast Colony Embedding Method

Patterning of different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms also display patterns of cell types. For example, in the yeast S. cerevisiae, sporulated cells and pseudohyphal cells are not uniformly distributed in colonies. The functional importance of patterning and the molecular mechanisms that underlie these patterns are still poorly understood.
One challenge with respect to investigating patterns of cell types in fungal colonies is that unlike metazoan tissue, cells in colonies are relatively weakly attached to one another. In particular, fungal colonies do not contain the same extensive level of extracellular matrix found in most tissues . Here we report on a method for embedding and sectioning yeast colonies that reveals the interior patterns of cell types in these colonies. The method can be used to prepare thick sections (0.5 &mu;) useful for light microscopy and thin sections (0.1 &mu;) suitable for transmission electron microscopy. Asci and pseudohyphal cells can easily be distinguished from ovoid yeast cells by light microscopy , while the interior structure of these cells can be visualized by EM.
The method is based on surrounding colonies with agar, infiltrating them with Spurr's medium, and then sectioning. Colonies with a diameter in the range of 1-2 mm are suitable for this protocol. In addition to visualizing the interior of colonies, the method allows visualization of the region of the colony that invades the underlying agar.

A method for embedding yeast colonies allowing sectioning for light and electron microscopy. This protocol allows determination of the distribution of sporulated cells and pseudohyphal cells within colonies providing a new tool toward understanding the organization of cell types within a fungal community.

酵母殖民地嵌入方法

Patterning of  different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms ...

Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova &amp; Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009).
Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition.

We present a public computational web site for the analysis of genomic sequences. It detects DNA sequence patterns with various non-random nucleotide compositions. This resource also generates randomized sequences with diverse levels of complexity.

磁共振成像基因 - 公共资源为研究基因组DNA序列内模式

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in ...

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic redundancy affecting simple genetics in higher plants. The novel model organism Ostreococcus tauri is the smallest free-living eukaryote known to date, and possesses a greatly reduced genome size and cellular complexity1,2, manifested by the presence of just one of most organelles (mitochondrion, chloroplast, golgi stack) per cell, and a genome containing only ~8000 genes. Furthermore, the combination of unicellularity and easy culture provides a platform amenable to chemical biology approaches. Recently, Ostreococcus has been successfully employed to study basic mechanisms underlying circadian timekeeping3-6. Results from this model organism have impacted not only plant science, but also mammalian biology7. This example highlights how rapid experimentation in a simple eukaryote from the green lineage can accelerate research in more complex organisms by generating testable hypotheses using methods technically feasible only in this background of reduced complexity. Knowledge of a genome and the possibility to modify genes are essential tools in any model species. Genomic1, Transcriptomic8, and Proteomic9 information for this species is freely available, whereas the previously reported methods6,10 to genetically transform Ostreococcus are known to few laboratories worldwide.
In this article, the experimental methods to genetically transform this novel model organism with an overexpression construct by means of electroporation are outlined in detail, as well as the method of inclusion of transformed cells in low percentage agarose to allow selection of transformed lines originating from a single transformed cell. Following the successful application of Ostreococcus to circadian research, growing interest in Ostreococcus can be expected from diverse research areas within and outside plant sciences, including biotechnological areas. Researchers from a broad range of biological and medical sciences that work on conserved biochemical pathways may consider pursuing research in Ostreococcus, free from the genomic and organismal complexity of larger model species.

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

This article describes genetic transformation of the unicellular marine alga Ostreococcus tauri by electroporation. This eukaryotic organism is an effective model platform for higher plants, possesing greatly reduced genomic and cellular complexity and being readily amenable to both cell culture and chemical biology.

基因改造的Picoeukaryote<em&gt; Ostreococcus金牛</em

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

从基因组DNA选择性捕获的5  - 羟甲基

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Assessing Differences in Sperm Competitive Ability in Drosophila

Competition among conspecific males for fertilizing the ova is one of the mechanisms of sexual selection, i.e. selection that operates on maximizing the number of successful mating events rather than on maximizing survival and viability 1. Sperm competition represents the competition between males after copulating with the same female 2, in which their sperm are coincidental in time and space. This phenomenon has been reported in multiple species of plants and animals 3. For example, wild-caught D. melanogaster females usually contain sperm from 2-3 males 4. The sperm are stored in specialized organs with limited storage capacity, which might lead to the direct competition of the sperm from different males 2,5.
Comparing sperm competitive ability of different males of interest (experimental male types) has been performed through controlled double-mating experiments in the laboratory 6,7. Briefly, a single female is exposed to two different males consecutively, one experimental male and one cross-mating reference male. The same mating scheme is then followed using other experimental male types thus facilitating the indirect comparison of the competitive ability of their sperm through a common reference. The fraction of individuals fathered by the experimental and reference males is identified using markers, which allows one to estimate sperm competitive ability using simple mathematical expressions 7,8. In addition, sperm competitive ability can be estimated in two different scenarios depending on whether the experimental male is second or first to mate (offense and defense assay, respectively) 9, which is assumed to be reflective of different competence attributes.
Here, we describe an approach that helps to interrogate the role of different genetic factors that putatively underlie the phenomenon of sperm competitive ability in D. melanogaster.

Assessing Differences in Sperm Competitive Ability in Drosophila

Differential sperm competitive ability among Drosophila males with distinct genotypes can be ascertained through double-mating experiments. Each of these experiments involves one of the males of interest and a reference male. Readily identifiable markers in the progeny allow inference of the fraction of individuals fathered by each male.

评估精子竞争能力的差异<em&gt;果蝇</em

Competition among conspecific males for fertilizing the ova is one of the mechanisms of sexual selection, i.e. selection that operates on maximizing the ...