Name: large scale zebrafish-based in vivo small molecule screen
Uploaded: 2010-12-30T17:00:00
Description: Watch this Scientific Journal Video about Large Scale Zebrafish-Based In vivo Small Molecule Screen at JoVE.com

1）斑马鱼的卵收集<ol><li>化学屏幕一天前的下午，成立10到20的斑马鱼的繁殖坦克。每个水箱装满水从水产养殖系统。使用渔网，将一个成年男性和一到两个成年女性在每个养殖池的内部容器。独立分频器从对方的男性和女性的鱼。标签的笼子，并把他们的盖子。 </li><li>在屏幕上午上，删除从养殖池的分隔，使斑马鱼进行交配。在未来1小时的课程，让受精卵通过电网秋天在每个内部容器的底部?...

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	<li>Bayliss, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+modulation+of+receptor+signaling+inhibits+regenerative+angiogenesis+in+adult+zebrafish.">Chemical modulation of receptor signaling inhibits regenerative angiogenesis in adult zebrafish.</a> Nature chemical biology. 2, 265-273 (2006).</li><li>Burns, C. G. <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+assay+for+small+molecules+that+modulate+zebrafish+embryonic+heart+rate.">High-throughput assay for small molecules that modulate zebrafish embryonic heart rate.</a> Nature chemical biology. 1, 263-264 (2005).</li><li>Hao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+structure-activity+relationship+study+of+dorsomorphin+analogues+identifies+selective+VEGF+and+BMP+inhibitors.">In vivo structure-activity relationship study of dorsomorphin analogues identifies selective VEGF and BMP inhibitors.</a> ACS chemical biology. 5, 245-253 (2010).</li><li>Hong, C. C., Peterson, Q. P., Hong, J. Y., Peterson, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artery/vein+specification+is+governed+by+opposing+phosphatidylinositol-3+kinase+and+MAP+kinase/ERK+signaling.">Artery/vein specification is governed by opposing phosphatidylinositol-3 kinase and MAP kinase/ERK signaling.</a> Curr Biol. 16, 1366-1372 (2006).</li><li>Milan, D. J., Peterson, T. A., Ruskin, J. N., Peterson, R. T., MacRae, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drugs+that+induce+repolarization+abnormalities+cause+bradycardia+in+zebrafish.">Drugs that induce repolarization abnormalities cause bradycardia in zebrafish.</a> Circulation. 107, 1355-1358 (2003).</li><li>Peterson, R. T., Link, B. A., Dowling, J. E., Schreiber, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule+developmental+screens+reveal+the+logic+and+timing+of+vertebrate+development.">Small molecule developmental screens reveal the logic and timing of vertebrate development.</a> Proceedings of the National Academy of Sciences of the United States of America. 97, 12965-12969 (2000).</li><li>Yu, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsomorphin+inhibits+BMP+signals+required+for+embryogenesis+and+iron+metabolism.">Dorsomorphin inhibits BMP signals required for embryogenesis and iron metabolism.</a> Nature chemical biology. 4, 33-41 (2008).</li><li>Zon, L. I., Peterson, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+drug+discovery+in+the+zebrafish.">In vivo drug discovery in the zebrafish.</a> Nature reviews. 4, 35-44 (2005).</li><li>Stern, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecules+that+delay+S+phase+suppress+a+zebrafish+bmyb+mutant.">Small molecules that delay S phase suppress a zebrafish bmyb mutant.</a> Nature chemical biology. 1, 366-370 (2005).</li><li>Peterson, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+suppression+of+a+genetic+mutation+in+a+zebrafish+model+of+aortic+coarctation.">Chemical suppression of a genetic mutation in a zebrafish model of aortic coarctation.</a> Nature biotechnology. 22, 595-599 (2004).</li></ol>

规划斑马鱼为基础的化学屏幕时，必须特别注意​​正在审议的表型，这种表型的背景率的稳健性。诱导表型的化学抑制器的屏幕，这一点尤为重要。例如，对于引起热休克诱导转基因表型，导致表型的条件下再现，必须精确地映射出启动屏幕前，以避免不可接受的高误报率。通过精心规划，斑马鱼化工屏幕，这就需要适度的资本投资，可能会导致的细胞信号转导和生理的新型调制器的发现，以?...

<ol><li>最小的20对成年斑马鱼所需的基因型。 </li><li>鱼网，养殖坦克，带有可拆卸的内胆和除法（水生境）。 </li><li>培养皿（10厘米）。 </li><li>塑料滤茶器。 </li><li>洗瓶containg胚胎水。 </li><li>一次性聚乙烯转移移液器。 </li><li>聚苯乙烯96圆底实验板（康宁COSTAR;洛厄尔，马）。 </li><li>玻璃巴斯德吸管（尔科技）。 </li><li>手动泵吸液管，10毫升（倍儿最先进的产品，Pequannock，新泽西州）。 </li><li> E3胚胎中期：5毫米氯化钠，0.17 mM的氯化钾， 氯化钙 0.33毫米，0.33毫米硫酸镁4，含有0.003％机动部队（苯，尿素，Sigma公司;圣路易斯，密苏里州）。溶解在1 L E3胚胎媒体0.3 - G机动部队，机动部队可以准备作为一个10倍的解决方案。解决方案包含机动部队应避光，用铝箔覆盖。 </li><li> 12通道移液器2-20μL（Eppendorf公司）。 </li><li> 12通道移液器，3-300μL（Eppendorf公司）。 </li><li>一次性聚苯乙烯吸管盆地，50毫升（尔科技）。 </li><li>小分子结构不同的化合物在96 - WLL格式排列在DMSO中的10毫米股票库。每个主板是分装成96孔聚丙烯存储板（康宁），并储存于-80 ° C，直到使用。 </li><li>铝箔封口磁带96孔板（NUNC，纽约州罗彻斯特）。 </li><li> DMSO（Sigma公司，圣路易斯，密苏里州）。 </li><li>基本孵化器，28.5℃（尔科技）。 </li><li>体视显微镜与透射光基地（徕卡显微系统，班诺克本，IL）。 </li></ol>

large scale zebrafish-based in vivo small molecule screen

鉴于他们的小胚胎的大小，快速发展，透明度，繁殖力，以及众多的分子，形态和生理上的相似性，对哺乳动物，已经成为一个强大的斑马鱼<em&gt;在体内</em&gt;表型为基础的药物屏幕和化学遗传分析平台。在这里，我们展示了一个简单，实用的方法大规模使用斑马鱼的胚胎小分子筛选。

Watch this Scientific Journal Video about Large Scale Zebrafish-Based In vivo Small Molecule Screen at JoVE.com

Large Scale Zebrafish-Based In vivo Small Molecule Screen

斑马鱼已成为一个强大的<em&gt;在体内</em&gt;表型为基础的药物屏幕和化学遗传分析平台。在这里，我们展示了一个简单，实用的方法大规模使用斑马鱼的胚胎小分子筛选。

斑马鱼为基础的大型在体内小分子屏幕

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to ...

large-scale-zebrafish-based-in-vivo-small-molecule-screen

Research

JoVE Journal

Biology

Large Scale Zebrafish-Based In vivo Small Molecule Screen

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

Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the expression pattern of a target protein across the entire brain are relatively easy to answer when using IHC in small brains, such as those of rodents. However, answering the same questions in large and convoluted brains, such as those of primates presents a number of challenges. Here we present a systematic approach for immunodetection of target proteins in an adult monkey brain. This approach relies on the tissue embedding and sectioning methodology of NeuroScience Associates (NSA) as well as tools developed specifically for batch-staining of free-floating sections. It results in uniform staining of a set of sections which, at a particular interval, represents the entire brain. The resulting stained sections can be subjected to a wide variety of analytical procedures in order to measure protein levels, the population of neurons expressing a certain protein.

Large-scale immunodetection of target proteins across the entire primate brain is possible by employing novel tissue embedding and sectioning methods combined with the use of creative apparatus for batch staining of multiple free-floating sections at a given time.

在整个猴脑进行大规模蛋白质检测的批次免疫染色

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the ...

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

成人内皮细胞集落形成祖细胞的分离和大型规模扩张

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries

A great deal of progress in understanding gene expression has been made using in vitro systems. For most studies, functional assays are carried out using extracts that are prepared in bulk from 10-50 or more liters of cells grown in suspension. However, these large-scale preparations are not amenable to rapidly testing in vitro effects that result from a variety of in vivo cellular treatments or conditions. This journal video article shows a method for preparing functional small-scale nuclear extracts, using HeLa cells as an example. This method is carried out using as few as three 150 mm plates of cells grown as adherent monolayers. To illustrate the efficiency of the small-scale extracts, we show that they are as active as bulk nuclear extracts for coupled RNA Polymerase II transcription/splicing reactions. To demonstrate the utility of the extract protocol, we show that splicing is abolished in extracts prepared from HeLa cells treated with the splicing inhibitor drug E7107. The small-scale protocol should be generally applicable to any process or cell type that can be investigated in vitro using cellular extracts. These include patient cells that are only available in limited quantities or cells exposed to numerous agents such as drugs, DNA damaging agents, RNAi, or transfection, which require the use of small cell populations. In addition, small amounts of freshly grown cells are convenient and/or required for some applications.

A protocol for preparation of robust, small-scale HeLa nuclear extracts is described. This protocol is valuable for assays that require use of small populations of cells, such as cells treated with drugs or RNAi. The method should be applicable to a wide variety of gene expression assays and other cell types, including patient cells.

小规模的核提取物，基因表达机具功能分析

A great deal of progress in understanding gene expression has been made using in vitro systems.  For most studies, functional assays are carried out using ...

Infinium Assay for Large-scale SNP Genotyping Applications

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of variants within families or populations can facilitate identification of the genetic factors of disease. Illumina&#39;s panel of genotyping BeadChips allows investigators to genotype thousands or millions of single nucleotide polymorphisms (SNPs) or to analyze other genomic variants, such as copy number, across a large number of DNA samples. These SNPs can be spread throughout the genome or targeted in specific regions in order to maximize potential discovery. The Infinium assay has been optimized to yield high-quality, accurate results quickly. With proper setup, a single technician can process from a few hundred to over a thousand DNA samples per week, depending on the type of array. This assay guides users through every step, starting with genomic DNA and ending with the scanning of the array. Using propriety reagents, samples are amplified, fragmented, precipitated, resuspended, hybridized to the chip, extended by a single base, stained, and scanned on either an iScan or Hi Scan high-resolution optical imaging system. One overnight step is required to amplify the DNA. The DNA is denatured and isothermally amplified by whole-genome amplification; therefore, no PCR is required. Samples are hybridized to the arrays during a second overnight step. By the third day, the samples are ready to be scanned and analyzed. Amplified DNA may be stockpiled in large quantities, allowing bead arrays to be processed every day of the week, thereby maximizing throughput.

A protocol is described that uses Illumina&#39;s Infinium assays to perform large-scale genotyping. These assays can reliably genotype millions of SNPs across hundreds of individual DNA samples in three days. Once generated, these genotypes can be used to check for associations with a variety of different diseases or phenotypes.

Infinium检测的大型SNP基因分型应用

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of ...

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously.&#160; Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. Utilizing normal-phased liquid chromatography coupled to high-resolution mass spectrometry with polarity switching and a rapid duty cycle, we describe a protocol to analyze the polar metabolic composition of biological material with high sensitivity, accuracy, and resolution.

敏感，大规模定量代谢组学的策略

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

手册小分子屏幕接近高通量使用斑马鱼胚胎

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

Novel Production Protocol for Small-scale Manufacture of Probiotic Fermented Foods

A novel dried bacterial consortium of Lactobacillus rhamnosus yoba 2012 and Streptococcus thermophilus C106 is cultured in 1 L of milk. This fresh starter can be used for the production of fermented milk and other fermented foods either at home or at small-scale in rural settings. For the fresh starter, 1 L of milk is pasteurized in a pan that fits into a larger pan containing water, placed on a source of heat. In this water bath, the milk is heated and incubated at 85 &#176;C for 30 min. Thereafter, the milk is cooled down to 45 &#176;C, transferred to a vacuum flask, inoculated with the dried bacteria and left for at least 16 hr between 30 &#176;C and 45 &#176;C. For the purpose of frequent home production, the fresh starter is frozen into ice cubes, which can be used for the production of small volumes of up to 2 L of fermented milk. For the purpose of small-scale production in resource-poor countries, pasteurization of up to 100 L of milk is conducted in milk cans that are placed in a large sauce pan filled with water and heated on a fire at 85 &#176;C for 30 min, and subsequently cooled to 45 &#176;C. Next, the 100 L batch is inoculated with the 1 L freshly prepared starter mentioned before. To assure an effective fermentation at a temperature between 30 and 45 &#176;C, the milk can is covered with a blanket for 12 hr. For the production of non-dairy fermented foods, the fresh starter is left in a cheese cloth for 12 hr, and the drained-off whey can be subsequently used for the inoculation of a wide range of food raw materials, including vegetables and cereal-based foods.

A production protocol is described for the small-scale production of probiotic fermented dairy drink with the aid of a novel bacterial starter culture.

对于益生菌发酵食品的小规模生产新生产协议

A novel dried bacterial consortium of Lactobacillus rhamnosus yoba 2012 and Streptococcus thermophilus C106 is cultured in 1 L of milk. This fresh starter ...

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water deficit or pathogens plays a crucial role. Identifying small molecules that regulate stomatal movement can therefore contribute to understanding the physiological basis by which plants adapt to their environment. Large-scale screening approaches that have been used to identify regulators of stomatal movement have potential limitations: some rely heavily on the abscisic acid (ABA) hormone signaling pathway, therefore excluding ABA-independent mechanisms, while others rely on the observation of indirect, long-term physiological effects such as plant growth and development. The screening method presented here allows the large-scale treatment of plants with a library of chemicals coupled with a direct quantification of their transpiration by thermal imaging. Since evaporation of water through transpiration results in leaf surface cooling, thermal imaging provides a non-invasive approach to investigate changes in stomatal conductance over time. In this protocol, Helianthus annuus seedlings are grown hydroponically and then treated by root feeding, in which the primary root is cut and dipped into the chemical being tested. Thermal imaging followed by statistical analysis of cotyledonary temperature changes over time allows for the identification of bioactive molecules modulating stomatal aperture. Our proof-of-concept experiments demonstrate that a chemical can be carried from the cut root to the cotyledon of the sunflower seedling within 10 minutes. In addition, when plants are treated with ABA as a positive control, an increase in leaf surface temperature can be detected within minutes. Our method thus allows the efficient and rapid identification of novel molecules regulating stomatal aperture.

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

We provide a method for identifying modulators of foliar transpiration by large-scale screening of a compound library.

赫连图斯安努斯大型热成像筛选装置新稳压器的鉴定

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water ...

A Semiautomated ChIP-Seq Procedure for Large-scale Epigenetic Studies

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is a powerful and widely used approach to profile chromatin DNA associated with specific histone modifications, such as H3K27ac, to help identify cis-regulatory DNA elements. The manual process to complete a ChIP-Seq is labor intensive, technically challenging, and often requires large-cell numbers (&#62;100,000 cells). The method described here helps to overcome those challenges. A complete semiautomated, microscaled H3K27ac ChIP-Seq procedure including cell fixation, chromatin shearing, immunoprecipitation, and sequencing library preparation, for batch of 48 samples for cell number inputs less than 100,000 cells is described in detail. The semiautonomous platform reduces technical variability, improves signal-to-noise ratios, and drastically reduces labor. The system can thereby reduce costs by allowing for reduced reaction volumes, limiting the number of expensive reagents such as enzymes, magnetic beads, antibodies, and hands-on time required. These improvements to the ChIP-Seq method suit perfectly for large-scale epigenetic studies of clinical samples with limited cell numbers in a highly reproducible manner.

This paper describes a semiautomated, microscaled ChIP-Seq protocol of DNA regions associated with a specific histone modification (H3K27ac) using a ChIP liquid-handler platform, followed by library preparation using tagmentation. The procedure includes control assessment for quality and quantity purposes and can be adopted to other histone modifications or transcription factors.

用于大规模表观遗传学研究的半自动ChIP-Seq程序

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is a powerful and widely used approach to profile chromatin DNA associated with specific ...