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。 </li><li> 1小时后，返回成年斑马鱼永久储存罐，取出内部容器，收集鸡蛋，使劲在每个养殖池的水通过塑料茶滤网。 </li><li>反转培养皿中的过滤器和冲洗过滤网轻轻培养皿中，通过使用一个洗瓶的E3媒体冲入鸡蛋。 </li><li>所有未受精卵，从而出现不透明的，应予删除，使用一次性塑料吸管。每次交配交应产生大约200个胚胎。 </li></ol> 2）阵列胚胎在96孔板<ol><li>约5到每一个使用玻璃巴斯德吸管96孔板以及胚胎在E3介质的转移。 </li><li>一旦胚胎在96孔板上摆着，消除可能使用12个通道（30 - 300μL）井出的E3媒体的吸管，小心不要穿刺胚胎。使用12道移液器，可提供250μL的E3中每个含有0.5μg/ml卡那霉素以及尽快，以免使胚胎干起来。 </li><li> 28.5 °彗星孵化器放入96孔板时要添加的化合物，直到他们达到预期的阶段。 </li></ol> 3）转让的小分子库而化合物转移可自动化与机器人的传输方法，我们将介绍的手动传输方式。 <ol><li>小分子库通常在96孔板格式提供，与存储在DMSO为10毫米的股票每个化合物。约60分钟前胚胎到达时，该化合物是无以复加的阶段，解冻所需数量的96等分的小分子（源盘）板。请注意源板的串行或其他识别号码。为了尽量减少对板凝结，解冻可以发生在一个干燥室，含Drierite（WA HAMMOND DRIERITE，森雅，）。 </li><li>在配备多孔板适配器桌面离心机简单自旋向下的板块。 </li><li>从源盘取出铝箔封口胶带。使用12通道移液器，稀释浓度为0.5毫米（例如，如果250 NL等分10mm的股票开始，添加4.75μL二甲基亚砜，每孔）在源盘的化合物。 </li><li>当胚胎在96孔板（收件人板）达到预期的阶段，使用12个通道（2-20μL）移液器从源头上板转移到收件人板块2.5μL化合物（0.5MM）胚胎。 </li><li>记录收件人胚胎板源板的识别号码。现在包含有盖的胚胎和化合物包括收件人板，轻轻混匀，轻轻摇动板，并放置在28.5 ° C培养箱。 </li><li>封面每个源板，铝箔封口胶带和一个-80℃长期储存的冷冻含有未使用的小分子（0.5毫米）。 </li></ol> 4）为小分子的影响，通过目视检查表型的筛选<ol><li>执行屏幕前，制定一个什么构成一个“打”的具体标准。 </li><li>在发展中所需的时间从孵化器中含有复合处理的胚胎，取出96孔板和立体显微镜下检查每口井。微妙的变化，如在循环模式的转变，对于更好的可视化，一个相衬倒置显微镜下可以使用。荧光显微镜可用于研究组织特异性启动子的绿色荧光蛋白或DsRed的蛋白表达的扰动。 </li><li>快速扫描以及其中至少3 5胚胎展出规定“砸”表型的96孔板。记录板，和每一个潜在的冲击以及位置的身份。 </li><li>再次确认由复检几个剂量（1微米，5微米，10μm和50微米）的复合型的影响，一个潜在的冲击。每个剂量，10个胚胎在0.5毫升在48孔板格式的E3媒体测试。复方此外复检的时间应该是相同的的原筛查。一击确认复检的compoun转载时引起的表型ð </li><li>确定从的小分子化学库中的数据库的命中化合物。 </li></ol>

<ol>
	<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

13.4K 次观看.  Vanderbilt University School of Medicine. 斑马鱼已成为一个强大的在体内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。

视频: 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 ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

高效的推导人类心脏的前体和从多能干的小分子诱导人类胚胎干细胞的心肌细胞

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

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 ...

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 ...