这项工作是由惠普ALS研究中心的约翰霍普金斯（ADG），美国国立卫生研究院主任的新的创新奖1DP2OD004417 - 01（ADG），美国国立卫生研究院R01 NS065317（ADG），丽塔艾伦基金会学者奖授予的支持。助理总干事，是由皮尤慈善信托基金支持的“皮尤研究中心的学者，在生物医学科学”。

1。酵母转化的筹备工作此协议是专为10个96孔板，但可以向上或向下扩展。我们发现，这个协议不超过二96孔盘每一轮的转型工作。整个改造过程（步骤I.3），将需要约8小时。 <ol><li>分装到每一个圆底96孔板Biorobot RapidPlate液体处理程序以及FLEXGene从酵母的ORF库的质粒DNA（为100 ng /μL）5 -10μL。放置在洁净工作台，通风柜一夜之间干破获96孔板。我们已经发现类似与CEN和2μ酵母质粒转化效率。 </li><li>查询应变，在500毫升莫名其妙锥形瓶中，接种酵母葡萄糖蛋白胨（YPD）媒体（10G / L酵母提取物，20G / L蛋白胨，20G / L葡萄糖）的200毫升。在30 ° C孵育过夜用颤抖的（200转）。如果需要，有选择性的媒体也可以被用于这些起动的文化。 </li><li>翌日清晨，淡化查询应变外径600 = 0.1的YPD 2L。摇4-6小时，在30 ° C（200转），直到文化达到外径600 = 0.8-1.0。为最佳生长，长大了两个独立的2.8大号莫名其妙烧瓶1L文化。如果需要，选择性的媒体也可以被用来代替YPD。 </li></ol> 2。酵母转化<ol><li>离心收集细胞。在3000RPM（〜1800 XG）填写四个一次性225mL锥形管与酵母培养和自旋为5分钟在台式离心机（Eppendorf公司5810R）。倒掉上清液，重复，直到所有的细胞都收获。 </li><li>在200毫升灭菌蒸馏水H 2 O洗细胞悬浮细胞沉淀，摇晃或震荡。结合在一个225mL的锥形管的细胞。在3000RPM旋转5分钟。去除上清液。 </li><li>准备0.1MLiOAc/1XTE溶液（0.1M LiOAc，10MM三，为1mM EDTA，灭菌蒸馏水的pH值在8）。在100毫升0.1MLiOAc/1XTE清洗细胞。在3000RPM旋转5分钟。去除上清液。 </li><li>悬浮细胞沉淀在70mL 0.1M LiOAc/1xTE。 Shake和/或涡流，以确保完全重悬沉淀。 </li><li>孵育30分钟（200转）在30 ° C震动。 </li><li>添加β-巯基乙醇0.1M。孵育30分钟（200转）在30 ° C震动。注意：此步骤可以省略，如果使用比较敏感的β-巯基乙醇的酵母菌株。我们发现，这一步是成功转型的重要，但它确实提高转化效率。 </li><li> 2ML超声鲑鱼精子DNA（10mg/ml的），煮沸15分钟，然后在冰上寒意。 </li><li>将水煮鲑鱼精子DNA 2ML添加到细胞的混合物。注意：沉淀，可能会形成另外的鲑鱼精子DNA的细胞混合物后。使用pipetman和200μL提示，小心地取出任何沉淀的形式，因为这可能会干扰下游吹打。 </li><li>分装50μL细胞的混合物以及每96孔板使用BioRobot Rapidplate（也可以使用多通道移液器）。不要混用。 </li><li>在室温30分钟孵育无晃动。 </li><li>准备200毫升的40％PEG-3350/10％DMSO/0.1M LiOAc（160毫升50％的PEG - 3350，二甲基亚砜20ML，20ML 1M LiOAc）。使用前准备的解决方案。 </li><li>添加PEG / LioAc / DMSO溶液，每孔125μL。混合吸气和配药细胞悬液与RapidPlate八次。 </li><li>在室温30分钟孵育无晃动。 </li><li>热休克的细胞在42℃，在干燥的孵化器15分钟。请勿堆叠板。注：我们发现，这种热休克步骤是没有成功转型的必要条件。对于某些酵母菌株（例如，温度敏感突变体），热休克是有害的。我们以前已减少了热休克时间一分钟，已经能够成功地改造了窝藏一个ypt1使用此协议的温度敏感突变的酵母菌株。 </li><li>转速2500RPM（〜1100 XG）板5分钟，用离心机适配器，以适应96孔板。卸下的废物桶反相板PEG解决方案。纸巾吸干倒板除去残留的液体（细胞将保持井底）。 </li><li>冲洗细胞，加入200μL最小的媒体（如SD / -市建局）与RapidPlate每口井。 </li><li>转速2500RPM板5分钟，取出上清液反相的废物桶和纸巾印迹。 </li><li>基本培养基中加入200μL（例如SD /浦），每孔RapidPlate。 </li><li>在30℃培养2天，无晃动。术后第2天，小菌落的细胞应该在每个成功转型以及的底部开始形成。 </li></ol> 3。合模检测<ol><li>棉子糖的基本培养基（如SRaf /市建局）免除到每个井200μL一个新的平底96孔板。 </li><li>混合吸气和配药细胞悬液与Rapidplate八次每个改造板以及。 </li><li>接种细胞的混合物5μL改造板SRaf板。 </li><li>在30 ° C孵育一天。一天后的小菌落，应在每口井的底部。 </li><li> SD / -市建局和SGal /市建局在罩板现货殖民地。消毒燃烧96螺栓复制（青蛙过河）。混合前斑点选择性培养基平板上的菌落与青蛙过河。察觉后，允许在5-10分钟的防护罩板干。 </li><li>在30 ° C培养2-3天。 </li></ol>板与数码相机的照片，并直观地比较SGal /浦板菌落生长菌落查询菌株毒性增强（增长放缓/密度较小的菌落）或抑制（增长更快/更密集的殖民地）。 4。代表性的成果： <img src="/files/ftp_upload/2836/2836fig1.jpg" alt="图1"/> 图1。酵母质粒过度表达屏幕的TDP - 43的毒性，以确定抑制器和增强剂。 TDP - 43已在肌萎缩性脊髓侧索硬化症（卢伽雷氏症）的发病机制有牵连的人类蛋白质。 TDP - 43的细胞质体积累在大脑和脊髓神经元的ALS患者17。在酵母细胞聚集和细胞毒性18中表达TDP - 43 。我们用这个模型系统的机制来定义的TDP - 43的毒性19,20。 repesentative板的例子，从我们的酵母TDP - 43的毒性修饰符屏幕显示。这些板块显示一个综合的半乳糖诱导的TDP - 43质粒与FLEXGene ORF表达文库的质粒也改变殖民地。左边的板含有葡萄糖，压抑TDP - 43或FLEXGene质粒的表达。在右边的板块包含半乳糖诱导的TDP - 43的表达和FLEXGene质粒的ORF。绿色箭头表示抑制TDP - 43的毒性与质粒转化殖民地。红色箭头表示增强TDP - 43的毒性质粒转化的一个殖民地。

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在这里，我们提出了一个协议来执行一种高通量质粒在酵母中的过度表达屏幕。这种方法可以让许多不同的细胞表型的遗传修饰词的迅速和公正的筛选。使用这种方法，研究人员可以在几个星期内屏幕的酵母基因组中的一个重要部分。这种不偏不倚的方式也可以修饰词的识别，预测基于以前的研究结果可能没有被。我们已经用这种方法来确定与人类神经退行性疾病的蛋白质的聚集 19,21-23?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th></tr><tr><td> BioRobot RapidPlate </td><td> Qiagen公司</td><td> 9000490 </td></tr><tr><td> 96螺栓复制（青蛙过河） </td><td> V＆P科学</td><td> VP404 </td></tr><tr><td> FLEXGene ORF的图书馆</td><td>蛋白质组学研究所，哈佛医学院</td><td></td></tr><tr><td>台式离心机</td><td> Eppendorf公司</td><td> 5810R </td></tr><tr><td> 500毫升莫名其妙烧瓶</td><td> Bellco </td><td> 2543-00500 </td></tr><tr><td> 2.8L三重莫名其妙费恩巴赫烧瓶</td><td> Bellco </td><td> 2551-02800 </td></tr><tr><td> 100μLRapidplate枪头</td><td>爱思进</td><td> ZT - 100 - RS </td></tr><tr><td> 200μLRapidplate枪头</td><td>爱思进</td><td> ZT - 200 - RS </td></tr></tbody></table>

high-throughput yeast plasmid overexpression screen

芽殖酵母， 酿酒酵母 ，是一个强大的模型系统，用于定义许多重要的细胞过程，包括那些与人类疾病直接相关的基本机制。由于其短的一代人的时间和特点是基因组，实验酵母模型系统的一个主要优势是能够进行遗传筛选，以确定基因和途径，是在一个给定的过程中涉及的。在过去的三十多年，这种遗传屏幕已被用来阐明细胞周期，分泌途径，以及更高度保守的许多方面的真核细胞生物学1-5。在过去的几年里中，几个酵母菌株和质粒基因组库已产生 6-10 。这些藏品现在允许对基因功能的系统，使用增益和损失功能的方法11-16的审讯。在这里，我们提供了一个详细的协议，与液体处理机器人执行质粒的过度表达屏幕使用一种高通量的酵母转化协议使用5500酵母质粒摆着库。我们一直在使用这些画面，找出容易发生聚集人类神经退行性疾病的蛋白质的积累相关的遗传毒性修饰符。这里介绍的方法很容易适应其他感兴趣的细胞表型的研究。

Watch this Scientific Journal Video about High-throughput Yeast Plasmid Overexpression Screen at JoVE.com

High-throughput Yeast Plasmid Overexpression Screen

在这里，我们描述了一个质粒的过度表达屏幕<em&gt;酿酒酵母</em&gt;，使用液体处理机器人摆着质粒库和高通量酵母转化协议。

高通量酵母质粒过表达屏幕

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

high-throughput-yeast-plasmid-overexpression-screen

Research

JoVE Journal

Biology

16.3K 次观看.  University of Pennsylvania School of Medicine. 在这里，我们描述了一个质粒的过度表达屏幕酿酒酵母，使用液体处理机器人摆着质粒库和高通量酵母转化协议。

视频: High-throughput Yeast Plasmid Overexpression Screen

A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel production 1. Chemical methods to modify or degrade cellulose typically require strong acids and high temperatures. As such, enzymatic methods have become prominent in the bioconversion process. While the identification of active cellulases from bacterial and fungal isolates has been somewhat effective, the vast majority of microbes in nature resist laboratory cultivation. Environmental genomic, also known as metagenomic, screening approaches have great promise in bridging the cultivation gap in the search for novel bioconversion enzymes. Metagenomic screening approaches have successfully recovered novel cellulases from environments as varied as soils 2, buffalo rumen 3 and the termite hind-gut 4 using carboxymethylcellulose (CMC) agar plates stained with congo red dye (based on the method of Teather and Wood 5). However, the CMC method is limited in throughput, is not quantitative and manifests a low signal to noise ratio 6. Other methods have been reported 7,8 but each use an agar plate-based assay, which is undesirable for high-throughput screening of large insert genomic libraries. Here we present a solution-based screen for cellulase activity using a chromogenic dinitrophenol (DNP)-cellobioside substrate 9. Our library was cloned into the pCC1 copy control fosmid to increase assay sensitivity through copy number induction 10. The method uses one-pot chemistry in 384-well microplates with the final readout provided as an absorbance measurement. This readout is quantitative, sensitive and automated with a throughput of up to 100X 384-well plates per day using a liquid handler and plate reader with attached stacking system.

This protocol describes a high throughput screen for cellulolytic activity from a metagenomic library expressed in Escherichia coli. The screen is solution based and highly automated, and uses one-pot chemistry in 384 well microplates with the final readout as an absorbance measurement.

一个Biomining从宏基因组库的纤维素酶的活性高通量筛选

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel ...

科研

生物学

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

木质纤维素材料的高通量检测糖化

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

高通量的亲和标签的重组蛋白的纯化

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

使用高通量形状的RNA二级结构预测

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.

Microinjection is a common technique used to deliver DNA constructs, mRNAs, morpholino antisense oligonucleotides or other treatments into eggs, embryos, and cells of various species.

海胆受精卵的高通量显微注射

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

萤光素酶表达的重组病毒的高通量滴定

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

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 ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

恢复成年斑马鱼的心脏的高通量应用

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

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 ...

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

高通量<em&gt;斑马鱼</em&gt;能量消耗分析

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...