Este trabalho foi financiado por uma doação do Centro de Packard de Investigação em ALS Johns Hopkins (ADG), um diretor Award NIH Innovator New 1DP2OD004417-01 (ADG), R01 NIH NS065317 (ADG), a Rita Allen Foundation Scholar Award. ADG é um Scholar Pew em Ciências Biomédicas, apoiado pelo Pew Charitable Trusts.

1. Preparações para a transformação de levedura Este protocolo é projetado para dez placas de 96 poços, mas podem ser ampliados ou reduzidos em conformidade. Descobrimos que este protocolo não funciona bem por mais de vinte placas de 96 poços por rodada de transformação. O procedimento de transformação inteira (a partir do passo I.3) levará cerca de oito horas. <ol><li> Alíquota de 5 10μL de DNA plasmídeo (100 ng / mL) a partir da levedura biblioteca ORF FLEXGene em todos os poços de fundo redondo-96-bem placa com Biorobot manipulador RapidPlate líquido. Lugar descoberto placas de 96 poços na capa da bancada limpa fume durante a noite para secar. Nós descobrimos que a eficiência de transformação semelhante com o CEN e plasmídeos de levedura 2μ. </li><li> Inocular 200 ml de levedura peptona dextrose (YPD) media (10g / L de extrato de levedura, 20g / L de peptona, 20g / L de glicose) com a tensão de consulta em um Erlenmeyer de 500mL perplexo. Incubar durante a 30 ° C com agitação (200 rpm). Se necessário, meios seletivos também pode ser usado para essas culturas starter. </li><li> Na manhã seguinte, dilua a tensão consulta para OD 600 = 0,1 em 2L de YPD. Agitar durante 4-6 horas a 30 ° C (200 rpm) até que a cultura atinge OD 600 = 0,8-1,0. Para o crescimento ideal, crescem duas culturas separadas na 1L 2,8 L frascos perplexo. Se necessário, meios seletivos também pode ser usado em vez de YPD. </li></ol> 2. Transformação de levedura <ol><li> Colheita de células por centrifugação. Encher quatro descartáveis ​​225mL tubos cônicos com cultura de levedura, girando em 3000rpm (~ xg 1800) por 5 minutos em centrífuga de mesa (Eppendorf 5810R). Deitar fora o sobrenadante e repetir até que todas as células são colhidas. </li><li> Lavar as células em 200ml de autoclavado destilada H 2 O. Ressuspender pellet celular por agitação ou vórtex. Combine as células em um tubo cônico 225mL. Giram a 3000rpm por 5 minutos. Remover o sobrenadante. </li><li> Preparar a solução 0.1MLiOAc/1XTE (0,1 M LiOAc, 10mM Tris, 1mM EDTA, pH 8 em água destilada autoclavada). Lavar as células 0.1MLiOAc/1XTE 100mL. Giram a 3000rpm por 5 minutos. Remover o sobrenadante. </li><li> Ressuspender celular em 70ml 0,1 M LiOAc/1xTE. Agitar e / ou vortex para garantir pellet ressuspenso é completamente. </li><li> Incube com agitação a 30 ° C por 30 minutos (200 rpm). </li><li> Adicionar β-mercaptoetanol a 0,1 M. Incube com agitação a 30 ° C por 30 minutos (200 rpm). Nota: Este passo pode ser omitido se estiver usando cepas de leveduras que são sensíveis à β-mercaptoetanol. Descobrimos que este passo não é essencial para a transformação bem-sucedida, no entanto, faz melhorar a eficiência de transformação. </li><li> Ferva 2 ml de DNA sonicado salmão esperma (10mg/mL) por 15 minutos, e depois relaxar no gelo. </li><li> Adicionar 2 ml de DNA de esperma de salmão cozido à mistura de células. Atenção: A precipitação pode formar com a adição do DNA do esperma de salmão com a mistura de células. Usando um Pipetman e 200 mL ponta, retire cuidadosamente qualquer precipitado que se forma, pois isso poderá interferir com pipetagem a jusante. </li><li> 50μL alíquota da mistura de células para cada poço da placa de 96 poços usando o Rapidplate BioRobot (também pode usar multi-canal de pipeta). Não se misturam. </li><li> Incubar à temperatura ambiente por 30 minutos sem agitar. </li><li> Prepare 200mL de 40% PEG-3350/10% DMSO/0.1M LiOAc (160ml de 50% PEG-3350, DMSO 20mL, 20mL LiOAc 1M). Preparar a solução imediatamente antes da utilização. </li><li> Adicionar 125μL de PEG / LioAc / DMSO solução para cada poço. Mix, aspirando e dispensando suspensão de células oito vezes com RapidPlate. </li><li> Incubar à temperatura ambiente por 30 minutos sem agitar. </li><li> Choque térmico das células a 42 ° C por 15 minutos em uma incubadora de seca. Não empilhar chapas. Nota: Nós descobrimos que essa etapa de choque térmico não é essencial para a transformação bem sucedida. Para certas cepas de leveduras (por exemplo, os mutantes sensíveis à temperatura), choque térmico é deletéria. Nós já reduziu o tempo de choque de calor para um minuto e ter sido capaz de transformar uma cepa de levedura abrigar uma mutação sensível a temperatura ypt1 usando esse protocolo. </li><li> Placas giram em 2500rpm (~ xg 1100) por 5 minutos, usando adaptadores centrífuga para acomodar placas de 96 poços. Remoção da solução de PEG por placas de inversão da caçamba de resíduos. Blot placa invertida sobre papel toalha para remover o líquido residual (células permanecerão no fundo de poços). </li><li> Enxágüe células adicionando 200μL de meio mínimo (por exemplo SD /-Ura) para cada poço com RapidPlate. </li><li> Girar placas a 2500rpm por 5 minutos e retire sobrenadante por inversão balde de lixo ao longo e nas toalhas de papel mata-borrão. </li><li> Adicionar 200μL de meio mínimo (por exemplo SD /-Ura) para cada poço com RapidPlate. </li><li> Incubar a 30 ° C por 2 dias sem agitar. Depois de dois dias, pequenas colônias de células deve começar a se formar na parte inferior de cada transformado com sucesso também. </li></ol> 3. Spotting ensaio <ol><li> Dispensar 200μL de rafinose mídia baseada mínima (por exemplo SRAF /-Ura) em cada poço deuma placa de fundo chato de 96 poços novos. </li><li> Mix cada poço da placa de transformação, aspirando e dispensando suspensão de células oito vezes com Rapidplate. </li><li> Inocular placas SRAF com 5μL da mistura de células de placa transformação. </li><li> Incubar a 30 ° C por um dia. Pequenas colônias devem estar presentes na parte inferior de cada poço depois de um dia. </li><li> Colônias Spot on SD /-Ura e / Ura-SGAL placas no capô. Esterilizar 96-parafuso replicador (frogger) por chamas. Mix colônias com frogger antes de spotting em placas de meios seletivo. Depois de spotting, permitem que as placas para secar na capa por 5-10 minutos. </li><li> Incubar a 30 ° C por 2-3 dias. </li></ol> Placas de fotografia com câmera digital, e visualmente comparar o crescimento das colônias em SGAL /-Ura placas para encontrar colônias em que a toxicidade tensão consulta é melhorada (um crescimento mais lento / colônias menos densas) ou suprimido (um crescimento mais rápido / mais densas colônias). 4. Resultados representativos: <img src="/files/ftp_upload/2836/2836fig1.jpg" alt="Figura 1"/> Figura 1. Yeast tela superexpressão plasmídeo para identificar supressores e estimuladores da TDP-43 toxicidade. TDP-43 é uma proteína humana que tem sido implicado na patogênese da esclerose lateral amiotrófica (doença de Lou Gehrig). Agregados citoplasmáticos de TDP-43 acumula no cérebro e neurônios da medula espinhal de pacientes com ELA 17. Expressando TDP-43 em células de levedura resultados na agregação e citotoxicidade 18. Temos usado este sistema modelo para definir mecanismos de toxicidade TDP-43 19,20. Mostrados são exemplos de placas representativo de nossa levedura TDP-43 tela do modificador de toxicidade. Estas placas mostrar colônias com um sistema integrado de galactose induzível-TDP-43 plasmídeo e também transformada com os plasmídeos da biblioteca expressão FLEXGene ORF. A placa do lado esquerdo contém glicose, que reprime a expressão de TDP-43 ou os plasmídeos FLEXGene. A placa da direita contém galactose, que induz a expressão de TDP-43 e as ORFs na plasmídeos FLEXGene. A seta verde indica uma colônia transformada com um plasmídeo que suprime a toxicidade da TDP-43. A seta vermelha indica uma colônia transformada com um plasmídeo que aumenta a toxicidade da TDP-43.

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Aqui apresentamos um protocolo para realizar um alto throughput tela superexpressão plasmídeo em levedura. Esta abordagem permite a detecção rápida e imparcial de modificadores genéticos de diferentes fenótipos celulares. Usando essa abordagem, um pesquisador pode selecionar uma porção significativa do genoma da levedura em questão de semanas. Esta abordagem imparcial também permite a identificação de modificadores, que podem não ter sido previsto com base em resultados anteriores. Nós temos usado essa ab...

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th></tr><tr><td> BioRobot RapidPlate </td><td> Qiagen </td><td> 9000490 </td></tr><tr><td> 96 parafuso replicador (frogger) </td><td> V &amp; P Scientific </td><td> VP404 </td></tr><tr><td> FLEXGene ORF Biblioteca </td><td> Instituto de Proteomics, Harvard Medical School </td><td></td></tr><tr><td> Centrífuga de mesa </td><td> Eppendorf </td><td> 5810R </td></tr><tr><td> Frasco perplexo 500mL </td><td> Bellco </td><td> 2543-00500 </td></tr><tr><td> 2.8L triple-perplexo frasco Fernbach </td><td> Bellco </td><td> 2551-02800 </td></tr><tr><td> Rapidplate 100μL ponteiras </td><td> Axygen </td><td> ZT-100-RS </td></tr><tr><td> Rapidplate 200μL ponteiras </td><td> Axygen </td><td> ZT-200-RS </td></tr></tbody></table>

high-throughput yeast plasmid overexpression screen

A levedura de brotamento, Saccharomyces cerevisiae, é um sistema poderoso modelo para a definição de mecanismos fundamentais de muitos processos celulares importantes, incluindo aquelas com relevância direta para a doença humana. Por causa de seu tempo de geração curto e bem caracterizadas do genoma, uma grande vantagem experimental do sistema de modelo de levedura é a capacidade de realizar triagem genética para identificar genes e caminhos que estão envolvidos em um determinado processo. Nos últimos 30 anos como telas genética têm sido usadas para elucidar o ciclo celular, via de excreção, e muitos outros aspectos altamente conservadas da biologia das células eucarióticas 1-5. No últimos anos, diversas bibliotecas do genoma de cepas de leveduras e plasmídeos foram gerados 6-10. Estas colecções permitem agora para o interrogatório sistemático da função do gene usando-ganho e perda de função abordagens 11-16. Aqui nós fornecemos um protocolo detalhado para o uso de um protocolo de transformação de alto rendimento fermento com um robô para manipulação de líquidos para executar uma tela superexpressão plasmídeo, usando uma biblioteca de vestidos de 5.500 plasmídeos de levedura. Temos vindo a utilizar estas telas para identificar modificadores genéticos de toxicidade associado ao acúmulo de agregação propensas proteínas humanas doença neurodegenerativa. Os métodos aqui apresentados são facilmente adaptáveis ​​para o estudo de outros fenótipos celulares de interesse.

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

High-throughput Yeast Plasmid Overexpression Screen

Aqui nós descrevemos uma tela superexpressão plasmídeo em<em&gt; Saccharomyces cerevisiae</em&gt;, Usando uma biblioteca vestiu plasmídeo e uma alta taxa de transferência de protocolo de transformação de levedura com um robô para manipulação de líquidos.

Alto rendimento Yeast Tela superexpressão Plasmid

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 visualizações.  University of Pennsylvania School of Medicine. Aqui nós descrevemos uma tela superexpressão plasmídeo em Saccharomyces cerevisiae, Usando uma biblioteca vestiu plasmídeo e uma alta taxa de transferência de protocolo de transformação de levedura com um robô para manipulação de líquidos.

Vídeo: 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.

A tela de elevada capacidade para Biomining Atividade da celulase de Bibliotecas metagenomic

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

Biologia

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.

Alto rendimento Assay Sacarificação para materiais lignocelulósicos

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.

De alto rendimento de purificação de afinidade com tag Proteínas Recombinantes

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.

RNA Secundária Previsão Estrutura Usando SHAPE de alta capacidade

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.

High Throughput microinjeções de ouriço do mar zigotos

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.

De alta capacidade de Titulação de luciferase expressando recombinantes Vírus

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.

Um manual de tela pequena molécula Aproximando de alta capacidade Usando embriões Zebrafish

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.

Recuperação de Zebrafish Adulto Corações para aplicações de alto rendimento

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.

Rastreio de alto rendimento para moduladores químicos de Genes Post-transcriptionally Regulamentados

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.

High Throughput<em&gt; Danio rerio</em&gt; Energia Despesas 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 ...