Ce travail a été soutenu par une subvention du Centre pour la recherche sur la SLA Packard à la Johns Hopkins (ADG), Prix d&#39;un directeur des NIH Innovateur Nouveau 1DP2OD004417-01 (ADG), NIH R01 NS065317 (ADG), la Fondation Rita Allen Scholar Award. ADG est une bourse d&#39;études Pew dans les sciences biomédicales, soutenu par le Pew Charitable Trusts.

1. Les préparatifs pour la transformation de la levure Ce protocole est conçu pour les dix plaques de 96 puits, mais peut être agrandie ou réduite en conséquence. Nous avons trouvé que ce protocole ne fonctionne pas bien depuis plus de vingt plaques de 96 puits par tour de transformation. La procédure de transformation complète (de l&#39;étape I.3) prendra environ huit heures. <ol><li> Aliquote de 5-10 ul d&#39;ADN plasmidique (100 ng / uL) de la bibliothèque de levure FLEXGene ORF dans chaque puits d&#39;un fond rond-plaque de 96 puits avec gestionnaire Biorobot RapidPlate liquide. Placez découverts plaques de 96 puits dans le capot banc propre fumées sécher une nuit. Nous avons trouvé des efficacités de transformation similaire avec le CEN et plasmides de levure 2μ. </li><li> Inoculer 200ml de levure, peptone et de dextrose (YPD) médias (extrait de levure 10g / L, 20g / L de peptone, 20 g / L de glucose) avec la souche de requête dans un erlenmeyer de 500 ml dérouté. Incuber une nuit à 30 ° C avec agitation (200 rpm). Si nécessaire, des milieux sélectifs peuvent également être utilisés pour ces ferments. </li><li> Le lendemain matin, diluer la souche de requête à DO600 = 0,1 en 2L de YPD. Agiter pendant 4-6 heures à 30 ° C (200 rpm) jusqu&#39;à ce que la culture atteint DO600 = 0,8 à 1,0. Pour une croissance optimale, grandir entre deux cultures séparées de 1L 2,8 L flacons dérouté. Si nécessaire, des milieux sélectifs peuvent également être utilisés au lieu de YPD. </li></ol> 2. Transformation de la levure <ol><li> Récolte des cellules par centrifugation. Remplissez quatre jetables 225ml tubes coniques avec la culture de levures et de spin à 3000rpm (~ 1800 xg) pendant 5 minutes dans la centrifugeuse de table (Eppendorf 5810R). Décanter le surnageant et répéter jusqu&#39;à ce que toutes les cellules sont récoltées. </li><li> Laver les cellules dans 200 ml d&#39;autoclave distillée H 2 O. Resuspendre le culot cellulaire en secouant ou en vortex. Combinez les cellules dans un tube conique 225ml. Spin à 3000rpm pendant 5 minutes. Retirer le surnageant. </li><li> Préparer la solution 0.1MLiOAc/1XTE (0,1 M LiOAc, 10 mM Tris, 1mM EDTA, pH 8 dans l&#39;eau distillée à l&#39;autoclave). Laver les cellules dans 0.1MLiOAc/1XTE 100mL. Spin à 3000rpm pendant 5 minutes. Retirer le surnageant. </li><li> Resuspendre le culot cellulaire dans 70 mL 0,1 M LiOAc/1xTE. Agiter et / ou vortex pour assurer culot est resuspendu complètement. </li><li> Incuber sous agitation à 30 ° C pendant 30 minutes (200 rpm). </li><li> Ajouter β-mercaptoéthanol à 0,1 M. Incuber sous agitation à 30 ° C pendant 30 minutes (200 rpm). Remarque: Cette étape peut être omise si l&#39;aide des souches de levure qui sont sensibles aux β-mercaptoéthanol. Nous avons constaté que cette étape n&#39;est pas essentielle pour la transformation réussie, mais il ne améliorer l&#39;efficacité de transformation. </li><li> Faites bouillir 2 mL de l&#39;ADN de sperme de saumon soniqué (10mg/ml) pendant 15 minutes, puis réfrigérer sur la glace. </li><li> Ajouter 2 mL de l&#39;ADN de sperme de saumon cuit au mélange de la cellule. Attention: Un précipité peut se former lors de l&#39;addition de l&#39;ADN de sperme de saumon dans le mélange de cellules. L&#39;utilisation d&#39;un Pipetman et 200 ul pointe, retirez soigneusement tout précipité qui se forme, car cela pourrait interférer avec le pipetage en aval. </li><li> 50 pl aliquote de mélange de cellules dans chaque puits de la plaque à 96 puits en utilisant la Rapidplate BioRobot (peut également utiliser une pipette multi-canaux). Ne pas mélanger. </li><li> Incuber à température ambiante pendant 30 minutes sans agitation. </li><li> Préparer 200ml de 40% PEG-3350/10% DMSO/0.1M LiOAc (160ml de 50% de PEG-3350, le DMSO 20mL, 20mL LiOAc 1M). Préparer la solution immédiatement avant utilisation. </li><li> Ajouter 125μL de PEG / LioAc / DMSO solution à chaque puits. Mélanger en aspirant et la distribution suspension cellulaire à huit reprises avec RapidPlate. </li><li> Incuber à température ambiante pendant 30 minutes sans agitation. </li><li> Choc thermique des cellules à 42 ° C pendant 15 minutes dans un incubateur sec. Ne pas empiler les assiettes. Note: Nous avons constaté que cette étape de choc thermique n&#39;est pas essentielle pour la transformation réussie. Pour certaines souches de levures (par exemple, des mutants sensibles à la température), le choc thermique est délétère. Nous avons déjà réduit le temps de choc thermique à une minute et ont été capables de transformer avec succès une souche de levure présentant une mutation ypt1 sensibles à la température en utilisant ce protocole. </li><li> Spin plaques à 2500rpm (~ 1100 xg) pendant 5 minutes, en utilisant des adaptateurs centrifugeuse pour accueillir les plaques 96 puits. Retirer la solution de PEG par des plaques inversant avec godet déchets. Blot plaque retournée sur du papier absorbant pour enlever le liquide résiduel (cellules restera sur fond de puits). </li><li> Rincer les cellules en ajoutant 200 pl d&#39;un milieu minimal (par exemple SD /-Ura) pour chaque puits avec RapidPlate. </li><li> Spin plaques à 2500rpm pendant 5 minutes et retirez surnageant par seau de déchets et plus inversant buvard sur du papier absorbant. </li><li> Ajouter 200 ul de milieu minimal (par exemple SD /-Ura) pour chaque puits avec RapidPlate. </li><li> Incuber à 30 ° C pendant 2 jours sans agitation. Après 2 jours, de petites colonies de cellules devrait commencer à se former au bas de chaque transformé avec succès bien. </li></ol> 3. Spotting test <ol><li> Distribuer 200 pl de raffinose médias basés sur un minimum (par exemple SRAF /-Ura) dans chaque puits deun nouveau fond plat plaque de 96 puits. </li><li> Mélanger chaque puits de la plaque de transformation par l&#39;aspiration et la distribution de cellules en suspension à huit reprises avec Rapidplate. </li><li> Inoculer plaques SRAF avec 5uL de mélange de cellules de la plaque de transformation. </li><li> Incuber à 30 ° C pendant une journée. Les petites colonies doivent être présents au fond de chaque puits après une journée. </li><li> Colonies spot sur carte SD /-Ura et SGal / Ura-plaques dans le capot. Stériliser 96-boulons réplicateur (Frogger) par flambage. Mélanger les colonies avec Frogger, avant de taches sur les plaques des milieux sélectifs. Après avoir repéré, permet de sécher les plaques dans la hotte pendant 5-10 minutes. </li><li> Incuber à 30 ° C pendant 2-3 jours. </li></ol> Plaques photographie avec un appareil photo numérique, et de comparer visuellement la croissance des colonies sur SGal / Ura-plaques de trouver des colonies dans lesquelles la toxicité souche requête est renforcée (ralentissement de la croissance / colonies moins denses) ou supprimée (une croissance plus rapide / plus de colonies denses). 4. Les résultats représentatifs: <img src="/files/ftp_upload/2836/2836fig1.jpg" alt="Figure 1"/> Figure 1. Surexpression d&#39;écran plasmide de levure pour identifier des suppresseurs et exhausteurs de TDP-43 toxicité. TDP-43 est une protéine humaine a été impliquée dans la pathogenèse de la sclérose latérale amyotrophique (maladie de Lou Gehrig). Agrégats cytoplasmiques de TDP-43 s&#39;accumulent dans le cerveau et les neurones de la moelle épinière des patients SLA 17. Exprimant TDP-43 dans les résultats des cellules de levure dans l&#39;agrégation et la cytotoxicité 18. Nous avons utilisé ce modèle pour définir les mécanismes de la toxicité de TDP-43 19,20. Présents sont des exemples de plaques repesentative de nos levures TDP-43 modificateur de l&#39;écran de toxicité. Ces plaques d&#39;affichage des colonies avec un système intégré de galactose-inductible TDP-43 plasmide et transformées avec les plasmides d&#39;expression de la bibliothèque FLEXGene ORF. La plaque sur la gauche contient du glucose, qui réprime l&#39;expression de la protéine TDP-43 ou les plasmides FLEXGene. La plaque de droite contient du galactose, qui induit l&#39;expression de TDP-43 et l&#39;ORF dans les plasmides FLEXGene. La flèche verte indique une colonie transformée avec un plasmide qui supprime la toxicité de la protéine TDP-43. La flèche rouge indique une colonie transformée avec un plasmide qui augmente la toxicité de TDP-43.

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Nous présentons ici un protocole pour effectuer un écran haut débit surexpression plasmide dans la levure. Cette approche permet le criblage rapide et impartiale pour les modificateurs génétiques de beaucoup de différents phénotypes cellulaires. En utilisant cette approche, un chercheur peut filtrer une partie importante du génome de la levure dans une affaire de semaines. Cette approche impartiale permet également d&#39;identifier les modificateurs, qui peuvent ne pas avoir été prédit sur la base des conclu...

<table border="1"><tbody><tr><th> Nom de réactif </th><th> Société </th><th> Le numéro de catalogue </th></tr><tr><td> BioRobot RapidPlate </td><td> Qiagen </td><td> 9000490 </td></tr><tr><td> 96 boulons réplicateur (Frogger) </td><td> V &amp; P scientifique </td><td> VP404 </td></tr><tr><td> FLEXGene ORF Bibliothèque </td><td> Institut de la protéomique, la Harvard Medical School </td><td></td></tr><tr><td> Centrifugeuse de table </td><td> Eppendorf </td><td> 5810R </td></tr><tr><td> 500 ml flacon dérouté </td><td> Bellco </td><td> 2543-00500 </td></tr><tr><td> 2.8L triple dérouté flacon Fernbach </td><td> Bellco </td><td> 2551-02800 </td></tr><tr><td> 100 ul Rapidplate pipette </td><td> Axygen </td><td> ZT-100-RS </td></tr><tr><td> 200 pl Rapidplate pipette </td><td> Axygen </td><td> ZT-200-RS </td></tr></tbody></table>

high-throughput yeast plasmid overexpression screen

Le bourgeonnement de levure Saccharomyces cerevisiae, est un système puissant modèle pour définir les mécanismes fondamentaux de nombreux processus cellulaires importants, y compris ceux en rapport direct avec la maladie humaine. En raison de son temps de génération court et bien caractérisés génome, un avantage majeur d&#39;expérimentation du système modèle la levure est la capacité à effectuer des criblages génétiques pour identifier les gènes et les voies qui sont impliquées dans un processus donné. Au cours des trente dernières années tels cribles génétiques ont été utilisés pour élucider le cycle cellulaire, voie de sécrétion, et de nombreux autres aspects de la biologie hautement conservée cellule eucaryote 1-5. Au cours des dernières années, plusieurs bibliothèques genomewide des souches de levures et plasmides ont été générés 6-10. Ces collections permettent maintenant de l&#39;interrogation systématique de la fonction des gènes en utilisant gain et la perte de fonction des approches 11-16. Ici nous fournissons un protocole détaillé pour l&#39;utilisation d&#39;un protocole de haut débit de la levure de transformation avec un robot de manipulation de liquides pour effectuer un écran surexpression plasmidique, en utilisant une bibliothèque de 5.500 vêtu plasmides de levure. Nous avons utilisé ces écrans pour identifier modificateurs génétiques de la toxicité associée à l&#39;accumulation de l&#39;agrégation des protéines humaines exposées aux maladies neurodégénératives. Les méthodes présentées ici sont facilement adaptables à l&#39;étude d&#39;autres phénotypes cellulaires d&#39;intérêt.

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

High-throughput Yeast Plasmid Overexpression Screen

Nous décrivons ici un écran surexpression plasmide dans<em&gt; Saccharomyces cerevisiae</em&gt;, En utilisant une bibliothèque vêtu plasmide et un protocole de haut débit de la levure de transformation avec un robot de manipulation de liquides.

Haut débit écran surexpression plasmide de levure

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 vues.  University of Pennsylvania School of Medicine. Nous décrivons ici un écran surexpression plasmide dans Saccharomyces cerevisiae, En utilisant une bibliothèque vêtu plasmide et un protocole de haut débit de la levure de transformation avec un robot de manipulation de liquides.

Vidéo: 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.

Un écran à haut débit pour la bioprospection minière activité cellulase à partir des bibliothèques métagénomique

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

Recherche

Biologie

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.

Assay saccharification haut débit pour les matériaux lignocellulosiques

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.

À haut débit Affinity Purification de protéines recombinantes-étiquette

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.

ARN prédiction de la structure secondaire via SHAPE à haut débit

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.

À haut débit microinjections oursins 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 ...

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.

Le titrage à haut débit de la luciférase-virus recombinants exprimant

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.

Un petit écran Molecule Manuel approchant à haut débit utilisant embryons de poissons zèbres

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.

Recouvrement des adultes coeurs de poisson zèbre à haut débit pour des applications

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.

Haut-débit criblage de modulateurs chimique des gènes post-transcriptionnelle réglementées

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; Dépense énergétique 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 ...