Este trabajo fue apoyado por una beca del Centro de Investigación Packard para la ELA en la Universidad Johns Hopkins (ADG), Premio a la Innovación Nuevo un director de los NIH 1DP2OD004417-01 (ADG), NIH R01 NS065317 (ADG), el Premio de la Fundación Rita Allen Académico. ADG es una beca Pew en las ciencias biomédicas, con el apoyo de The Pew Charitable Trusts.

1. Los preparativos para la transformación de la levadura Este protocolo está diseñado para diez placas de 96 pozos, pero se puede escalar hacia arriba o hacia abajo según corresponda. Hemos encontrado que este protocolo no funciona bien desde hace más de veinte placas de 96 pozos por cada ronda de la transformación. El procedimiento de transformación completa (desde el paso I.3) tendrá una duración aproximada de ocho horas. <ol><li> Alícuota de 5 10μL de ADN plásmido (100 ng / l) de la biblioteca de la levadura FLEXGene ORF en cada pocillo de un fondo redondo de 96 pocillos con el manejador de RapidPlate Biorobot líquido. Lugar descubierto placas de 96 pozos en la campana de humos durante la noche banco limpio para secar. Hemos encontrado eficacia similar de transformación con el CEN y plásmidos 2μ levadura. </li><li> Inocular 200 ml de levadura peptona dextrosa (YPD) los medios de comunicación (10 g / L de extracto de levadura, peptona 20 g / L, 20 g / l de glucosa) con la cepa de la consulta en un matraz Erlenmeyer de 500 ml desconcertado. Incubar toda la noche a 30 ° C con agitación (200 rpm). Si es necesario, los medios de comunicación selectiva también se puede utilizar para estos cultivos iniciadores. </li><li> A la mañana siguiente, se diluye el esfuerzo de consulta a OD 600 = 0,1 en 2 litros de YPD. Agitar durante 4-6 horas a 30 ° C (200 rpm) hasta que el cultivo llegue a OD 600 = 0,8 hasta 1,0. Para un óptimo crecimiento, crecen dos culturas separadas 1L en un recipiente de 2,8 L desconcertado. Si es necesario, los medios de comunicación selectiva también se puede utilizar en lugar de YPD. </li></ol> 2. La levadura de transformación <ol><li> Las células por centrifugación de la cosecha. Llenar cuatro tubos cónicos desechables 225ml con levadura de cerveza y giran a 3.000 rpm (~ 1800 xg) durante 5 minutos en centrífuga de mesa (Eppendorf 5810R). Elimínese el líquido sobrenadante y repetir hasta que todas las células se cosechan. </li><li> Lavar las células en 200 ml de autoclave destilada H 2 O. Resuspender el sedimento celular por agitación o agitación. Combine las células en un tubo cónico de 225 ml. Giran a 3.000 rpm durante 5 minutos. Eliminar el sobrenadante. </li><li> Preparar la solución 0.1MLiOAc/1XTE (0,1 M LiOAc, 10 mM Tris, 1 mM EDTA, pH 8 en agua destilada en autoclave). Lavar las células en 100 ml 0.1MLiOAc/1XTE. Giran a 3.000 rpm durante 5 minutos. Eliminar el sobrenadante. </li><li> Resuspender el sedimento celular en 70 ml 0,1 M LiOAc/1xTE. Agitar y / o vórtice de asegurar pellet es completamente resuspendido. </li><li> Incubar con agitación a 30 ° C durante 30 minutos (200 rpm). </li><li> Añadir β-mercaptoetanol 0,1 M a. Incubar con agitación a 30 ° C durante 30 minutos (200 rpm). Nota: Este paso puede omitirse si la utilización de cepas de levadura que son sensibles a los β-mercaptoetanol. Hemos encontrado que este paso no es esencial para la transformación exitosa, sin embargo, no mejora la eficiencia de transformación. </li><li> Hervir 2 ml de ADN de esperma de salmón sonicado (10mg/ml) durante 15 minutos, y luego se enfría en hielo. </li><li> Añadir 2 ml de ADN de esperma de salmón cocido a la mezcla de la célula. Precaución: puede formarse un precipitado con la adición de ADN de esperma de salmón a la mezcla de células. El uso de un Pipetman y 200 l punta, retire con cuidado cualquier precipitado que se forma debido a que esto puede interferir con la pipeta abajo. </li><li> 50μL alícuota de la mezcla de células en cada pocillo de la placa de 96 pocillos con el Rapidplate BioRobot (también puede utilizar una pipeta multicanal). No se mezclan. </li><li> Incube a temperatura ambiente durante 30 minutos sin agitación. </li><li> Preparar 200 ml de 40% PEG-3350/10% DMSO/0.1M LiOAc (160ml de 50% de PEG-3350, DMSO 20 ml, 20 ml LiOAc 1M). Preparar la solución inmediatamente antes de su uso. </li><li> Añadir 125μL de PEG / LioAc / DMSO a cada pocillo. Mezclar por aspiración y dispensación de suspensión de células en ocho ocasiones con RapidPlate. </li><li> Incube a temperatura ambiente durante 30 minutos sin agitación. </li><li> Choque térmico de las células a 42 ° C durante 15 minutos en un incubador seco. No apile los platos. Nota: Hemos encontrado que este paso de choque térmico no es esencial para la transformación exitosa. Para ciertas cepas de levaduras (por ejemplo, los mutantes sensibles a la temperatura), golpe de calor es perjudicial. Previamente hemos reducido el tiempo de golpe de calor a un minuto y han sido capaces de transformar con éxito una cepa de levadura albergar una mutación temperatura ypt1 sensibles que utilizan este protocolo. </li><li> Giro placas a 2500rpm (~ 1100 xg) durante 5 minutos, el uso de adaptadores de centrífuga para dar cabida a placas de 96 pozos. Eliminar la solución de PEG por las placas de inversión de más de cubo de residuos. Blot placa invertida sobre papel absorbente para eliminar el líquido residual (las células que permanecen en el fondo de los pozos). </li><li> Enjuague las células mediante la adición de 200μL de medio mínimo (por ejemplo, SD /-Ura) a cada pocillo con RapidPlate. </li><li> Giro placas a 2500rpm durante 5 minutos y retirar el sobrenadante por el cubo de residuos invertir más y en toallas de papel secante. </li><li> Añadir 200μL de medio mínimo (por ejemplo, SD /-Ura) a cada pocillo con RapidPlate. </li><li> Se incuba a 30 ° C durante 2 días sin agitación. Después de 2 días, pequeñas colonias de células se empiezan a formar en la parte inferior de cada transformado con éxito así. </li></ol> 3. Manchado ensayo <ol><li> Dispensar 200μL de rafinosa medios basados ​​en mínimos (por ejemplo, sraf /-Ura) en cada pocillo deun nuevo fondo plano placa de 96 pocillos. </li><li> Mezcle cada pocillo de la placa de transformación mediante la aspiración y dispensación de suspensión de células en ocho ocasiones con Rapidplate. </li><li> Inocular las placas con sraf 5μL de mezcla de células de la placa de la transformación. </li><li> Se incuba a 30 ° C durante un día. Pequeñas colonias deben estar presentes en la parte inferior de cada bien después de un día. </li><li> Colonias lugar en tarjetas SD /-Ura y SGal / Ura-placas en campana. Esterilizar de 96 perno replicador (Frogger) por fuego. Mezcla de colonias con Frogger antes de manchas en las placas de medios selectivos. Después de ver, permite secar las placas en campana durante 5-10 minutos. </li><li> Se incuba a 30 ° C durante 2-3 días. </li></ol> Placas de fotografía con cámara digital, y comparar visualmente el crecimiento de colonias en SGal / Ura-placas para encontrar colonias en las que la toxicidad se incrementa la tensión de consulta (un crecimiento más lento / colonias menos densas) o suprimida (un crecimiento más rápido / más densas colonias). 4. Los resultados representativos: <img src="/files/ftp_upload/2836/2836fig1.jpg" alt="Figura 1"/> Figura 1. Levadura pantalla sobreexpresión plásmido para identificar supresores y potenciadores de la toxicidad TDP-43. TDP-43 es una proteína humana que ha sido implicada en la patogenia de la esclerosis lateral amiotrófica (enfermedad de Lou Gehrig). Agregados citoplasmáticos de TDP-43 acumulan en el cerebro y las neuronas de médula espinal de pacientes con ELA 17. Expresando TDP-43 en células de levadura resultados de la agregación y la citotoxicidad de 18 años. Hemos utilizado este modelo de sistema para definir los mecanismos de toxicidad de la TDP-43 19,20. Se muestran ejemplos representativo de platos de nuestra levadura TDP-43 pantalla modificador de toxicidad. Estas placas colonias con una pantalla integrada de galactosa-inducible TDP-43 plásmido y se transforman también con plásmidos de la biblioteca de expresión FLEXGene ORF. La placa de la izquierda contiene glucosa, que reprime la expresión de TDP-43 o los plásmidos FLEXGene. La placa de la derecha contiene galactosa, que induce la expresión de TDP-43 y el ORF en los plásmidos FLEXGene. La punta de flecha verde indica una colonia transformada con un plásmido que suprime la toxicidad de la TDP-43. La punta de flecha roja indica una colonia transformada con un plásmido que aumenta la toxicidad de la TDP-43.

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Aquí se presenta un protocolo para llevar a cabo una pantalla de alto rendimiento de la sobreexpresión plásmido en la levadura. Este método permite la detección rápida e imparcial de modificadores genéticos de muchos fenotipos celulares distintos. Con este enfoque, un investigador puede filtrar una parte importante del genoma de la levadura en cuestión de semanas. Este enfoque no sesgado también permite la identificación de los modificadores, que pueden no haber sido predicho sobre la base de los resultados an...

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th></tr><tr><td> BioRobot RapidPlate </td><td> Qiagen </td><td> 9000490 </td></tr><tr><td> 96 perno replicador (Frogger) </td><td> V &amp; P Científico </td><td> VP404 </td></tr><tr><td> FLEXGene ORF biblioteca </td><td> Instituto de Proteómica de Harvard Medical School </td><td></td></tr><tr><td> Centrífuga de mesa </td><td> Eppendorf </td><td> 5810R </td></tr><tr><td> Frasco de 500 ml desconcertado </td><td> Bellco </td><td> 2543-00500 </td></tr><tr><td> 2,8 litros de tres desconcertado Fernbach frasco </td><td> Bellco </td><td> 2551-02800 </td></tr><tr><td> 100μL Rapidplate puntas de pipeta </td><td> Axygen </td><td> ZT-100-RS </td></tr><tr><td> 200μL Rapidplate puntas de pipeta </td><td> Axygen </td><td> ZT-200-RS </td></tr></tbody></table>

high-throughput yeast plasmid overexpression screen

La incipiente levadura, Saccharomyces cerevisiae, es un modelo de sistema de gran alcance para la definición de los mecanismos fundamentales de muchos procesos celulares importantes, incluidos los que tienen relación directa con la enfermedad humana. Debido a su corto tiempo de generación y bien caracterizado del genoma, una gran ventaja experimental del sistema de modelo de la levadura es la capacidad de realizar exámenes genéticos para identificar los genes y las vías que están involucrados en un proceso determinado. En los últimos treinta años, tales exámenes genéticos se han utilizado para dilucidar el ciclo celular, la vía secretora, y muchos aspectos más altamente conservadas de la biología de las células eucariotas 1-5. En los últimos años, varias bibliotecas genoma de cepas de levadura y plásmidos se han generado 6.10. Estas colecciones permiten ahora a la interrogación sistemática de la función genética utilizando la ganancia y la pérdida de la función de los enfoques 11-16. Aquí les ofrecemos un protocolo detallado para el uso de un protocolo de levadura de alto rendimiento de transformación con un robot de manejo de líquidos para llevar a cabo una pantalla sobreexpresión plásmido, vestida con una biblioteca de 5.500 plásmidos de levadura. Hemos estado utilizando estas pantallas para identificar modificadores genéticos de toxicidad asociados con la acumulación de la agregación de las proteínas humanas expuestas a la enfermedad neurodegenerativa. Los métodos que aquí se adaptan fácilmente al estudio de otros fenotipos celulares de interés.

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

High-throughput Yeast Plasmid Overexpression Screen

Aquí se describe una pantalla de la sobreexpresión del plásmido en<em&gt; Saccharomyces cerevisiae</em&gt;, Con una biblioteca dispuestos plásmido y un protocolo de transformación de alto rendimiento de la levadura con un robot de manejo de líquidos.

La levadura de alto rendimiento de la pantalla sobreexpresión plásmido

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 vistas.  University of Pennsylvania School of Medicine. Aquí se describe una pantalla de la sobreexpresión del plásmido en Saccharomyces cerevisiae, Con una biblioteca dispuestos plásmido y un protocolo de transformación de alto rendimiento de la levadura con un robot de manejo de líquidos.

Video: 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.

Una pantalla de alto rendimiento para Biominería actividad de celulasa de bibliotecas metagenómicas

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

Investigación

Biología

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.

De alto rendimiento de sacarificación de ensayo para materiales 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 rendimiento de purificación de afinidad de proteínas marcadas con 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.

ARN de predicción de estructura secundaria Utilizando SHAPE de alto rendimiento

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 microinyecciones de Sea Urchin cigotos

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 Alto Rendimiento La titulación de luciferasa que expresan virus recombinantes

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 manual de la pantalla pequeña molécula Acercarse De Alto Rendimiento Utilizando embriones de pez cebra

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.

Recuperación de adultos de pez cebra Corazones para aplicaciones de alto rendimiento

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.

Selección de alto rendimiento para moduladores químicos de genes post-transcripcionalmente regulados

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; Ensayo gasto energético

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