The authors would like to thank Drs. Roger Cone and Chao Zhang for their contributions to validating the application of this assay as previously published. This work was supported by NIH 1F32DK082167-01 (BJR).

Nota: Este protocolo segue as diretrizes da Universidade do Arizona Gabinete de Conduta Responsável em Pesquisa e foi aprovado pelo Comitê de Cuidado e Uso do animal Institucional 1. Criação de peixe-zebra e Embryo Manutenção <ol><li> Prepare meio embrião E3 8. Fazer 60x solução estoque, dissolver 34,8 g de NaCl, 1,6 g de KCl, 5,8 g de CaCl2 ● 2H 2 O, e 9,78 g de MgCl2 ● 6H 2 O em 1,95 L ddiH 2 O. Ajustar o pH para 7,2 usando NaOH e HCl Ajustar o volume para 2 L, utilizando ddiH 2 O. </li><li> Para preparar o meio embrião E3 1x, diluir 16,5 ml 60x estoque em 1 L ddiH20. Adicionar 100 mL de 1% de azul de metileno. </li><li> Reprodução <ol><li> Casa Masculino e Feminino reprodutores (7 - 18 meses de idade) separadamente para maximizar a fecundidade. </li><li> Desligue as luzes 1 - 2 horas antes, no dia anterior ovos são desejados. Configure o acasalamento (1 macho e 1 - 2 fêmeas) em cgaiolas Rossing 9 como previamente descrito. Cruzando gaiolas para permitir que os ovos a predação escapar à medida que passam através da base perfurada da peça inserta na qual o tanque de peixes adultos são realizadas. </li><li> Opcional:. Para reprodução programada, (como seria necessário para estudos envolvendo morfolino injeção), separe a peixes machos e fêmeas por um divisor de plástico transparente, Remover esta manhã para resultar na colocação cronometrado dos ovos e fertilização. Para morfolino estudos, realizar cálculos de energia para avaliar o número de peixes embrionário exigido por tratamento. Sem uma estimativa da magnitude do efeito, ou variação, um ensaio preliminar pode ser executado com uma N = 8. </li></ol></li><li> Despeje os ovos a partir do cruzamento de gaiola em uma placa de Petri (100 - 150 embriões / 10 centímetros placa de Petri). Permita que os ovos se depositam no fundo do prato e despeje a água. Retire toda a água restante usando uma ponta fina transferência descartável da pipeta. Adicionar costas meio E3 embrião feita na etapa 1.1. </li><li> Faça afpipeta descartável polido lame para o ovo futuro e transferência de peixes. Com uma tesoura, corte uma pipeta de transferência descartável formado pela graduação de 0,25 ml. Para remover arestas, polonês chama a ponta corte sobre um bico de Bunsen. </li><li> Manter peixes embrionárias para 4 dias após a fertilização (DPF) em 10 cm placas de Petri em meio E3 embrião de peixe-zebra a 28,5 ○ C. Duas vezes por dia, use uma chama polido formou pipeta de transferência descartável para remover quaisquer ovos não fertilizados ou embriões mortos (aparência branca opaca). Uma vez por dia, substitua meio embrião E3. Deitar fora meio embrião E3, remover o líquido restante usando uma ponta fina transferência descartável da pipeta, e adicionar novamente pré-aquecido (28,5 ○ C) meio embrião E3. </li></ol> 2. Preparar a Solução de Ensaio <ol><li> Preparar a solução de ensaio de acordo com a Tabela 1. Solução de ensaio de filtração estéril utilizando um filtro de 0,22 um. Antes do ensaio de iniciação, aquecer a solução de ensaio estéril numa 28,5 ○ Cbanho d&#39;água. </li></ol><table fo:keep-together.within-page="1"><tbody><tr><td> Ingrediente </td><td> % Do Total </td><td> Volume (mL / 10 mL) </td></tr><tr><td> 60x E3 Embryo Médio </td><td> 1.667 </td><td> 166,7 </td></tr><tr><td> Duplo deionizada (DDI) H 2 0 </td><td> 96,233 </td><td> 9623,3 </td></tr><tr><td> Alamar Blue </td><td> 1 </td><td> 100 </td></tr><tr><td> 400 mM de NaOH </td><td> 1 </td><td> 100 </td></tr><tr><td> DMSO </td><td> 0,1 </td><td> 10 </td></tr></tbody></table> Tabela 1. Ensaio Médio 3. Execute Assay <ol><li> Enxágüe embriões de peixe-zebra 3 vezes com meio embrião E3 filtrada estéril. Misture todos os embriões de peixe-zebra viáveis ​​do embreagem para um copo. Filtro estéril 75 ml meio embrião E3. Usando uma ponta fina transferência descartável pipeta, remover-se tanto a forma de embrião E3 a partir dos embriões de peixe-zebra quanto possível. Adicionar 20 ml de volta médio embrião estéril filtrada E3. Remover e substituir meio embrião E3 3 vezes. </li><li> Placa 1 peixe para cada um dos 93 poços de uma de 96 poços preto sided placa clara de fundo. Transferir o peixe embrionária para a placa de captura individualmente por um embrião dentro de uma chama de plástico graduado lustrado pipeta descartável (passo 1.5) e ejectar suavemente para dentro do poço. Transferir meio embrião E3 retirado da proveta contendo os peixes enxaguado em 3 poços em branco. Isto assegura que os poços em branco foram expostas a água da mesma exacta como os poços que contêm peixes. Ambos os poços que contêm peixes e poços de branco deve ser de, pelo menos, ½ completo (200 ul) à conclusão deste passo. </li><li> Substituir meio embrião E3 com solução de ensaio em todos os 96 poços, incluindo poços em branco. Esta etapa deve ser realizada uma coluna de cada vez para cada uma das 12 colunas de uma placa de 96 poços. Usando uma ponta fina descartável transfer pipeta, remover o meio embrião E3 de cada poço numa coluna de a placa (8 poços). Tome cuidado para não tocar o embrião de peixe-zebra. Adicionar 300 ul de solução de ensaio a cada poço a partir do qual a água foi removida. Repetir a remoção do meio de embrião E3 e substituição com a solução de ensaio para todas as 12 colunas da placa. </li><li> Opcional: Para testar a alteração da taxa metabólica em resposta a um composto, fazer uma solução que é 100 vezes (100X) a concentração de tratamento desejado. De nota, assegurar que a solução 100x não exceda 10% DMSO para limitar a concentração de DMSO final a não mais do que 0,1%. Adicionar 3 mL da solução de 100x a cada um dos poços de tratamento. NOTA: Executar cálculos de potência para avaliar o número de poços de tratamento. No entanto, sem uma estimativa da grandeza da resposta ou variação, um ensaio preliminar pode ser executado em 8 tratado com composto e 8 tratados com veículo poços de controlo que contêm peixes. <ol><li> Adicionar 3 mL de controlo do veículo para o controlo (sem Drutratamento g) poços que contêm peixes. Para assegurar que a resposta ao composto fluorescente é um resultado das acções dentro do peixe, aplicam-se tratamentos com drogas e 3 do veículo para poços em branco em placa de 96 poços. </li></ol></li><li> Leia peixes cheio placa fluorescente leitor de placas com comprimento de onda de excitação a 530 nm e comprimento de onda de emissão a 590 nm. </li><li> Colocar a placa na incubadora umidificado definida para 28,5 ○ C (placa de manter na escuridão durante todo ensaio para evitar a fotodegradação de solução de ensaio). O ensaio pode ser executado por períodos de tempo que variam desde 1 h a 24 h. A duração do ensaio depende do objectivo do estudo. Para testar a resposta de um composto não-estável de curta duração de curta duração pode ser mais adequado. No entanto, para os testes composto estável ou testes de efeitos genéticos, maximizando a duração do ensaio é preferível explorar as vantagens associadas com o sinal cumulativo. </li><li> Em um ponto de tempo pré-determinado (veja o passo 3.6 para ajudar a determinar sincronismo)leia fluorescência de placa cheio de peixe novamente com as mesmas configurações do passo 3.5. </li></ol> 4. Avaliar a mudança relativa na fluorescência associada ao tratamento <ol><li> Calcular alteração na fluorescência de cada cavidade subtraindo a fluorescência no tempo 0 da fluorescência após a conclusão do estudo. Wells sem peixe (espaços em branco) deverá ter valores próximos de 0, muito abaixo dos valores dos poços que contêm peixe. Alteração na fluorescência = Fluorescência na conclusão - fluorescência no tempo 0 </li><li> Para determinar a alteração relativa da fluorescência, calcular a variação média da fluorescência para os poços de controlo, em seguida, dividir a alteração na fluorescência de cada poço pela variação média da fluorescência dos poços de controlo. De nota, controles poderia ser ou comandos do veículo ou controles genéticos. <img alt="Equação 1" src="/files/ftp_upload/53297/53297eq1.jpg" /></li></ol>

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The authors have no competing financial interests to disclose.

A contaminação com bactérias ou fungos limita grandemente a aplicação deste ensaio. O azul de metileno no meio embrião E3 limita a possibilidade de contaminação por fungos. Ao longo embrião criação é fundamental que o cuidado é tomado para remover embriões mortos duas vezes por dia. Além disso, é essencial para lavar cuidadosamente os embriões com meio embrião E3 estéril no dia do início do ensaio. Estes passos 2 limitar o potencial para a contaminação bacteriana dos embriões. A inclusão de poço...

O mouse é atualmente o modelo predominante para a investigação de obesidade. O curto intervalo de geração e ferramentas genéticas disponíveis no rato têm sido inigualável até à data. No entanto, o peixe-zebra também tem um intervalo de geração curto (3 - 4 meses) e supera até mesmo o mouse na facilidade de manipulação genética 1,2. O peixe-zebra mantém quase 90% de genes de mamíferos, enquanto ultrapassando o mouse no número de descendentes e potencial para uso em telas genéticos e medicamentos 3. Para explorar o potencial do modelo peixe-zebra para estudos em obesidade, os ensaios devem ser desenvolvidos para investigar os fatores que influenciam a regulação do peso corporal, incluindo as despesas de energia. Como metabolitos são processados ​​através β-oxidação e o ácido tricarboxílico ciclo oxigénio é consumido NADH e 2 é produzido. Assim, NADH 2 é um indicador directo do fluxo de metabolitos através de vias metabólicas (oxidação metabolito). Em poikilotherms, H + vazar através da membrana mitocondrial interna, que desacopla o NADH 2 de oxidação a partir da síntese de ATP, é de 4 - 5 vezes menor do que em homeotérmicos 4. Assim, em NADH 2 peixe-zebra é muito bem ligados à produção de ATP através da fosforilação oxidativa. Aqui, descrevemos um ensaio que mede a produção de NADH 2 em zebrafish larval como um proxy para o gasto energético 5. O consumo de oxigênio é o padrão ouro para medir o gasto de energia. No entanto, para melhor aproveitar o potencial de alto rendimento do peixe-zebra, ensaios de gasto de energia devem ser passíveis de high-throughput. Sistemas de consumo de oxigênio que dependem de uma câmara fechada, sistema de circulação são limitados em taxa de transferência pelo número de câmaras disponíveis 6. Open air consumo de O 2 / CO 2 ensaios de produção também foram aplicadas no peixe-zebra 5,7. Estes ar livre de 96 poços placa de baseD Systems são passíveis de alto rendimento. Infelizmente, as trocas gasosas com o ambiente de limites de sensibilidade destes ensaios. Nós publicada recentemente a aplicação de um ensaio que monitora NADH 2, utilizando o indicador redox alamarBlue 5. Este ensaio supera as limitações na taxa de transferência e sensibilidade comum às análises de consumo de oxigênio no peixe-zebra. O peixe-zebra está se tornando um modelo cada vez mais importante para estudos da homeostase energética do corpo inteiro. Em parte, por causa do peixe-zebra são passíveis de uso em telas genéticos para a frente e telas de drogas. Além disso, orientada manipulação genética, incluindo knockdown e knock-in, pode ser aplicada rapidamente. Nós mostramos anteriormente que este ensaio pode ser combinado com a aplicação de drogas e banho de knockdown ou nocaute genético para identificar compostos e genes que alteram a taxa metabólica 5. Além disso, este ensaio foi concebido para explorar as vantagens de elevada capacidade inerentes ao peixe-zebra. </p&gt;

<table border="0" cellpadding="0" cellspacing="0" width="966">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">AlamarBlue</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">10-230-103</td>
			<td style="width:307px;">Manufactured by Thermo Scientific</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Fine Tip DisposableTransfer Pipette</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td>13-711-26</td>
			<td>Manufactured by Thermo Scientific</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Fisherbrand Graduated Disposable Transfer Pipette</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td>13-771-9AM</td>
			<td>Manufactured by Thermo Scientific</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Black sided clear bottom 96-well plates</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">50-320-785</td>
			<td>Manufactured by E&#38;K Scientific Products Inc.</td>
		</tr>
	</tbody>
</table>

high throughput danio rerio energy expenditure assay

Peixe-zebra são um modelo importante organismo com vantagens inerentes que têm o potencial para fazer zebrafish um modelo amplamente aplicado para o estudo da homeostase da energia e da obesidade. O pequeno tamanho do peixe-zebra permite ensaios sobre os embriões para ser conduzido num formato de placa de 96 ou 384 poços, morfolino e tecnologias baseadas CRISPR promover a facilidade de manipulação genética, e no tratamento de droga por aplicação de banho é viável. Além disso, peixe-zebra são ideais para telas genéticos para a frente, permitindo a descoberta do gene para a novela. Dada a relativa novidade do peixe-zebra como um modelo para a obesidade, é necessário desenvolver ferramentas que exploram plenamente esses benefícios. Aqui, nós descrevemos um método para medir o gasto de energia em milhares de peixes-zebra embrionária simultaneamente. Nós desenvolvemos uma plataforma de microplacas animal completo no qual usamos placas de 96 poços para isolar cada peixe e avaliar cumulativa de produção de NADH usando a 2 disponível comercialmente cultura celular viabilidade reagente alamarBlue. Em poiquilotérmicos a relação entre NADH 2 produção e gasto de energia está intimamente ligado. Este ensaio gasto energético cria o potencial para a tela rapidamente manipulações farmacológicas ou genéticas que alteram diretamente o gasto de energia ou alteram a resposta a um fármaco aplicada (por exemplo, sensibilizadores de insulina).

Solução de ensaio não aumenta de fluorescência na ausência de peixe embrionário (Figura 1). No entanto, o ensaio é altamente sensível a pequenas alterações na taxa metabólica dentro do poço. Um peixe dentro de um único poço cria um sinal significativamente diferente de poços em branco dentro de 1 h (P &lt;0,0001). A Figura 2 proporciona uma representação visual de sinais gerados pelo peixe embrionário após 24 h de incubação. Para a finalidade da imagem foram usadas ...

Watch this Scientific Journal Video about High Throughput Danio Rerio Energy Expenditure Assay at JoVE.com

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 ...

high-throughput-danio-rerio-energy-expenditure-assay

Research

JoVE Journal

Biology

High Throughput Danio Rerio Energy Expenditure Assay

12.6K visualizações.  Vanderbilt University Medical Center. 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. 

Vídeo: High Throughput Danio Rerio Energy Expenditure Assay

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss.
Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish Danio rerio

The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.

Dissecação bruta e Multa de Ouvido Interno Epitélio sensorial em adultos Zebrafish ( Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the ...

Biologia

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

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, ...

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

Dissecção do coração em larval, Zebrafish Juvenil e Adulto, Danio rerio</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

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 ...

Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and behavioural research. As a vertebrate, zebrafish share considerable genetic sequence similarity with humans and are being used as an animal model for various human disease conditions. The advantages of zebrafish in comparison to other common vertebrate models include high fecundity, low maintenance cost, transparent embryos, and rapid development. Due to the spur of interest in zebrafish research, the need to establish and maintain a productive zebrafish housing facility is also increasing. Although literature is available for the maintenance of a zebrafish laboratory, a concise video protocol is lacking. This video illustrates the protocol for regular housing, feeding, breeding and raising of zebrafish larvae. This process will help researchers to understand the natural behaviour and optimal conditions of zebrafish husbandry and hence troubleshoot experimental issues that originate from the fish husbandry conditions. This protocol will be of immense help to researchers planning to establish a zebrafish laboratory, and also to graduate students who are intending to use zebrafish as an animal model.

Regular Care and Maintenance of a Zebrafish Danio rerio Laboratory: An Introduction

This protocol outlines regular maintenance and care to maintain optimal conditions for zebrafish husbandry. The video illustrates the protocol for system maintenance, regular housing, feeding, breeding, and raising of zebrafish larvae.

Cuidados e manutenção regular de um peixe-zebra (<em&gt; Danio rerio</em&gt;) Laboratório: Uma Introdução

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

De alta capacidade de Triagem funcional usando um caseiro Dual-brilho ensaio de luciferase

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

In Vivo Modelagem do Morbid Genoma Humano utilizando Danio rerio

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

Um Ensaio de Ligação de Alto Rendimento II do MHC para análise quantitativa de Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

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 ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

Detecção de alvos miRNA em métodos de alta capacidade utilizando o ensaio 3&#39;LIFE

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

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