Este trabalho é financiado pelo NICHD / NIH (1R01HD053287) para Hukriede, NCI / NIH (P01 CA78039) para Vogt, e NHLBI / NIH (1R01HL088016) para Tsang.

1) Configure acasalamentos zebrafish e tratamento BCI. <ol><li> Dois dias antes do tratamento, identificar Tg homozigotos do sexo masculino (dusp6: d2eGFP) PT6 zebrafish e coloque individualmente em tanques de acasalamento 1. Adicionar separador e um tipo selvagem de peixe * AB feminino e deixar em gaiolas de acasalamento durante a noite. </li><li> Na manhã seguinte, remover o separador, e coletar embriões após 1 hora. Transferência de embriões para pratos 100mm com E3 (5 mM NaCl, 0,17 mM KCl, 0,33 mM CaCl2, 0,33 mM MgSO4), solução e incubar a 28 ° C por 6 horas. Remover mortos e não fertilizados embriões, adicione solução E3 fresco, e incubar durante a noite. </li><li> Ordenar embriões transgênicos para a fase similar (24hpf) e expressão de GFP uniforme. Embriões matriz individualmente em cada poço de uma placa de 96 poços. </li><li> Remoção da solução E3 excesso com uma micropipeta. Adicionar 200μl de solução E3 com uma pipeta multi-canal em cada poço. </li><li> Adicionar os seguintes compostos, conforme listado: <table border="1"><tbody><tr><td></td><td> 1 </td><td> 2 </td><td> 3 </td><td> 4 </td><td> 5 </td><td> 6 </td><td> 7 </td><td> 8 </td><td> 9 </td><td> 10 </td><td> 11 </td><td> 12 </td></tr><tr ><td> A </td><td> 2μl DMSO </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1mM) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2.5mm) </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1mM) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2.5mm) </td><td> 2μl DMSO </td></tr><tr ><td> B </td><td> 2μl DMSO </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1mM) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2.5mm) </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1mM) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2.5mm) </td><td> 2μl DMSO </td></tr><tr ><td> C </td><td> 2μl DMSO </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1mM) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2.5mm) </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1mM) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2.5mm) </td><td> 2μl DMSO </td></tr><tr ><td> D </td><td> 2μl DMSO </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1mM) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2.5mm) </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1mM) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2.5mm) </td><td> 2μl DMSO </td></tr><tr ><td> E </td><td> 2μl DMSO </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1mM) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2.5mm) </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1mM) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2.5mm) </td><td> 2μl DMSO </td></tr><tr ><td> F </td><td> 2μl DMSO </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1mM) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2.5mm) </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1mM) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2.5mm) </td><td> 2μl DMSO </td></tr><tr ><td> G </td><td> 2μl DMSO </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1mM) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2.5mm) </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1mM) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2,5 mM) </td><td> 2μl DMSO </td></tr><tr ><td> H </td><td> 2μl DMSO </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1mM) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2.5mm) </td><td> 2μl BCl (0.1mm) </td><td> 2μl BCl (0.5mm) </td><td> 2μl BCl (1mM) </td><td> 2μl BCl (2mm) </td><td> 2μl BCl (2.5mm) </td><td> 2μl DMSO </td></tr></tbody></table> Concentrações finais são 1 Hm, 5m, 10μM, 20Hm, 25μM BCI em DMSO 1%. Cobrir a placa em papel alumínio e incubar por 6 horas a 28 ° C. </li></ol> 2) imagem automática de placas de 96 poços e análise. <ol><li> Adicionar 10μl de tricaina (0.61mM) a cada poço para anestesiar embriões 30hpf. </li><li> Carga em placa Molecular Devices Imagexpress Ultra. </li><li> Software open Metaxpress. Escolher uma configuração com um objetivo de 4x e diâmetro máximo de pinhole, configure a laser autofocus para detectar placa de fundo, e determinar o melhor plano Z para detectar regiões de interesse. Adquirir imagens de excitação / emissão de comprimentos de onda de 488/525 nm (GFP). Definir scan área como um único site de 2000x2000 pixels (4 x 4 mm) sem binning. Configure potência do laser e ganho de tal forma que as regiões mais brilhantes nas imagens do controlo positivo não saturar. Confirmar a aquisição da imagem adequada em vários poços de controlo positivo e negativo. </li><li> Start scan prato. O instrumento irá automaticamente adquirir imagens de todos os poços e arquivá-los em um banco de dados SQL server. </li><li> Upload de imagens em Definiens desenvolvedor usando o módulo de aplicação Cellenger e um filtro de importação que reconhece o formato de imagem e estrutura do arquivo gerado pelo Dispositivos Imagexpress Molecular plataforma Ultra. </li><li> Processar imagens com um algoritmo projetado Cognição personalizado Technology Network (também chamado de um conjunto de regras). O algoritmo, que personalizado, desenvolvido com base em um conjunto de regras recentemente publicado 2, foi projetado para automaticamente identificar embrião, gema, e na cabeça, e de quantificar o brilho ea área de GFP-estruturas expressar cabeça. Wells em que nenhum embrião é detectado ou quando a cabeça não pode ser inequivocamente atribuída (por exemplo, com toxicidade composto de alta ou fora de foco imagens) são excluídos da análise. Configure o algoritmo usando várias imagens controle negativo (veículo tratados) e imagens de controle positivo (BCI tratados), tais que a cabeça ea gema são corretamente identificados em ambos. Definir uma &quot;cabeça estruturas&quot; limiar de detecção em múltiplos de intensidade média de gema, até a detecção de estruturas de cabeça brilhantes partidas de observação visual. Definir parâmetros para exportação. Neste exemplo, o parâmetro mais informativo foi o brilho total integrada de GFP-positivos regiões dentro da cabeça, a seguir denominado &quot;total cabeça brilho estruturas&quot; (Figura 1). <img src="/files/ftp_upload/1900/1900fig2.jpg" alt="figura 1" /> Figura 1 Gráfico depicitng os efeitos dose-dependente da BCI na GFP expressão em embriões transgênicos. </li><li> Executar o algoritmo em todas as imagens usando a placa integrada agendador de tarefa. O software Definiens usa computação distribuída em vários processadores (&quot;motores&quot;), permitindo análise simultânea de várias imagens. O algoritmo calcula medidas numéricas com base nos resultados da análise de imagens, que são guardados no banco de dados juntamente com uma cópia da imagem processada, com a análise aplicada. </li></ol> Resultados representativos: Depois de todas as imagens são analisadas, os dados podem ser visualizados dentro Cellenger ou exportados para o software de terceiros (Excel, GraphPad Prism) para posterior análise e representação gráfica. A Figura 2 mostra arquivados digitalizar imagens a partir de um veículo e um tratado BCI-bem tratados, com e sem CNT análise aplicados. Figura 1 documenta um efeito dose-dependente da BCI na expressão do gene GFP repórter em Tg (dusp6: d2EGFP) PT6 embriões. A concentração necessária para provocar uma resposta meia máxima (EC50) foi de aproximadamente 12 mM. <img src="/files/ftp_upload/1900/1900fig1.jpg" alt="figura 2" /> Figura 2 Captura de imagem e análise automatizada de embriões zebrafish transgênicos. (A) veículo DMSO tratados Tg (dusp6: d2eGFP) PT6 embrião no 30hpf. (B) embriões transgênicos tratados com 20Hm mostrar BCI aumentos na expressão GFP. (C) Imagem do embrião a partir de (A) após a execução Definiens conjunto de regras para encontrar expressão GFP na cabeça. Manchas laranja denotam onde o software mede a intensidade GFP. (D) Imagem do embrião a partir de (B) após a execução Definiens conjunto de regras para encontrar expressão GFP na cabeça. Manchas laranja denotam onde o software mede a intensidade GFP. Note-se que o conjunto de regras foi capaz de encontrar um espaço alargado de expressão GFP no embrião tratado.

<ol>
	<li>Molina, G. A., Watkins, S. C., Tsang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+FGF+reporter+transgenic+zebrafish+and+their+utility+in+chemical+screens.">Generation of FGF reporter transgenic zebrafish and their utility in chemical screens.</a> BMC Dev Biol. 7, 62-62 (2007).</li><li>Vogt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+image-based+phenotypic+analysis+in+zebrafish+embryos.">Automated image-based phenotypic analysis in zebrafish embryos.</a> Dev Dyn. 238, 656-663 (2009).</li><li>Molina, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+chemical+screening+reveals+an+inhibitor+of+Dusp6+that+expands+cardiac+cell+lineages.">Zebrafish chemical screening reveals an inhibitor of Dusp6 that expands cardiac cell lineages.</a> Nat Chem Biol. 5, 680-687 (2009).</li></ol>

Um fator importante limitar a taxa de telas química zebrafish é a falta de metodologia para processar sistematicamente placas de 96 poços para a imagem latente e análise. Porque as imagens de organismos multicelulares são contraste, complexos de baixa, e de natureza heterogénea, algoritmos de análise de imagem existentes não conseguem detectar e quantificar estruturas específicas dentro do organismo. A maioria das telas química publicada zebrafish, portanto, envolverá a análise visual de poços individuais por pessoal dedicado durante todo o procedimento. Isso geralmente impede a geração de leituras numéricas e um completo arquivo de imagens da tela e dados. Além disso, avaliação manual elimina várias vantagens importantes da imagem baseada em triagem, ou seja, a capacidade de realizar análises retrospectivas de desempenho da tela, para examinar as mudanças fenotípicas que não eram o foco principal da campanha de triagem (por exemplo, toxicidade, ou defeitos de desenvolvimento) e para cruzar -dados de referência de triagem com telas de passado ou futuro usando a biblioteca mesmo composto. Neste relatório, destacam-se metodologia para geração de imagens e análise automatizada de embriões em placas de zebrafish vários poços sem intervenção do usuário. Nós captura de imagem automatizados em um laser Ultra ImageXpress varredura confocal leitor (dispositivos Molecular, Sunnyvale, CA) para a imagem Tg (dusp6: d2EGFP) PT6 embriões em 96 placas bem e desenvolveu um algoritmo de análise de imagem com base em Rede Definiens &quot;Cognição Tecnologia que GFP quantificados expressão nas cabeças dos embriões transgênicos. O método fornecido respostas classificados e quantificados a atividade in vivo de uma pequena molécula ativador da FGF sinalização. Resultados semelhantes foram obtidos com o epifluorescência baseado ArrayScan II (Cellomics Inc., Pittsburgh PA) documenta que é possível implementar a captura de imagem automatizado de embriões zebrafish em uma variedade de leitores de placas disponíveis comercialmente 2. O desenvolvimento de metodologia para analisar automaticamente imagens organismo multicelular eliminou a necessidade de marcar visual por um observador humano e permitiu a geração e arquivamento de dados numéricos e imagens para análise retrospectiva e comparações com telas futuro.

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development of automated imaging and analysis for zebrafish chemical screens.

Nós demonstramos a aplicação de baseada em imagem de alta teor de metodologia de triagem (HCS) para identificar as moléculas pequenas que podem modular a via de FGF / RAS / MAPK em embriões de peixe-zebra. O embrião do zebrafish é um sistema ideal para in vivo telas de alto conteúdo químico. O embrião de 1 dia de idade é de aproximadamente um milímetro de diâmetro e pode ser facilmente dispostas em placas de 96 poços, um formato padrão para a triagem de alto rendimento. Durante o primeiro dia de desenvolvimento, os embriões são transparentes com a maioria dos principais órgãos presentes, possibilitando a visualização da formação de tecido durante a embriogênese. A automação completa de telas química zebrafish ainda é um desafio, no entanto, particularmente no desenvolvimento de aquisição de imagem e análise automatizada. Nós já gerou uma linha de repórter transgênico que expressa a proteína verde fluorescente (GFP) sob o controle da atividade FGF e demonstraram sua utilidade em telas de química<sup&gt; 1</sup&gt;. Estabelecer metodologia para a alta taxa de telas organismo inteiro, desenvolvemos um sistema automatizado para geração de imagens e análise de embriões zebrafish em 24-48 horas após a fertilização (hpf) em placas de 96 poços<sup&gt; 2</sup&gt;. Neste vídeo podemos destacar os procedimentos para arraying embriões transgênicos em placas de vários poços em 24hpf ea adição de uma pequena molécula (BCI) que hyperactivates sinalização FGF<sup&gt; 3</sup&gt;. As placas são incubadas por 6 horas seguido pela adição de tricaina para anestesiar as larvas antes da imagem automatizados em um Molecular Devices ImageXpress Ultra confocal de varredura a laser HCS leitor. As imagens são processadas pelo software Definiens desenvolvedor usando um algoritmo de Cognition Network Technology que desenvolveu para detectar e quantificar a expressão de GFP na cabeça de embriões transgênicos. Neste exemplo, destacamos a capacidade do algoritmo para medir efeitos dose-dependentes da BCI na expressão do gene GFP repórter em embriões tratados.

Watch this Scientific Journal Video about Development of automated imaging and analysis for zebrafish chemical screens. at JoVE.com

Development of automated imaging and analysis for zebrafish chemical screens.

Relata-se o desenvolvimento de um sistema automatizado para geração de imagens e análise de zebrafish embriões transgênicos em placas de vários poços. Isso demonstra a capacidade de medir os efeitos dose-dependente de uma pequena molécula, BCI, na expressão do gene Fator de Crescimento de Fibroblastos repórter e fornecer tecnologia para o estabelecimento de high-throughput telas química zebrafish.

Desenvolvimento de imagem automatizada e análise química para as telas de zebrafish.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK ...

development-automated-imaging-analysis-for-zebrafish-chemical

Research

JoVE Journal

Biology

11.3K visualizações.  University of Pittsburgh Drug Discovery Institute. Relata-se o desenvolvimento de um sistema automatizado para geração de imagens e análise de zebrafish embriões transgênicos em placas de vários poços. Isso demonstra a capacidade de medir os efeitos dose-dependente de uma pequena molécula, BCI, na expressão do gene Fator de Crescimento de Fibroblastos repórter e fornecer tecnologia para o estabelecimento de high-throughput telas química zebrafish.

Vídeo: Development of automated imaging and analysis for zebrafish chemical screens.

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

A Neuronal e astrocitária Ensaio Co-Cultura de Análise de Conteúdo de Alta de neurotoxicidade

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Biologia

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

Semi-automático de análise de pulsação óptica de pequenos corações

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

Transplante de GFP-expressando Blastômeros for Imaging ao vivo de Desenvolvimento da retina e do cérebro em embriões Zebrafish Quimérico

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharides1, 2. The modified monosaccharides, processed by the glycan biosynthetic machinery, are incorporated into cell surface glycoconjugates. The bioorthogonal azide or alkyne tags then allow covalent conjugation with fluorescent probes for visualization, or with affinity probes for enrichment and glycoproteomic analysis. This protocol describes the procedures typically used for noninvasive imaging of fucosylated glycans in zebrafish embryos, including: 1) microinjection of one-cell stage embryos with GDP-5-alkynylfucose (GDP-FucAl), 2) labeling fucosylated glycans in the enveloping layer of zebrafish embryos with azide-conjugated fluorophores via biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and 3) imaging by confocal microscopy3. The method described here can be readily extended to visualize other classes of glycans, e.g. glycans containing sialic acid4 and N-acetylgalactosamine5, 6, in developing zebrafish and in other living organisms.

A click-chemistry based method that allows for the rapid, noninvasive, and robust labeling of alkyne-tagged glycans in zebrafish embryos is described. Fucosylated glycans in the enveloping layer of zebrafish embryos in the late gastrulation stage were imaged in this study.

Glicanos imagem em embriões Zebrafish por Labeling Metabólica e Química Clique Bioorthogonal

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or ...

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

Quantitativos e automatizada de alto rendimento de todo o genoma Telas de RNAi em C. elegans</em

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

Rastreamento de celular utilizando proteínas Photoconvertible Durante o Desenvolvimento Zebrafish

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

Imagem semi-automatizada de Fluorescência de tecidos específicos em embriões Zebrafish

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in &#181;Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

We present an open source high content analysis (HCA) instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts. Data acquisition for this openFLIM-HCA instrument is controlled by software written in &#181;Manager and data analysis is undertaken in FLIMfit.

Open Source Análise alta Conteúdo Utilizando Automated microscopia de fluorescência Lifetime Imagiologia

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

Análise da função de células Beta utilizando imagens de cálcio de célula única resolução em ilhotas de Zebrafish

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

Automated Detection and Analysis of Exocytosis

Timelapse TIRF microscopy of pH-sensitive GFP (pHluorin) attached to vesicle SNARE proteins is an effective method to visualize single vesicle exocytic events in cell culture. To perform an unbiased, efficient identification and analysis of such events, a computer-vision based approach was developed and implemented in MATLAB. The analysis pipeline consists of a cell segmentation and exocytic-event identification algorithm. The computer-vision approach includes tools for investigating multiple parameters of single events, including the half-life of fluorescence decay and peak &#916;F/F, as well as whole-cell analysis of the frequency of exocytosis. These and other parameters of fusion are used in a classification approach to distinguish distinct fusion modes. Here a newly built GUI performs the analysis pipeline from start to finish. Further adaptation of Ripley&#39;s K function in R Studio is used to distinguish between clustered, dispersed, or random occurrence of fusion events in both space and time.

We developed automated computer vision software to detect exocytic events marked by pH-sensitive fluorescent probes. Here, we demonstrate the use of a graphical user interface and RStudio to detect fusion events, analyze and display spatiotemporal parameters of fusion, and classify events into distinct fusion modes.

Detecção e Análise Automatizada de Exocistose

Timelapse TIRF microscopy of pH-sensitive GFP (pHluorin) attached to vesicle SNARE proteins is an effective method to visualize single vesicle exocytic ...