Gostaríamos de agradecer a Erika Taylor para edição deste manuscrito. Este trabalho foi financiado por subvenções provenientes do National Institutes of Health (P01 HL098053, P01 AG033561 e HL054732 R01) de R.B., um suplemento de investigação pós-doutoramento (R01 HL085481) e companheirismo (AAUW) para S.B.D., concessões e da associação americana do coração para S.B.D. e RGB

1. HFD alimentação protocolo <img alt="Table 1" src="/files/ftp_upload/56029/56029table1.jpg" /> tabela 1. Receita de comida de mosca. 
Esta tabela resume os diferentes ingredientes usados para preparar nosso controle alimentar. Uma vez feita, 10 mL do alimento é vertido em frascos, refrigerado e estocado a 4 ° C para armazenamento a longo prazo.
<ol><li>Preparação HFD <ol><li>para fazer 1 kg de alimento gordo alto, pesa 700 g de comida de mosca normal pre-feita (ver tabela 1) e 300 g de óleo de coco em dois recipientes separados usando uma balança analítica.</li>
		<li>Calor cada recipiente individualmente no microondas, até que o conteúdo derrete completamente. Despeje o óleo de coco o recipiente de alimento de voar. Mexa com um batedor até que o óleo é bem misturado a comida da mosca. 
		Nota: Não há pedaços de comida ou óleo devem ser visíveis. Quando a comida de mosca está derretendo no microondas, parar em intervalos de 1 minuto para misturar os alimentos e evitar ferver.</li>
		<li>Re-pesar o alimento de gordura alto. Se o peso total é inferior a 1 kg (700 g de comida + 300 g de óleo de coco), adicionar água (cerca de 10 mL) para compensar a evaporação durante o aquecimento e para trazer de volta o peso de 1 kg...</li>
		<li>Pour cerca de 10 mL bem misturado alta gordura comida por frasco (cerca de 25% do volume do frasco). Em seguida, cubra com gaze para evitar moscas &#39; contaminação. Deixe esfriar por 1h à temperatura ambiente, em seguida, transferir os frascos a 4 ° C para armazenamento acima de 4 semanas. 
		Nota: A comida Normal (NF) é usada como um alimento de controle. Também despeje 10 mL (cerca de 25% do volume do frasco) de NF por frasco. Para a comida HFD, substitua 30% da comida NF com óleo de coco. Para controle e condições experimentais, as moscas receberam a mesma quantidade de NF e HFD (10ml de comida por frasco).</li>
	</ol></li></ol> <img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56029/56029fig1.jpg" /> Figura 1 . HFD alimentação em Drosophila. 
O esquema mostra as diferentes etapas de alimentação para as moscas em um alimento de controle (dieta normal sem adição de óleo de coco-NF) ou HFD (com a adição de óleo de coco). O processo todo leva 10 dias após a coleta inicial de moscas adultas. <a href="//ecsource.jove.com/files/ftp_upload/56029/56029fig1large.jpg" target="_blank"> clique aqui para ver uma versão maior desta figura.</a>
<ol start="2"> <li>HFD alimentação de d. melanogaster Nota: Este protocolo inclui controle alimentar e HFD alimentando os procedimentos que são compatíveis com qualquer linha de voar. Controle e experimentais moscas são colocadas em frascos contendo a mesma comida de quantidade (NF ou HFD).
	<ol><li>Uso de CO 2 para anestesiar moscas. 
		Nota: Para os neófitos, por favor leia referência 21 para uma visão geral sobre criação e manipulação de d. melanogaster.</li>
		<li>Recolher 0-5 moscas dia velho de frascos e transferência (lançando) as moscas em novos frascos contendo NF (previamente aquecido à temperatura ambiente). Deixe a idade de moscas por 5 dias adicionais no 21 ° C.</li>
		<li>No final dos 5 dias, retire os frascos que contêm HFD e NF de 4 ° C. 
		Nota: como temperaturas frias podem salientar vivos moscas, deixe os frascos descansar por 30 min à temperatura ambiente para se aquecer antes de usar.</li>
		<li>Usando lenços, limpar e remover o excesso de líquido dos lados dos frascos.</li>
		<li>Inserir um pequeno pedaço de papel de filtro, sobre 1 cm x 3 cm, a comida da alta gorda para absorver todo o líquido excedente. 
		Nota: Este passo é essencial para prevenir as moscas de degola para a comida HFD.</li>
		<li>Em seguida, transferir as moscas sobre os alimentos de alta gordura (lançando). Colocar os frascos horizontalmente do lado deles, (não permanente) em 21-22 ° C durante 5 dias. 
		Nota: Evite temperaturas mais altas, como isto pode parcialmente derreter a comida gorda alta, fazendo com que as moscas manter os alimentos e morrer.</li>
		<li>Depois de 5 dias, transferir (lançando) as moscas de NF e HFD dentro dos frascos de novos sem comida para permitir que a sua coragem de vazio durante 30 min. Em seguida, proceder à pesagem moscas. 
		Nota: Uma das etapas críticas neste HFD protocolo de alimentação é preparar um HFD bem misturado, livre de quaisquer pedaços de comida de mosca ou óleo condensado. Mistura homogênea e refrigerar o HFD 45-30 ° c é aconselhável, antes de derramar dentro dos frascos e armazenar os alimentos na idade adequada de 4 ° C. correspondência de controle e experimentais moscas é importante, assim como prior de condições nutricionais e ambientais controladas para a alimentação de HFD. Durante a fase de alimentação, controle e experimentais moscas devem ser dada a mesma quantidade de NF e HFD. Nunca colete moscas de superlotadas frascos ou garrafas (para evitar efeitos de transgeracional). Sempre coloque um máximo de 25 moscas por frasco durante NF e HFD alimentação para evitar restrição dietética ou outros efeitos crowding. O HFD é composto de 30% de óleo de coco, assim, temperaturas acima de 22 ° C parcialmente podem derreter o HFD, fazendo com que as moscas enfiar a comida oleosa e morrer. Antes de colocar as moscas o HFD, limpe os frascos para remover qualquer resíduo de óleo e inserir um filtro de papel para absorver qualquer excesso de líquido no alimento. Durante a incubação das moscas em HFD, os frascos são colocados em seus lados para fornecer uma superfície horizontal sem gordura para as moscas.</li>
	</ol></li> <li>Pesando as moscas <ol><li>para cada genótipo e gênero a ser testado, usar uma escala ultra sensível para registrar os pesos de tubos de vazio, previamente rotulado 1,7 mL.</li>
		<li>Usando anestésico voar ou CO 2, anestesiar e recolher 36 moscas com o mesmo genótipo, sexo, e alimentar o status com um pincel em um pre-pesava 1.7 tubo mL.</li>
		<li>Pesa o tubo contendo moscas com a mesma escala ultra-sensíveis.</li>
		<li>Determinar o peso médio de voar esta fórmula a seguir: 
		<img alt="Equation 1" src="/files/ftp_upload/56029/56029eq1.jpg" /> Nota: O número de moscas neste protocolo é 36.</li>
		<li>Flash congela os tubos de 1,7 mL contendo moscas por mergulhar no nitrogênio líquido por 2 min. coletar e colocar os tubos em caixas. Em seguida, transferir as caixas a-80 ° C (temperatura preferível) para o armazenamento até o uso com o ensaio de TAG. 
		Atenção: O nitrogênio líquido é a temperatura extremamente baixa. Por favor, use equipamento de proteção pessoal apropriado.</li>
	</ol></li></ol> 2. TAG ensaio <ol><li>preparação das concentrações padrão TAG <ol><li>preparar 500 mL de PBT (PBS 1x, 0.05% Triton).</li>
		<li>Usando tubos de 0,5 mL e a solução de TAG (Tabela de materiais), preparam-se 100 µ l de espaço em branco (apenas PBT) e 100 µ l de seis concentrações de TAG padrão (2 µ g / µ l; 1 µ g / µ l; 0,5 µ g / µ l; 0,25 µ g / µ l; 0,125 µ g / µ l; 0.0625 µ g / µ l) com o PBT.</li>
		<li>Coloque os tubos no gelo e prosseguir com as moscas de moedura.</li>
	</ol></li> <li>Moagem as moscas <ol><li>tirar as moscas congeladas de-80 ° C (etapa 1.3). Transferir os tubos contendo as moscas no gelo</li>
		<li>Primeiro, coloque o metal de 2 milímetros, bolas de moagem em um dispensador de bola 96 poços, use um pincel pequeno para remover bolas extras.</li>
		Placa de <li>lugar da moagem de 96 poços de cabeça para baixo em cima do dispensador de bola e flip transferir as bolas de metal directamente para a placa de moedura. Deve haver uma bola por alvéolo.</li>
		<li>Usando uma pipeta multicanal, adicionar 600 µ l de PBT em cada linha da placa de 96 poços moedura.</li>
		<li>Com fórceps, adicionar 3 voa por bem. Indicar a condição de genótipo, sexo e comida das moscas em cada linha/poço da placa de 96 poços moedura.</li>
		<li>Firmemente Coloque as tampas para a placa de moedura de 96 poços. Fita pode ser colocada na parte superior para assegurar que não há nenhum escapamento.</li>
		<li>, Corretamente, colocar as placas de 96 poços moagem para a máquina de moer. Ajuste a máquina para a velocidade mais alta e pressione &#34; executar &#34; para moer as moscas por 3 min.</li>
		<li>Após 3 min, parar a moagem e proceder com centrifugação de placa: centrifuGE para 15 min a 4.000 x g, 4 ° C. Após a centrifugação, gerenciar a placa de moedura cuidadosamente para evitar misturar o sobrenadante com a pelota.</li>
	</ol></li> <li>Carregamento de amostra e determinação de conteúdo de TAG <ol><li>tomar uma nova placa de 96 poços Espectrofotômetro. Usando uma pipeta multicanal, carregar 200 µ l de reagente a TAG em cada linha da placa.</li>
		<li>Carga 20 µ l de PBT dentro do primeiro poço da primeira linha, para criar um espaço em branco. Misture pipetando para cima e para baixo. Evitar a formação de bolhas.</li>
		Poços de <li>carga 20 µ l de cada diluição padrão (da etapa 2.1.2) para o restante da primeira linha e mistura pipetando para cima e para baixo. Evitar a formação de bolhas.</li>
		<li>Usando uma pipeta multicanal, transferência de 20 µ l do sobrenadante de cada linha da placa de moagem para a linha correspondente da placa de 96 poços Espectrofotômetro. Pipete para cima e para baixo para misturar bem. Evitar a formação de bolhas.</li>
		<li>Em seguida, coloque uma folha de gás-permeável sobre a parte superior da placa de 96 poços.</li>
		<li>Incubar a placa a 37 ° C durante 10 min. 
		Nota: Se houver bolhas nos poços, centrifugar a placa por 2 min a 2.000 x g para remover todas as bolhas. A presença de bolhas irá interferir com a leitura de absorvância.</li>
		<li>Leia a absorvância de cada poço da placa em 550 nm, utilizando um leitor de microplacas compatível. Em seguida, traçar a curva-padrão e determinar a concentração [µ g / µ l] das amostras desconhecidas.</li>
		<li>Para determinar a quantidade de conteúdo da marca por médio peso mosca, use a seguinte fórmula: 
		<img alt="Equation 2" src="/files/ftp_upload/56029/56029eq2.jpg" /> Nota: neste protocolo, 3 moscas foram picadas em 600 µ l de PBT; em consequência, o volume médio por mosca é 200 µ l.</li>
	</ol></li> <li>Ensaio de Bradford e normalização de conteúdo da marca com conteúdo de proteína <ol><li>preparar sete 100 µ l de albumina de soro bovino (BSA) padrão diluições (75 µ g/mL; 100 µ g/mL; 150 µ g/mL, 200 µ g/mL; 250 µ g/mL; 500 µ g/mL; 1.000 µ g/mL) com o PBT.</li>
		<li>Tomar uma nova placa de 96 poços e carregar 200 µ l de reagente de Bradford X 1 em cada linha da placa.</li>
		<li>No primeiro bem de primeira linha, adicionar 10 µ l de PBT para criar um espaço em branco. Misture pipetando para cima e para baixo. Evitar a formação de bolhas.</li>
		Poços de <li>Adicionar 10 µ l de cada diluição padrão para o restante da primeira linha e mistura pipetando para cima e para baixo. Evitar a formação de bolhas.</li>
		<li>Usando uma pipeta multicanal, transferir 10 µ l do sobrenadante a mosca de cada linha da placa de moagem para a linha correspondente da placa de 96 poços. Pipete para cima e para baixo para misturar bem. Evitar a formação de bolhas.</li>
		<li>Colocar uma película permeável ao gás por cima da placa de 96 poços.</li>
		<li>Incube a placa na temperatura de quarto por 5 min. Se houver bolhas nos poços, centrifugar a placa por 2 min a 2.000 x g para removê-los. 
		Nota: A presença de bolhas irá interferir com a leitura de absorvância.</li>
		<li>Uso de um espectrofotômetro para ler a absorbância do branco, padrões e amostras desconhecidas em 595 nm. Use a curva padrão para determinar as concentrações [µ g / µ l] das amostras em cada linha.</li>
		<li>Normalizar a TAG conteúda com nível de proteína usando a seguinte fórmula: 
		<img alt="Equation 3" src="/files/ftp_upload/56029/56029eq3.jpg" /></li></ol></li></ol>

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+between+class+III+obesity+(BMI+of+40-59+kg/m2)+and+mortality:+a+pooled+analysis+of+20+prospective+studies.">Association between class III obesity (BMI of 40-59 kg/m2) and mortality: a pooled analysis of 20 prospective studies.</a> PLoS Med. 11, 1001673 (2014).</li><li>Reis, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+five+lipid+extraction+solvent+systems+for+lipidomic+studies+of+human+LDL.">A comparison of five lipid extraction solvent systems for lipidomic studies of human LDL.</a> J Lipid Res. 54, 1812-1824 (2013).</li><li>Turne, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supercritical+fluid+extraction+and+chromatography+for+fat-soluble+vitamin+analysis.">Supercritical fluid extraction and chromatography for fat-soluble vitamin analysis.</a> J Chromatogr A. 936, 215-237 (2001).</li><li>Na, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+model+of+high+sugar+diet-induced+cardiomyopathy.">Drosophila model of high sugar diet-induced cardiomyopathy.</a> PLoS Genet. 9, 1003175 (2013).</li></ol>

Os autores não têm nada para divulgar.

Indução de obesidade em ratos pode levar meses19,20. Nas moscas, este HFD alimentando o protocolo permite a indução de acúmulo de gordura em excesso em questão de dias ou menos, causando aumentos no acúmulo de gordura somente após 18 h (ver Figura 2). HFD alimentando com o protocolo descrito aumenta o conteúdo de glicose 12 e Bmm lipase e PGC-1 expressão24. Isto está...

Estamos em um tempo onde a obesidade e seus associados encargos económicos, é um problema mundial1. Dois em cada três americanos estão com sobrepeso ou obesos com patologias do coração relacionadas, a principal causa de morte dentro da população adulta2. Novos métodos eficientes são necessários para investigar adequadamente os componentes genéticos e moleculares implicados na regulação da síndrome metabólica, usando organismos modelo. Por esta razão, nós escolhemos o mosca de fruta Drosophila modelo porque ele compartilha os processos biológicos mais básicos com mamíferos, incluindo ratos e seres humanos3,4,5,6. Genoma de drosophilaé altamente conservadas durante a evolução, mas no geral é muito menor com menos duplicação de genes e complexidade metabólica, tornando-a ideal para compreender os mecanismos fundamentais implicados em muitas doenças humanas4 , 7 , 8. Além disso, característico de processos realizados pelo tecido adiposo, intestino e pâncreas são representados na mosca e mediar funções reguladoras no metabolismo glicose e lipídios, por exemplo, que são semelhantes aos seres humanos9, 10,11. Além disso, as vias moleculares básicas envolvidos no controle da obesidade, resistência à insulina e diabetes em seres humanos são funcionalmente conservadas em Drosophila melanogaster12,13,14 , 15 , 16. como organismos superiores, Drosophila tem um coração que é formado durante o desenvolvimento de processos semelhantes do coração dos mamíferos3,17. Assim, o desenvolvimento de um confiável HFD alimentação protocolo e ensaio de TAG alto throughput, adaptado para fins de triagem eficiente usando a caixa de ferramenta genética da Drosophila, fornecem um meio importante para estudar e entender a base genética fundamental doenças metabólicas complexas subjacentes.
O próprio alimento HFD é feito a partir de um alimento mosca de laboratório padrão suplementado com óleo de coco, que é composto principalmente de ácidos graxos saturados, conhecidos por ser associado com a síndrome metabólica18. Enquanto induzir obesidade em modelos de mamíferos, como roedores, pode levar meses19,20, nosso HFD otimizado protocolo de alimentação em Drosophila eficazmente e reproducibly aumenta a gordura em questão de dias12,14. Este protocolo, em conjunto com um ensaio de alto throughput TAG, permite eficiente triagem em massa para os efeitos de fatores genéticos, influências ambientais e candidatos a fármacos para descobrir novos moduladores do metabolismo das gorduras. Em consequência, estes protocolos são prováveis relevantes para entender e/ou combater a obesidade e patologias humanas associadas a obesidade.
O protocolo de alimentação é versátil e pode ser aplicado para estudar os efeitos metabólicos e funcionais do único de ácidos gordos saturados ou insaturados. O uso deste teste de TAG de alta taxa de transferência não está limitado a d. melanogaster, mas pode ser adaptado a uma variedade de organismos modelo pequeno com cutícula ou matrizes extracelulares duros (por exemplo, outras espécies de Drosophila , c. elegans e outros emergentes organismos invertebrados modelo) para medir o teor de gordura em diferentes condições ambientais, genéticas ou fisiológicas, em qualquer fase de desenvolvimento, maturidade ou fase da doença metabólica. O ensaio de TAG é baseado em uma medição colorimétrica de uma série de reações enzimáticas que degradam as TAGs em ácidos graxos livres, glicerol, o glicerol-3-fosfato e finalmente H2O2 , que reage com 4-aminoantipyrine (4-AAP) e 3,5- dicloro-2-hidroxibenzeno sulfonato (3,5 DHBS) para produzir um produto de cor vermelho que é medido utilizando um Espectrofotômetro de 96 poços.

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high fat diet feeding and high throughput triacylglyceride assay in drosophila melanogaster

Doença cardíaca é a principal causa de morte humana no mundo. Numerosos estudos têm demonstrado forte ligação entre obesidade e mau funcionamento cardíaco em humanos, mas mais ferramentas e esforços de pesquisa são necessários para melhor elucidar os mecanismos envolvidos. Para mais de um século, o modelo geneticamente altamente tratável de Drosophila tem sido fundamental na descoberta de genes-chave e vias moleculares que provou ser altamente conservadas em toda a espécie. Muitos processos biológicos e mecanismos de doença são funcionalmente conservados na mosca, como desenvolvimento (por exemplo, plano de corpo, coração), câncer e doenças neurodegenerativas. Recentemente, o estudo da obesidade e patologias secundárias, tais como doença em organismos-modelo, tem desempenhado um papel altamente crítico na identificação dos principais reguladores envolvidos na síndrome metabólica em seres humanos.
Aqui, propomos usar esse organismo modelo como uma ferramenta eficiente para induzir a obesidade, ou seja, acumulação excessiva de gordura e desenvolver um protocolo eficiente para monitorar o teor de gordura sob a forma de acumulação de TAGs. Além de altamente conservadas, mas menos complexo genoma, a mosca também tem uma vida curta para a experimentação rápida, combinada com a relação custo-eficácia. Este artigo fornece um protocolo detalhado para a alimentação de alta gordura dieta (HFD) em Drosophila para induzir a obesidade e um ensaio de triacilglicerol (TAG) de alto rendimento para medir o associado aumento no teor de gordura, com o objectivo de ser altamente reprodutível e eficiente para rastreio genético ou químico em larga escala. Estes protocolos oferecem novas oportunidades para eficientemente investigar mecanismos reguladores envolvidos na obesidade, bem como fornecer uma plataforma padronizada para a pesquisa de descoberta de drogas para testes rápidos do efeito de candidatos da droga sobre o desenvolvimento ou prevenção da obesidade, diabetes e doenças metabólicas associadas.

No d. melanogaster, como é o caso com outras espécies, existe dimorfismo sexual entre machos e fêmeas,22. É sabido que as fêmeas são maiores, com mais gordura em seus abdomens, do que os machos22. Para testar a eficácia do nosso protocolo, realizamos ensaios de TAG para determinar as diferenças de conteúdo da marca entre machos e fêmeas de sua padrão de laboratório (w1118) voa. Os dados mostram que as...

Watch this Scientific Journal Video about High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster at JoVE.com

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

Dieta de alta gordura alimentar e ensaio de triacilglicerol alto Throughput em Drosophila Melanogaster.

Esta é uma dieta de alta gordura protocolo para induzir obesidade em Drosophila, um modelo para a compreensão fundamental molecular os mecanismos implicados na lipotoxicity de alimentação. Ele também fornece um ensaio de triacilglicerol alto throughput para medir o acúmulo de gordura em Drosophila e potencialmente outros modelos (insetos) sob diferentes condições de dieta, ambientais, genéticas ou fisiológicas.

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in ...

high-fat-diet-feeding-high-throughput-triacylglyceride-assay

Research

JoVE Journal

Genetics

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

11.1K visualizações.  Sanford Burnham Prebys Medical Discovery Institute. Esta é uma dieta de alta gordura protocolo para induzir obesidade em Drosophila, um modelo para a compreensão fundamental molecular os mecanismos implicados na lipotoxicity de alimentação. Ele também fornece um ensaio de triacilglicerol alto throughput para medir o acúmulo de gordura em Drosophila e potencialmente outros modelos (insetos) sob diferentes condições de dieta, ambientais, genéticas ou fisiológicas.

Vídeo: High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

A plataforma robótica de alta capacidade de protoplastos Isolamento e Transformação

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

Genética

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

Systematic identification and characterization of genetic perturbations have proven useful to decipher gene function and cellular pathways. However, the conventional approaches of permanent gene deletion cannot be applied to essential genes. We have pioneered a unique collection of ~70 temperature-sensitive (ts) lethal mutants for studying cell cycle regulation in the unicellular green algae Chlamydomonas reinhardtii1. These mutations identify essential genes, and the ts alleles can be conditionally inactivated by temperature shift, providing valuable tools to identify and analyze essential functions. Mutant collections are much more valuable if they are close to comprehensive, since scattershot collections can miss important components. However, this requires the efficient collection of a large number of mutants, especially in a wide-target screen. Here, we describe a robotics-based pipeline for generating ts lethal mutants and analyzing their phenotype in Chlamydomonas. This technique can be applied to any microorganism that grows on agar. We have collected over 3000 ts mutants, probably including mutations in most or all cell-essential pathways, including about 200 new candidate cell cycle mutations. Subsequent molecular and cellular characterization of these mutants should provide new insights in plant cell biology; a comprehensive mutant collection is an essential prerequisite to ensure coverage of a broad range of biological pathways. These methods are integrated with downstream genetics and bioinformatics procedures for efficient mapping and identification of the causative mutations that are beyond the scope of this manuscript.

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

Temperature-sensitive (ts) lethal mutants are valuable tools to identify and analyze essential functions. Here we describe methods to generate and classify ts lethal mutants in high throughput.

Alto Throughput Robotically Isolamento Assistida da temperatura sensível Lethal Mutantes em<em&gt; Chlamydomonas reinhardtii</em

Systematic identification and characterization of genetic perturbations have proven useful to decipher gene function and cellular pathways. However, the ...

Quantifying Abdominal Pigmentation in Drosophila melanogaster

Pigmentation is a morphologically simple but highly variable trait that often has adaptive significance. It has served extensively as a model for understanding the development and evolution of morphological phenotypes. Abdominal pigmentation in Drosophila melanogaster has been particularly useful, allowing researchers to identify the loci that underlie inter- and intraspecific variations in morphology. Hitherto, however, D. melanogaster abdominal pigmentation has been largely assayed qualitatively, through scoring, rather than quantitatively, which limits the forms of statistical analysis that can be applied to pigmentation data. This work describes a new methodology that allows for the quantification of various aspects of the abdominal pigmentation pattern of adult D. melanogaster. The protocol includes specimen mounting, image capture, data extraction, and analysis. All the software used for image capture and analysis feature macros written for open-source image analysis. The advantage of this approach is the ability to precisely measure pigmentation traits using a methodology that is highly reproducible across different imaging systems. While the technique has been used to measure variation in the tergal pigmentation patterns of adult D. melanogaster, the methodology is flexible and broadly applicable to pigmentation patterns in myriad different organisms.

Quantifying Abdominal Pigmentation in Drosophila melanogaster

This work presents a method to quickly and precisely quantify the abdominal pigmentation of Drosophila melanogaster using digital image analysis. This method streamlines the procedures between phenotype acquisition and data analysis and includes specimen mounting, image acquisition, pixel value extraction, and trait measurement.

Quantificando pigmentação abdominal em<em&gt; Drosophila melanogaster</em

Pigmentation is a morphologically simple but highly variable trait that often has adaptive significance. It has served extensively as a model for ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution based analysis methodology (nucleosome density ChIP-seq; ndChIP-seq) that enables the generation of combined measurements of micrococcal nuclease (MNase) accessibility with histone modification genome-wide. Nucleosome position and local density, and the posttranslational modification of their histone subunits, act in concert to regulate local transcription states. Combinatorial measurements of nucleosome accessibility with histone modification generated by ndChIP-seq allows for the simultaneous interrogation of these features. The ndChIP-seq methodology is applicable to small numbers of primary cells inaccessible to cross-linking based ChIP-seq protocols. Taken together, ndChIP-seq enables the measurement of histone modification in combination with local nucleosome density to obtain new insights into shared mechanisms that regulate RNA transcription within rare primary cell populations.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) methodology for the generation of sequence datasets suitable for a nucleosome density ChIP-seq analytical framework integrating micrococcal nuclease (MNase) accessibility with histone modification measurements.

Geração de imunoprecipitação da cromatina nativa sequenciamento de bibliotecas para análise da densidade nucleossoma

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of tissues, we have developed numerous modifications and improvements to our original fluorescent in situ hybridization (FISH) protocol. To facilitate throughput and cost effectiveness, all steps, from probe generation to signal detection, are performed using exon 96-well microtiter plates. Digoxygenin (DIG)-labelled antisense RNA probes are produced using either cDNA clones or genomic DNA as templates. After tissue fixation and permeabilization, probes are hybridized to transcripts of interest and then detected using a succession of anti-DIG antibody conjugated to biotin, streptavidin conjugated to horseradish peroxidase (HRP) and fluorescently conjugated tyramide, which in the presence of HRP, produces a highly reactive intermediate that binds to electron dense regions of immediately adjacent proteins. These amplification and localization steps produce a robust and highly localized signal that facilitates both cellular and subcellular transcript localization. The protocols provided have been optimized to produce highly specific signals in a variety of tissues and developmental stages. References are also provided for additional variations that allow the simultaneous detection of multiple transcripts, or transcripts and proteins, at the same time.

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

The described RNA in situ hybridization protocol allows the detection of RNA in whole Drosophila embryos or dissected tissues. Using 96-well microtiter plates and tyramide signal amplification, transcripts can be detected at high resolution, sensitivity, and throughput, and at a relatively low cost.

Alta resolução hibridação fluorescente In Situ em embriões da drosófila e tecidos usando amplificação de sinal Tyramide

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

This article demonstrates a detailed protocol for DNA isolation and high-throughput sequencing library construction from herbarium material including rescue of exceptionally poor-quality DNA.

Isolamento do DNA robusto e construção de biblioteca de sequenciamento de alto rendimento para espécimes de herbário

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our entire society's ability to treat systemic infections. In the United States alone, antibiotic-resistant infections kill approximately 23,000 people a year and cost around 20 billion USD in additional healthcare. One approach to combat antimicrobial resistance is combination therapy, which is particularly useful in the critical early stage of infection, before the infecting organism and its drug resistance profile have been identified. Many antimicrobial treatments use combination therapies. However, most of these combinations are additive, meaning that the combined efficacy is the same as the sum of the individual antibiotic efficacy. Some combination therapies are synergistic: the combined efficacy is much greater than additive. Synergistic combinations are particularly useful because they can inhibit the growth of antimicrobial drug resistant strains. However, these combinations are rare and difficult to identify. This is due to the sheer number of molecules needed to be tested in a pairwise manner: a library of 1,000 molecules has 1 million potential combinations. Thus, efforts have been made to predict molecules for synergy. This article describes our high-throughput method for predicting synergistic small molecule pairs known as the Overlap2 Method (O2M). O2M uses patterns from chemical-genetic datasets to identify mutants that are hypersensitive to each molecule in a synergistic pair but not to other molecules. The Brown lab exploits this growth difference by performing a high-throughput screen for molecules that inhibit the growth of mutant but not wild-type cells. The lab's work previously identified molecules that synergize with the antibiotic trimethoprim and the antifungal drug fluconazole using this strategy. Here, the authors present a method to screen for novel synergistic combinations, which can be altered for multiple microorganisms.

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Synergistic drug combinations are difficult and time-consuming to identify empirically. Here, we describe a method for identifying and validating synergistic small molecules.

Identificação de alta produtividade de combinações de drogas sinérgica pelo método de sobreposição2

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our ...

High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short generation time, are easy to handle, display a high reproductive rate, low cost, and are easily amenable to genetic manipulations by microinjection of DNA in embryos. Recent advances in gene editing tools are enabling precise introduction of mutations and transgenes in zebrafish. Disease modeling in zebrafish often leads to larval phenotypes and early death which can be challenging to interpret if genotypes are unknown. This early identification of genotypes is also needed in experiments requiring sample pooling, such as in gene expression or mass spectrometry studies. However, extensive genotypic screening is limited by traditional methods, which in most labs are performed only on adult zebrafish or in postmortem larvae. We addressed this problem by adapting a method for the isolation of PCR-ready genomic DNA from live zebrafish larvae that can be achieved as early as 72 h post-fertilization (hpf). This time and cost-effective technique, improved from a previously published genotyping protocol, allows the identification of genotypes from microscopic fin biopsies. The fins quickly regenerate as the larvae develop. Researchers are then able to select and raise the desired genotypes to adulthood by utilizing this high-throughput PCR-based genotyping procedure.

Zebrafish have been used as reliable genetic model organisms in biomedical research, especially with the advent of gene-editing technologies. When larval phenotypes are expected, DNA extraction and genotype identification can be challenging. Here, we describe an efficient genotyping procedure for zebrafish larvae, by tail clipping, as early as 72-h post-fertilization.

Extração de DNA do elevado-throughput e genotipagem de larvas de Zebrafish 3dpf por Fin recorte

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short ...

High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often performed at low throughput, it is moreover time-consuming and error-prone, even more so when multiplexing several plasmids. We developed an easy, fast, and accurate method to perform cell transfection in a 384-well plate layout using acoustic droplet ejection (ADE) technology. The nanodispenser device used in this study&#160;is based on this technology and allows precise nanovolume delivery at high speed from a source well plate to a destination one. It can dispense and multiplex DNA and transfection reagent according to a predesigned spreadsheet. Here we present an optimal protocol to perform ADE-based high-throughput plasmid transfection which makes it possible to reach an efficiency of up to 90% and a nearly 100% cotransfection in cotransfection experiments. We extend initial work by proposing a user-friendly spreadsheet-based macro, able to manage up to four plasmids/wells from a library containing up to 1,536 different plasmids, and a tablet-based pipetting guide application. The macro designs the necessary template(s) of the source plate(s) and generates the ready-to-use files for the nanodispenser and tablet-based application. The four-steps transfection protocol involves i) a diluent dispense with a classical liquid handler, ii) plasmid distribution and multiplexing, iii) a transfection reagent dispense by the nanodispenser, and iv) cell plating on the prefilled wells. The described software-based control of ADE plasmid multiplexing and transfection allows even nonspecialists in the field to perform a reliable cell transfection in a fast and safe way. This method enables rapid identification of optimal settings for a given cell type and can be transposed to higher-scale and manual approaches. The protocol eases applications, such as human ORFeome protein (set of open reading frames [ORFs] in a genome) expression or CRISPR-Cas9-based gene function validation, in nonpooled screening strategies.

This protocol describes high-throughput plasmid transfection of mammalian cells in a 384-well plate using acoustic droplet ejection technology. The time-consuming, error-prone DNA dispensing and multiplexing, but also the transfection reagent dispensing, are software-driven and performed by a nanodispenser device. The cells are then seeded in these prefilled wells.

Multiplexação e transfecção de plasmídeo de DNA de alta produtividade usando tecnologia de Nanodosagem acústica

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often ...

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to perform and its ability to standardize the fitness of many strains to a wild-type organism. The technique is limited, however, by available phenotypic markers and the number of strains that can be assessed simultaneously, creating the need for a great number of replicate experiments. Concurrent with large numbers of experiments, the labor and material costs for quantifying bacteria based on phenotypic markers are not insignificant. To overcome these negative aspects while retaining the positive aspects, we have developed a molecular-based approach to directly quantify microorganisms after engineering genetic markers onto bacterial chromosomes. Unique, 25 base pair DNA barcodes were inserted at an innocuous locus on the chromosome of wild-type and mutant strains of Salmonella. In vitro competition experiments were performed using inocula consisting of pooled strains. Following the competition, the absolute numbers of each strain were quantified using digital PCR and the competitive indices for each strain were calculated from those values. Our data indicate that this approach to quantifying Salmonella is extremely sensitive, accurate, and precise for detecting both highly abundant (high fitness) and rare (low fitness) microorganisms. Additionally, this technique is easily adaptable to nearly any organism with chromosomes capable of modification, as well as to various experimental designs that require absolute quantification of microorganisms.

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

This molecular-based approach for determining bacterial fitness facilitates precise and accurate detection of microorganisms using unique genomic DNA barcodes that are quantified via digital PCR. The protocol describes calculating the competitive index for Salmonella strains; however, the technology is readily adaptable to protocols requiring absolute quantification of any genetically-malleable organism.

Índice competitivo baseado em PCR digital para análise de alta produtividade de aptidão em Salmonella

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to ...

Dieta de alta gordura alimentar e ensaio de triacilglicerol alto Throughput em Drosophila Melanogaster.

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