C. elegans transgenes foram fornecidos pelo Centro de Genética Caenorhabditis (University of Minnesota, Minneapolis, MN). Este trabalho foi financiado pelo NIH R21 conceder NS0667678-01 a KS. Todos os autores participaram da coleta, análise e interpretação dos dados. CKL, KS, e KPC participaram da elaboração e revisão do manuscrito.

1. Preparação de comida de verme bacteriana 1 dia <ol><li> Adicionar 5 ml de E. saturada coli ml caldo de cultura bacteriana OP50 a 500 Terrific suplementado com estreptomicina 50 mg / ml e crescer em uma incubadora de agitação (225 rpm) durante a noite a 37 ° C. </li></ol> Dia 2 <ol start="2"><li> Dividir a cultura de bactérias durante a noite em dez tubos de 50 ml e bactérias centrífuga em uma centrífuga refrigerada a 2500 rcf durante 20 minutos. </li><li> Decantar caldo LB e ressuspender cada pellet de bactérias em 10 ml de líquido media nematóide de crescimento (NGM). Agitar horizontalmente em um piso shaker por 15 minutos para voltar a suspender as bactérias. Para fazer tampão NGM, adicionar 3 g de NaCl a 1 L de água deionizada e autoclave. Esfriar a 55 ° C e adicionar em ordem as seguintes soluções estéreis: 1 ml de CaCl 2 1 M, 1 ml de 1 M MgSO 4, e 25 ml de fosfato de potássio 1 M, pH 6,0. </li><li> Centrifugar a cultura bacteriana em uma centrífuga refrigerada a 2500 rcf durante 20 minutos. </li><li> Decantar tampão NGM e pesar o pellet bacteriano. </li><li> Adicionar um volume igual de tampão NGM para ressuspender as pelotas, alíquota de 3 ml de bactérias se concentrar em tubos de 15 ml, e armazenar a -20 ° C. </li></ol> 2. Grande escala C. elegans cultura líquida Nós usamos uma linhagem transgênica VP596 (dvIs19 [pAF15 (gst-4:: GFP:: NLS)]; vsIs33 [dop-3:: RFP]) carregando duas construções fluorescente: Pgst -4:: GFP 14 para monitorar SKN-1 atividade e PDOP-3:: RFP 17 para servir como um padrão para a normalização de número worm. 1 dia <ol><li> Misture 150 ml NGM buffer, 1,5 ml de caldo LB, 150 mL de 5 mg de colesterol / ml (em etanol) e 75 mL de estreptomicina 100 mg / ml. Filtro de esterilizar a mistura e adicionar a um frasco de 1 litro estéril. NOTA: A adição de caldo LB 1% para tamponar NGM ajuda a evitar worms de degola para copos e plasticware, mas este montante não é suficiente para suportar o crescimento de bactérias. </li><li> Sincronizar worms com o procedimento padrão de hipoclorito de 18. Até 0,5 ml de vermes gravídico pode ser processado em um tubo única de 15 ml com 5 ml de solução de hipoclorito (3,75 ml de água estéril, uma lixívia ml, e 250 mL NaOH 10 N). Lave os ovos liberados com água estéril e ressuspender-los em 10 ml de tampão NGM. </li><li> Diluir uma amostra do ovos 100 vezes na NGM buffer (por exemplo, 100 l em 10 ml) e contar o número de ovos em três diferentes alíquotas 5 mL. Múltiplos, o número médio de 200.000 (cem fator de diluição x 2.000) para estimar o número total de ovos. </li><li> Adicionar 200 mil para 2 milhões de ovos para o frasco com tampão NGM e agitar a cultura em 100 rpm a 20 ° C. </li></ol> Dia 2 <ol start="5"><li> Pelo menos 16 h após a adição de ovos para o balão, use uma pipeta estéril sorológica para remover cerca de 0,5 ml da cultura verme suspenso. Pipetar 5 gotas três mL para a tampa estéril de uma placa de Petri. Coloque a tampa em um freezer -20 ° C por 1-2 minutos para paralisar worms. Conte o número médio de filhos nascidos worms por 5 mL e, em seguida, vários por 30.000 (150 ml / 5 mL) para estimar o número total de vermes nascidos. </li><li> Descongelar um tubo de cultura bacteriana OP50 congelados (Protocolo 1.6) e adicionar 3 ml de 50% OP50 na cultura verme por 500.000 nascidos worms. Agitar o frasco a 100 rpm a 20 ° C. Vermes vão se desenvolver em larvas L4 e jovens adultos em cerca de 51 horas. O OD600 de bactérias deve ser cerca de 2,200 no início. Monitorar a quantidade de bactérias por absorbância e adicionar mais se o OD cai abaixo de 0,900 600. </li></ol> 3. Coleta e distribuição Worm Dia 4 <ol><li> Use uma pipeta estéril sorológica para transferir cerca de 0,5 ml da cultura verme suspensos a uma placa padrão NGM agar. Ver os vermes com uma lupa para se certificar de que a maioria são L4 larvas ou adultos jovens. L4 larvas terá um ponto ventral clara no meio do corpo. Jovens adultos será um pouco maior e não terá um ponto claro. </li><li> Despeje a cultura verme em tubos de 50 ml estéril e colocar os tubos em um rack tubo de ensaio sobre a bancada. Permitir que os vermes que se contentar com cerca de 10 minutos. Retire o sobrenadante por aspiração ou pipetagem. NOTA: Esta etapa pode ser importante para remover os ovos unhatched ou worms que não se desenvolveu porque eles vão resolver mais lento do que L4 larvas e adultos jovens. </li><li> Colete todos os worms em um único tubo de 50 ml e lavar com tampão NGM com caldo LB 1% (NGM LB +) 3-4 vezes para remover bactérias. NOTA: Este é o melhor feito em uma centrífuga balançando-balde, recolhendo worms a 500 rcf durante 30 seg e substituindo o sobrenadante com NGM fresco + tampão LB. </li><li> Após a lavagem, encher o tubo com NGM + tampão LB a 50 ml e despeje em um frasco personalizado verme dispensa. Adicionar uma barra de agitação e mexa para manter os worms suspenso. º Condenúmero de vermes e em três mL 5 gotas como indicado acima. Diluir com NGM LB + até chegar 12-15 worms por 5 mL queda (~ 2,5-3,0 worms / mL). NOTA: Um mínimo de cerca de 20 ml (60.000 worms) é necessário para preencher o espaço morto do recipiente de dispensação e cassete dispenser. Cada placa 384 poços requer cerca de 35 mil worms ou 11,5 ml de vermes suspenso. </li><li> Carga de 10 mL de cassetes de dispensação e esterilizar em priming com etanol 70%. Enxaguar o etanol por priming com água estéril. </li><li> Insira a extremidade do cassete de distribuição para o balão de modo que seja logo acima da barra mexa sem interromper seu movimento. Certifique-se que os vermes continuam suspensas em todo o frasco inteiro. Programa o dispensador para dispensar a baixa velocidade com 2 pré-pulsos. Primeiro e executar pelo menos 10 ml de vermes suspenso. Preencha placas conforme necessário rapidamente para evitar que os vermes de se estabelecer na tubulação. </li><li> Selar as placas com fita respirável. </li><li> Coloque as placas em uma plataforma de agitação em uma incubadora à temperatura adequada para o seu ensaio. Não empilhar os pratos. </li></ol> 4. Análise de fluorescência <ol><li> Coloque a placa de alvo em um leitor de microplacas para medir a intensidade de fluorescência de cada bem com o comprimento de onda de emissão e excitação apropriada (Filtros para o nosso ensaio: GFP 485/20ex 528/20em; RFP 540/25ex 590/35em). </li><li> Calcular a proporção de GFP / RFP e normalizar com as leituras dos poços de controlo (sem composto ativar) para determinar a diferença exata dobra da intensidade de fluorescência derivados de tratamentos individuais. </li></ol> 5. Resultados representativos: Nosso SKN-1 ensaio utiliza uma cepa repórter cromossomicamente integrado dual (VP596). Conforme mostrado na Figura 1, o número de vermes é bem correlacionada com o volume dispensado. Como mostrado nas Figuras 2A e 2B, GFP total e RFP fluorescência por poço é altamente reprodutível de bem para o bem através de uma placa de 384 poços. Conforme mostrado na Figura 2C, un induzida Pgst-4:: GFP fluorescência é linearmente correlacionada com PDOP-3:: RFP em 384 poços. Quando expresso como uma razão de GFP / RFP, fluorescência torna-se altamente reprodutível de bem para o bem através de uma placa de 384 poços (compare o coeficiente de variação da Figura 2D para 2A), demonstrando a capacidade do PDOP-3:: RFP repórter para reduzir a variabilidade. Conforme mostrado na Figura 3, a indução de boas práticas agrícolas em relação a RFP com uma SKN-1 ativando xenobióticos (38 mM juglone) é robusto e altamente reprodutível através de uma placa de 384 poços. <img src="/files/ftp_upload/2745/2745fig1.jpg" alt="Figura 1"/> Figura 1. Número de worms versus volume dispensado. Worms foram diluídas a cerca de 2/μl e dispensado em uma placa de 24 poços para a contagem manual com um estereomicroscópio. N = 8 poços por volume. <img src="/files/ftp_upload/2745/2745fig2.jpg" alt="Figura 2"/> Figura 2. Fluorescência total de worms dispensado em uma placa de 384 poços é reproduzível. Worms foram diluídas a cerca de 2.5/μl mL e 30 foi dispensado em cada poço de uma placa 384 poços. GFP (A) e RFP fluorescência (B) de todos os poços tiveram um coeficiente de variação abaixo de 9%. (C) de fluorescência da GFP é altamente correlacionada com a RFP fluorescência. (D) Cálculo da proporção de GFP para RFP reduziu o coeficiente de variação abaixo de 6% (A, B e D) As linhas contínuas indicam os meios e linhas quebradas indicam três desvios-padrão acima ou abaixo da média. <img src="/files/ftp_upload/2745/2745fig3.jpg" alt="Figura 3"/> . Figura 3 Ativação do Pgst-4:: GFP é robusta e consistente. Aproximadamente 75 L4 worms foram dispensados ​​em todos os poços de uma placa de 384 poços e 38 juglone mM foi adicionado em cada outra coluna. GFP e RFP fluorescência foi medida após 21 h de incubação. A média relativa de fluorescência de todos os índices de controle (1.0) e juglone poços (8.9) são marcadas com linhas sólidas. Três desvios padrão acima da média dos controles e abaixo da média juglone são marcados com linhas quebradas.

<ol>
	<li>Simeonov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+high-throughput+screen+identifies+inhibitors+of+the+Schistosoma+mansoni+redox+cascade.">Quantitative high-throughput screen identifies inhibitors of the Schistosoma mansoni redox cascade.</a> PLoS Negl Trop Dis. 2, e127-e127 (2008).</li><li>Giacomotto, J., Segalat, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+and+small+animal+models,+where+are+we.">High-throughput screening and small animal models, where are we.</a> Br J Pharmacol. 160, 204-216 (2010).</li><li>Artal-Sanz, M., de Jong, L., Tavernarakis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans:+a+versatile+platform+for+drug+discovery.">Caenorhabditis elegans: a versatile platform for drug discovery.</a> Biotechnol J. 1, 1405-1418 (2006).</li><li>An, J. H., Blackwell, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SKN-1+links+C.+elegans+mesendodermal+specification+to+a+conserved+oxidative+stress+response.">SKN-1 links C. elegans mesendodermal specification to a conserved oxidative stress response.</a> Genes Dev. 17, 1882-1893 (2003).</li><li>Choe, K., Przybysz, A., Strange, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+WD40+repeat+protein+WDR-23+functions+with+the+CUL4/DDB1+ubiquitin+ligase+to+regulate+nuclear+abundance+and+activity+of+SKN-1+in+Caenorhabditis+elegans.">The WD40 repeat protein WDR-23 functions with the CUL4/DDB1 ubiquitin ligase to regulate nuclear abundance and activity of SKN-1 in Caenorhabditis elegans.</a> Mol Cell Biol. 29, 2704-2715 (2009).</li><li>Hasegawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acrylamide-responsive+genes+in+the+nematode+Caenorhabditis+elegans.">Acrylamide-responsive genes in the nematode Caenorhabditis elegans.</a> Toxicol. Sci. 101, 215-225 (2008).</li><li>Tullet, J. M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+inhibition+of+the+longevity-promoting+factor+SKN-1+by+insulin-like+signaling+in+C.+elegans.">Direct inhibition of the longevity-promoting factor SKN-1 by insulin-like signaling in C. elegans.</a> Cell. 132, 1025-1038 (2008).</li><li>Oliveira, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Condition-adapted+stress+and+longevity+gene+regulation+by+Caenorhabditis+elegans+SKN-1/Nrf.">Condition-adapted stress and longevity gene regulation by Caenorhabditis elegans SKN-1/Nrf.</a> Aging Cell. , (2009).</li><li>Park, S. -. K., Tedesco, P. M., Johnson, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+and+longevity+in+Caenorhabditis+elegans+as+mediated+by+SKN-1.">Oxidative stress and longevity in Caenorhabditis elegans as mediated by SKN-1.</a> Aging Cell. 8, 258-269 (2009).</li><li>Hayes, J. D., McMahon, M., Chowdhry, S., Dinkova-Kostova, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+chemoprevention+mechanisms+mediated+through+the+Keap1-Nrf2+pathway.">Cancer chemoprevention mechanisms mediated through the Keap1-Nrf2 pathway.</a> Antioxid Redox Signal. , (2010).</li><li>Kwak, M. K., Kensler, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+NRF2+signaling+for+cancer+chemoprevention.">Targeting NRF2 signaling for cancer chemoprevention.</a> Toxicol Appl Pharmacol. 244, 66-76 (2010).</li><li>Leiser, S. F., Miller, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nrf2+signaling,+a+mechanism+for+cellular+stress+resistance+in+long-lived+mice.">Nrf2 signaling, a mechanism for cellular stress resistance in long-lived mice.</a> Mol Cell Biol. 30, 871-884 (2010).</li><li>Link, C. D., Johnson, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reporter+transgenes+for+study+of+oxidant+stress+in+Caenorhabditis+elegans.">Reporter transgenes for study of oxidant stress in Caenorhabditis elegans.</a> Methods Enzymol. 353, 497-505 (2002).</li><li>Vanduyn, N., Settivari, R., Wong, G., Nass, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SKN-1/Nrf2+inhibits+dopamine+neuron+degeneration+in+a+Caenorhabditis+elegans+model+of+methylmercury+toxicity.">SKN-1/Nrf2 inhibits dopamine neuron degeneration in a Caenorhabditis elegans model of methylmercury toxicity.</a> Toxicol Sci. , (2010).</li><li>Hasegawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+inexpensive+method+to+screen+for+common+foods+that+reduce+the+action+of+acrylamide,+a+harmful+substance+in+food.">A rapid and inexpensive method to screen for common foods that reduce the action of acrylamide, a harmful substance in food.</a> Toxicol Lett. 175, 82-88 (2007).</li><li>Chase, D. L., Pepper, J. S., Koelle, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+extrasynaptic+dopamine+signaling+in+Caenorhabditis+elegans.">Mechanism of extrasynaptic dopamine signaling in Caenorhabditis elegans.</a> Nature Neuroscience. 7, 1096-1103 (2004).</li><li>Hope, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> C. elegans, a Practical Guide. , (2005).</li><li>Pulak, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+analysis,+sorting,+and+dispensing+of+C.+elegans+on+the+COPAS+flow-sorting+system.">Techniques for analysis, sorting, and dispensing of C. elegans on the COPAS flow-sorting system.</a> Methods Mol Biol. 351, 275-286 (2006).</li></ol>

Não há conflitos de interesse declarados.

Nós apresentamos um método para cultivo e distribuição de um grande número de nematóides transgênicos. O equipamento utilizado para vermes é padrão para laboratórios de clonagem molecular e da manipulação de líquidos e equipamentos de fluorescência é padrão para os laboratórios de processamento de um grande número de microplacas. Outros métodos de dispensar um grande número de viver C. elegans exigem partículas caros equipamentos de triagem 19. O Pgst-4:: ensaio de G...

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários (opcional) </th></tr><tr><td> LB </td><td> Research Products International Corp </td><td> L24066 </td><td></td></tr><tr><td> Terrific caldo </td><td> Research Products International Corp </td><td> T15100 </td><td></td></tr><tr><td> Synergy ™ HT Multi-Modo de Leitora </td><td> Biotek </td><td></td><td> Filtros: GFP 485/20ex 528/20em; RFP 540/25ex 590/35em </td></tr><tr><td> MicroFlo Dispenser Selecione </td><td> Biotek </td><td></td><td></td></tr><tr><td> Worm frasco de dispensação </td><td> Southern Scientific Inc., Micanopy, FL </td><td></td><td> Personalizado montada (352) 284-2531 </td></tr><tr><td> 384 microplacas </td><td> Greiner Bio-One </td><td> 5678-1209 </td><td></td></tr><tr><td> Fita de vedação respirável </td><td> Nunc </td><td> 241205 </td><td></td></tr><tr><td> (5-hidroxi-p-naphthoqinone) Juglone </td><td> ACROS </td><td> 121640010 </td><td> Juglone é dissolvido em DMSO </td></tr><tr><td> DMSO </td><td> Sigma-Aldrich </td><td> D-5879 </td><td></td></tr><tr><td> C. elegans transgênicos tensão </td><td> Laboratório autor </td><td> VP596 </td><td> Pgst-4:: GFP e PDOP-3:: RFP </td></tr></tbody></table>

high-throughput screening and biosensing with fluorescent c. elegans strains

High-throughput screening (HTS) é uma poderosa abordagem para a identificação de moduladores de química de processos biológicos. No entanto, muitos compostos identificados em telas usando modelos de cultura celular são encontrados frequentemente para ser tóxicos ou farmacologicamente inativas in vivo 1-2. Triagem em modelos animais inteiros pode ajudar a evitar estas armadilhas e agilizar o caminho para o desenvolvimento de drogas. C. elegans é um organismo multicelular modelo adequado para HTS. Ele é pequeno (&lt;1 mm) e pode ser economicamente cultivadas e distribuídas em líquidos. C. elegans é também um dos modelos animais mais experimentalmente tractable permitindo identificação rápida e detalhada de drogas modo-de-acção 3. Nós descrevemos um protocolo para cultivo e distribuição de linhagens fluorescentes de C. elegans para high-throughput screening de bibliotecas de química ou de detecção de contaminantes ambientais que alteram a expressão de um gene específico. Um grande número de worms developmentally sincronizado são cultivadas em cultura líquida, colhidas, lavadas e suspensas em uma densidade definida. Worms são então adicionados ao preto, de fundo chato de 384 poços placas utilizando um dispensador peristáltica líquido. Pequenas moléculas de uma biblioteca de produtos químicos ou amostras de teste (por exemplo, água, alimentos, solo ou) podem ser adicionados a poços com worms. In vivo, em tempo real intensidade de fluorescência é medida com um leitor de microplacas de fluorescência. Este método pode ser adaptado a qualquer gene induzível em C. elegans para o qual um repórter está disponível. Estresse induzível muitos caminhos e de desenvolvimento de transcrição são bem definidas em C. elegans e tensões repórter GFP transgênicos já existem em muitos deles 4. Quando combinado com os repórteres apropriado transgênicos, o nosso método pode ser utilizado para triagem de moduladores caminho ou para desenvolver robusto ensaios biosensor para os contaminantes ambientais. Demonstramos nossa C. elegans cultura e dispensando protocolo com um ensaio HTS que desenvolvemos para monitorar o C. fator &#39;n&#39; elegans cap transcrição colar SKN-1. SKN-1 e seu homólogo Nrf2 mamíferos ativar genes citoprotetora durante o estresse oxidativo e xenobióticos 50-10. Nrf2 protege mamíferos de várias doenças relacionadas à idade, tais como câncer, neurodegeneração, e inflamação crônica e se tornou um dos principais alvos quimioterápicos 11-13. Nosso ensaio é baseado em um repórter GFP transgênicos para o SKN-1 gene-alvo gst -4 14, que codifica a 6 glutationa-s-transferase. O repórter gst -4 é também um biosensor para produtos químicos xenobióticos e oxidativa que ativam SKN-1 e pode ser usado para detectar baixos níveis de contaminantes, como acrilamida e metil-mercúrio 15-16.

Watch this Scientific Journal Video about High-throughput Screening and Biosensing with Fluorescent C. elegans Strains at JoVE.com

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

Um procedimento para líquidos baseados em cultura e distribuição de<em&gt; C. elegans</em&gt; Cepas que expressam proteínas fluorescentes repórter é descrito que não requer equipamento caro classificação. Esta abordagem pode ser aplicada a inúmeras induzível<em&gt; C. elegans</em&gt; Genes para a descoberta de drogas ou biosensoriamento de contaminantes.

Alto rendimento Triagem e Biossensoriais com Fluorescente C. elegans Cepas

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

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Biology

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

17.8K visualizações.  University of Florida. Um procedimento para líquidos baseados em cultura e distribuição de C. elegans Cepas que expressam proteínas fluorescentes repórter é descrito que não requer equipamento caro classificação. Esta abordagem pode ser aplicada a inúmeras induzível C. elegans Genes para a descoberta de drogas ou biosensoriamento de contaminantes.

Vídeo: High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

High Throughput Screening da atividade endoglucanase de fungos em Escherichia coli</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

Biologia

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi de triagem para identificar fenótipos pós-embrionário em C. elegans</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

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. ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

De alto rendimento de purificação de afinidade com tag Proteínas Recombinantes

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

High-throughput screening para pequenas moléculas Moduladores de canais de potássio Interiores retificador

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

Bioquímica e Alto Rendimento Avaliação microscópica de massa gorda em<em&gt; Caenorhabditis elegans</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

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 ...

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 ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

Identificação de pares Kinase-substrato utilizando High Throughput Screening


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...