Agradecemos a D. Braendle, CL Kurz e F. Montañana-Sanchis por suas contribuições. Este projecto foi apoiado por doações da ANR e do Conseil Regional PACA e financiamento institucional de INSERM e do CNRS.

<ol>
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Biol. 278, 49-59 (2005).</li><li>Pujol, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-fungal+innate+immunity+in+C.+elegans+is+enhanced+by+evolutionary+diversification+of+antimicrobial+peptides.">Anti-fungal innate immunity in C. elegans is enhanced by evolutionary diversification of antimicrobial peptides.</a> PLoS Pathog. 4, e1000105-e1000105 (2008).</li><li>Simmer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-Wide+RNAi+of+C.+elegans+Using+the+Hypersensitive+rrf-3+Strain+Reveals+Novel+Gene+Functions.">Genome-Wide RNAi of C. elegans Using the Hypersensitive rrf-3 Strain Reveals Novel Gene Functions.</a> PLoS Biol. 1, e12-e12 (2003).</li><li>Couillault, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TLR-independent+control+of+innate+immunity+in+Caenorhabditis+elegans+by+the+TIR+domain+adaptor+protein+TIR-1,+an+ortholog+of+human+SARM.">TLR-independent control of innate immunity in Caenorhabditis elegans by the TIR domain adaptor protein TIR-1, an ortholog of human SARM.</a> Nat Immunol. 5, 488-494 (2004).</li><li>Morton, E., Lamitina, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+suite+of+MATLAB-based+computational+tools+for+automated+analysis+of+COPAS+Biosort+data.">A suite of MATLAB-based computational tools for automated analysis of COPAS Biosort data.</a> Biotechniques. 48, xxv-xxx (2010).</li><li>Rohlfing, A. K., Miteva, Y., Hannenhalli, S., Lamitina, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+physiological+activation+of+osmosensitive+gene+expression+mimics+transcriptional+signatures+of+pathogen+infection+in+C.+elegans.">Genetic and physiological activation of osmosensitive gene expression mimics transcriptional signatures of pathogen infection in C. elegans.</a> PLoS One. 5, e9010-e9010 (2010).</li><li>Lee, K. Z., Kniazeva, M., Han, M., Pujol, N., Ewbank, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fatty+acid+synthase+fasn-1+acts+upstream+of+WNK+and+Ste20&#47;GCK-VI+kinases+to+modulate+antimicrobial+peptide+expression+in+C.+elegans+epidermis.">The fatty acid synthase fasn-1 acts upstream of WNK and Ste20&#47;GCK-VI kinases to modulate antimicrobial peptide expression in C. elegans epidermis.</a> Virulence. 1, 113-122 (2010).</li><li>Duverger, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+semi-automated+high-throughput+approach+to+the+generation+of+transposon+insertion+mutants+in+the+nematode+Caenorhabditis+elegans.">A semi-automated high-throughput approach to the generation of transposon insertion mutants in the nematode Caenorhabditis elegans.</a> Nucleic Acids Res. 35, e11-e11 (2007).</li><li>Pierce-Shimomura, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+crawling+and+swimming+locomotory+patterns+in+C.+elegans.">Genetic analysis of crawling and swimming locomotory patterns in C. elegans.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 20982-20987 (2008).</li><li>Ruzanov, P., Riddle, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+SAGE+analysis+of+the+Caenorhabditis+elegans+transcriptome.">Deep SAGE analysis of the Caenorhabditis elegans transcriptome.</a> Nucleic Acids Res. 38, 3252-3262 (2010).</li><li>Kurz, C. L., Ewbank, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection+in+a+dish:+high-throughput+analyses+of+bacterial+pathogenesis.">Infection in a dish: high-throughput analyses of bacterial pathogenesis.</a> Curr. Opin. Microbiol. 10, 10-16 (2007).</li><li>Okoli, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+antifungal+compounds+active+against+Candida+albicans+using+an+improved+high-throughput+Caenorhabditis+elegans+assay.">Identification of antifungal compounds active against Candida albicans using an improved high-throughput Caenorhabditis elegans assay.</a> PLoS One. 4, e7025-e7025 (2009).</li><li>Moy, T. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+novel+antimicrobials+using+a+live-animal+infection+model.">Identification of novel antimicrobials using a live-animal infection model.</a> Proc. Natl. Acad. Sci. U. S. A. 103, 10414-10419 (2006).</li><li>Ballestriero, F., Thomas, T., Burke, C., Egan, S., Kjelleberg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+compounds+with+bioactivity+against+the+nematode+Caenorhabditis+elegans+by+a+screen+based+on+the+functional+genomics+of+the+marine+bacterium+Pseudoalteromonas+tunicata+D2.">Identification of compounds with bioactivity against the nematode Caenorhabditis elegans by a screen based on the functional genomics of the marine bacterium Pseudoalteromonas tunicata D2.</a> Appl. Environ. Microbiol. 76, 5710-5717 (2010).</li></ol>

O laboratório Ewbank colabora com União Biometrica e Modul-Bio.

<p class="jove_content"&gt; O protocolo atual permite grande escala de rastreamento em RNAi<em&gt; C. elegans</em&gt; Em meios sólidos, usando o nível de expressão de um gene repórter fluorescente como uma leitura para fora. Tem sido optimizada com instrumentos automatizados para facilitar alto rendimento ensaios, permitindo a manipulação fácil e rápido de grandes números de amostras, de uma maneira eficiente, fiável e reproduzível. Outros métodos têm sido descritos, que não dependa tanto manipulação automatizada da amostra líquida<sup&gt; 8</sup&gt;.</p&gt;<p class="jove_content"&gt; Um aspecto chave organizacional para o protocolo tem sido o desenvolvimento de um método robusto de acompanhamento amostra através da adaptação do software MBioLIMS comercial para o procedimento de tela RNAi em colaboração com o ModulBio empresa. Em cada fase do processo, as placas são agora identificados por códigos de barras geradas dentro do LIMS. À medida que o LIMS também regista a identidade dos clones em cada poço em cada placa, indo de bem ao gene putativo é simples. E, além disso, os links LIMS para o banco de dados WormBase comunidade, permitindo o acesso direto aos dados moleculares e funcionais sobre qualquer gene. O LIMS também facilita a análise a ser realizada na placa ou mesmo a nível dos cromossomas e genoma. Para estas análises, os dados são armazenados de uma maneira que é inteiramente compatível com as ferramentas previamente descritos, tais como COPAmulti<sup&gt; 16</sup&gt;.</p&gt;<p class="jove_content"&gt; Um ponto crítico experimental no método é o ajustamento da quantidade de alimento em relação ao número de vermes. Para muitos ensaios, é essencial que os vermes não fome no final da experiência, mas também é importante não ter muita comida no início. Observou-se que uma densidade elevada de alimentos diminui a indução de fluorescência no protocolo usando vermes que transportam o p<em&gt; Nlp-29</em&gt; :: Repórter GFP. Isto pode reflectir uma eficiência de infecção diminuída quando os vermes são cultivadas em bactérias demasiado (BS, resultados não publicados). Ao mesmo tempo, as condições de cultura das bactérias RNAi também é importante, uma vez que este determina se ARNdc é produzido a um nível óptimo. Alguns protocolos para grande escala RNAi requerem cultura de RNAi clones em placas de ágar antes de semear culturas líquidas<sup&gt; 8</sup&gt;. Foram obtidos silenciamento do gene robusto omitindo este passo. Optou-se para o crescimento de um excesso de cada cultura para assegurar resultados homogéneos. O protocolo descrito no facto fornece material suficiente para conduzir 2 ou 3 telas em paralelo. No interesse de obter os resultados mais robustos, nós favorecemos, no entanto, a realização de telas em duplicata, utilizando culturas independentes de vermes, bactérias e esporos de fungos de RNAi.</p&gt;<p class="jove_content"&gt; Outro passo crítico está relacionado ao uso de meio que não é nem muito úmido nem muito seco. Se as placas não são suficientemente seca, a bactéria RNAi não seca de forma adequada e vermes nadará durante o ensaio. Isto pode causar problemas. Por exemplo, os vermes não são eficientemente infectado por<em&gt; D. coniospora</em&gt; Em líquido (C. Couillault, resultados não publicados). Por outro lado, em placas secas, a osmolaridade dos aumentos de meio de cultura. Isso tem efeitos diretos sobre a fisiologia verme<sup&gt; 17</sup&gt;, Incluindo a indução de alguns genes de péptidos antimicrobianos, tais como<em&gt; Nlp 29</em&gt;<sup&gt; 18</sup&gt;. O volume utilizado para dispensar vermes e esporos é crítico para permitir a secagem eficaz e rápida. Se mais do que 10 uL de líquido é adicionada às placas frescas, os poços pode levar várias horas para secar. Isto pode significar que é difícil de utilizar métodos robóticos para adicionar vermes para as placas. Por exemplo, cada verme é dispensado pelo Biosort em cerca de 1 uL de líquido. Atualmente, distribuir os vermes manualmente, que também nos permite superar a tendência de vermes em solução de sedimentos.</p&gt;<p class="jove_content"&gt; Nós anteriormente relatado um ajustamento do Biosort que permitiu uma placa de 96 poços a serem analisados ​​em 36 minutos<sup&gt; 19</sup&gt;. A máquina do fabricante UBI agora oferece uma modificação do Biosort diminuir ainda mais o tempo de análise. Com o &quot;FastReFlex&quot;, as amostras são encaminhadas diretamente através da célula de fluxo para análise, ignorando o filtro bolha armadilha. Isto resulta numa redução significativa no tempo de aquisição de dados; uma placa inteira pode ser analisado em 22 min, permitindo a cerca de 20 placas a ser tratado por dia. Se as placas não foram congeladas, no entanto, não haveria um intervalo de 8 horas entre os primeiros e últimos. Dada a duração relativamente curta do experimento, com a infecção dura apenas 18 horas, isso iria introduzir uma variável inaceitável em placas. Não só as placas de congelação antes da análise resolver este problema, mas também é essencial no contexto de alto rendimento como análises, com o protocolo padrão actual e líquido de tratamento de robôs, 2 pessoas podem facilmente processar um máximo de 50 placas de uma semana. Atualmente, nós rotineiramente tela de 30 placas por semana, o que permite uma tela de genoma inteiroa ser conduzida uma vez em menos de 2 meses.</p&gt;<p class="jove_content"&gt; Uma característica interessante deste protocolo é o facto de que ele usa agar sólido, permitindo que os ensaios que não podem ser realizados em cultura líquida. Como fisiologia verme difere dependendo das condições de cultura<sup&gt; 20,21</sup&gt;, Sendo capaz de realizar ensaios em meio sólido pode também complementar habituais líquido à base de telas. Para certos fenótipos (por exemplo, movimento), a análise pode ser realizada directamente com placas de 96 poços em meio sólido. Para a tela atual, no entanto, como o Biosort não pode provar a partir de placas de meio sólido, worms são primeiro transferidos para líquido antes do congelamento. Por outro lado, o Biosort COPAS permite uma análise quantitativa multiparamétrico compatível com a medição de muitos diferentes fenótipos. É provável que para qualquer determinada tela nem todos os parâmetros medidos será o primário de leitura para fora. No entanto, estas medições podem ser úteis por várias razões. Eles permitem que um para determinar se o ensaio correu correctamente. Por exemplo, pode-se verificar o número e tamanho dos vermes em cada poço. Só quando um clone de RNAi deve bloquear o crescimento ou a reprodução deve haver um desvio dos valores esperados. Wells com menos de 20 vermes e / ou animais 30% menor (TOF) em comparação com um standard controle sem efeito sobre o crescimento (<em&gt; Por exemplo gfp (RNAi</em&gt;)) Pode ser excluída a partir de análises subsequentes. A função destes clones podem ser tratadas utilizando um protocolo alternativo, em que os vermes são expostos às bactérias RNAi após o seu desenvolvimento é bem avançado<sup&gt; 15</sup&gt;. Os parâmetros diferentes podem também ser utilizado para refinar gene candidato (batida) de selecção, por exemplo, limitando acessos apenas para os que afectam a fluorescência verde, mas não vermelho. O método preciso escolhido para a selecção acessos dependerá do tipo de tela e sua exacta de leitura para fora. Dada a complexidade dos dados gerados a partir de uma tela de todo o genoma quantitativa, a colaboração com um estatístico pode ser necessária.</p&gt;<p class="jove_content"&gt; O protocolo descrevemos pode ser prontamente adaptado a outros tipos de ensaios. Por exemplo, na fase de infecção<em&gt; D. coniospora</em&gt; Poderia ser substituído por qualquer outro agente patogénico capaz de infectar em meios sólidos. Isto é potencialmente útil como nós descobrimos que muitos dos factores de virulência importantes para Serratia marcescens infecção em cultura líquida não desempenham um papel durante a infecção em meio sólido (E. Pradel, comunicação pessoal). O protocolo é também totalmente compatível com os testes de compostos químicos<sup&gt; 22-24</sup&gt;, Ou para identificar compostos com bioactividade usando bibliotecas diferentes de bactérias<sup&gt; 25</sup&gt;, Então deve encontrar uma utilidade geral no<em&gt; C. elegans</em&gt; Comunidade de pesquisa.</p

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> BactoAgar </td><td> BD Diagnostic Systems </td><td> 214010 </td><td></td></tr><tr><td> Ampicilina </td><td> Sigma-Aldrich </td><td> A9518 </td><td></td></tr><tr><td> BactoPeptone </td><td> BD Diagnostic Systems </td><td> 211677 </td><td></td></tr><tr><td> CaCl 2 </td><td> Qualquer fornecedor </td><td></td><td></td></tr><tr><td> Película adesiva AeraSeal </td><td> Dutscher </td><td> 760214 </td><td></td></tr><tr><td> Isopropílico β-d-tiogalactopiranósido (IPTG) </td><td> Thermo Scientific </td><td> AB-0481 </td><td></td></tr><tr><td> K 2 HPO 4 </td><td> Qualquer fornecedor </td><td></td><td></td></tr><tr><td> KH 2 PO 4 </td><td> Qualquer fornecedor </td><td></td><td></td></tr><tr><td> MgSO4 </td><td> Qualquer fornecedor </td><td></td><td></td></tr><tr><td> NaCl </td><td> Qualquer fornecedor </td><td></td><td></td></tr><tr><td> Na 2 HPO 4 </td><td> Qualquer fornecedor </td><td></td><td></td></tr><tr><td> NaOH </td><td> Qualquer fornecedor </td><td></td><td></td></tr><tr><td> Placa de 96 poço profundo </td><td> Thermo Scientific </td><td> AB-0932 </td><td></td></tr><tr><td> Placa de 96 poços plana </td><td> FALCON </td><td> 353072 </td><td></td></tr><tr><td> Placa de 96 poços rodada </td><td> FALCON </td><td> 353077 </td><td></td></tr><tr><td> Tetraciclina </td><td> Sigma-Aldrich </td><td> T8032 </td><td></td></tr><tr><td> Triton X-100 </td><td> Qualquer fornecedor </td><td></td><td></td></tr><tr><td> TECAN robô </td><td> TECAN </td><td></td><td></td></tr><tr><td> Liquidator96 TM </td><td> RAININ </td><td></td><td></td></tr><tr><td> COPAS Biosort </td><td> União Biometrica </td><td></td><td></td></tr><tr><td> LIMS </td><td> Modul-Bio </td><td></td><td></td></tr></tbody></table>

quantitative and automated high-throughput genome-wide rnai screens in c. elegans

RNA de interferência é um poderoso método para compreender a função do gene, especialmente quando conduzida a uma escala do genoma inteiro-e num contexto quantitativa. Em C. elegans, a função do gene pode ser batido para baixo de maneira simples e eficiente pela alimentação de vermes com bactérias que expressam um ARNdc que corresponde a um gene específico 1. Embora a criação de bibliotecas de clones de RNAi cobrindo a maior parte do C. elegans genoma 2,3 abriu o caminho para estudos genômicos funcionais verdadeiros (ver, por exemplo 4-7), a maioria dos métodos estabelecidos são difíceis. Moy e colegas desenvolveram semi-automatizados protocolos que facilitem genome-wide telas 8. A abordagem baseia-se na imagem microscópica e análise de imagem. Descrevemos aqui um protocolo alternativo para um alto rendimento de tela de todo o genoma, com base no manuseamento robótico da bacteriana RNAi clones, a análise quantitativa utilizando o Biosort COPAS (União Biometrica (UBI)), e umasoftware integrado: o MBioLIMS (Laboratório de Sistema de Gestão da Informação da Modul-Bio) uma tecnologia que proporciona maior produtividade para gerenciamento de dados e rastreamento de amostra. O método permite que as telas para ser conduzido em placas de meio sólido. Isto é particularmente importante para alguns estudos, como as que tratam sistema patógeno-hospedeiro interações em C. elegans, uma vez que certos micróbios não infectam eficientemente vermes em meio líquido. Nós mostramos como o método pode ser utilizado para quantificar a importância de genes em anti-fúngica imunidade inata em C. elegans. Neste caso, a abordagem baseia-se na utilização de uma estirpe transgénicos portadores uma infecção epidérmica induzível-gene repórter fluorescente, com GFP sob o controlo do promotor do gene antimicrobiana péptido nlp 29 e um repórter vermelho fluorescente que é expressa constitutivamente na epiderme. Este último apresenta um controlo interno para a integridade funcional da epidermee transgene não específica silenciamento 9. Quando vermes de controlo são infectadas pelo fungo que fluorescem verde. Derrubar por RNAi um gene necessário para a PNL 29 resultados de expressão em fluorescência reduzida após a infecção. Actualmente, este protocolo permite que mais de 3.000 clones RNAi a ser testado e analisados ​​por semana, abrindo a possibilidade de peneiramento todo o genoma, em menos de 2 meses.

Watch this Scientific Journal Video about Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans at JoVE.com

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

Nós descrevemos um protocolo com<em&gt; C. elegans</em&gt; E bibliotecas de alimentação RNAi que permite a medição automatizada de vários parâmetros, tais como fluorescência, tamanho e opacidade de vermes individuais de uma população. Nós dar um exemplo de um ecrã para identificar genes envolvidos na imunidade anti-fúngica inato em<em&gt; C. elegans</em&gt;.

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. ...

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Biology

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

17.6K visualizações.  Université de la Méditerranée. Nós descrevemos um protocolo com C. elegans E bibliotecas de alimentação RNAi que permite a medição automatizada de vários parâmetros, tais como fluorescência, tamanho e opacidade de vermes individuais de uma população. Nós dar um exemplo de um ecrã para identificar genes envolvidos na imunidade anti-fúngica inato em C. elegans.

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

Development of automated imaging and analysis for zebrafish chemical screens. 

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens 1. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates 2. In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling 3. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos.

Development of automated imaging and analysis for zebrafish chemical screens.

We report the development of a system for automated imaging and analysis of zebrafish transgenic embryos in multiwell plates. This demonstrates the ability to measure dose dependent effects of a small molecule, BCI, on Fibroblast Growth Factor reporter gene expression and provide technology for establishing high-throughput zebrafish chemical screens.

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

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

Biologia

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3)1. The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes2. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me33. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform4. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner.
To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.

Here we are presenting a chromatin immunoprecipitation (ChIP) procedure for genome-wide location analysis of protein isoforms that differ in a histone-binding domain. We are applying it to ChIP-Seq analysis to identify the targets of the KDM5A/JARID1A/RBP2 histone demethylase.

Análise do genoma usando chip para identificar alvos isoforma específica Gene

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the ...

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

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

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

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

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 ...

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 ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

Alta capacidade mapeamento físico de cromossomos usando Automated<em&gt; In situ</em&gt; Hibridação

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

Do genoma deleções de genes em<em&gt; Streptococcus sanguinis</em&gt; Por PCR de Alto Rendimento

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

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 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 ...

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide&#8211;DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin

Identifying the direct targets of genome-targeting molecules remains a major challenge. To understand how DNA-binding molecules engage the genome, we developed a method that relies on crosslinking of small molecules to isolate chromatin (COSMIC).

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

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