Nous tenons à remercier D. Braendle, CL Kurz et F. Montañana-Sanchis pour leurs contributions. Ce projet a été soutenu par des subventions de l&#39;ANR et le Conseil Régional PACA, et le financement institutionnel de l&#39;INSERM et le CNRS.

<ol>
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R., Russel, S., Ruvkun, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genomic+analysis+of+C.+elegans+molting.">Functional genomic analysis of C. elegans molting.</a> PLoS Biol. 3, e312-e312 (2005).</li><li>Parry, D. H., Xu, J., Ruvkun, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+whole-genome+RNAi+Screen+for+C.+elegans+miRNA+pathway+genes.">A whole-genome RNAi Screen for C. elegans miRNA pathway genes.</a> Curr. Biol. 17, 2013-2022 (2007).</li><li>O'Rourke, E. J., Conery, A. L., 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=Whole-animal+high-throughput+screens:+the+C+elegans+model.">Whole-animal high-throughput screens: the C elegans model.</a> Methods Mol. Biol. 486, 57-75 (2009).</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=Distinct+innate+immune+responses+to+infection+and+wounding+in+the+C.+elegans+epidermis.">Distinct innate immune responses to infection and wounding in the C. elegans epidermis.</a> Curr. Biol. 18, 481-489 (2008).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> WormBook. , 1551-8507 (2006).</li><li>Powell, J. R., Ausubel, F. M., Ewbank, J., Vivier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+Caenorhabditis+elegans+Infection+by+Bacterial+and+Fungal+Pathogens.">Models of Caenorhabditis elegans Infection by Bacterial and Fungal Pathogens.</a> Methods Mol Biol. 415, 403-427 (2008).</li><li>Cardoso, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XNP-1/ATR-X+acts+with+RB,+HP1+and+the+NuRD+complex+during+larval+development+in+C.+elegans.">XNP-1/ATR-X acts with RB, HP1 and the NuRD complex during larval development in C. elegans.</a> Dev. 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>

Le laboratoire collabore avec l&#39;Union Ewbank Biometrica et Modul-Bio.

<p class="jove_content"&gt; Le protocole actuel permet dépistage à grande échelle dans l&#39;ARNi<em&gt; C. elegans</em&gt; Sur un milieu solide, en utilisant le niveau d&#39;expression d&#39;un gène rapporteur fluorescent en tant que lecture. Il a été optimisé avec des outils automatisés pour faciliter tests à haut débit, permettant une manipulation facile et rapide d&#39;un grand nombre d&#39;échantillons, d&#39;une manière efficace, fiable et reproductible. D&#39;autres méthodes ont été décrites qui ne reposent pas si lourdement sur la manipulation automatisée des échantillons de liquide<sup&gt; 8</sup&gt;.</p&gt;<p class="jove_content"&gt; Un aspect clé de l&#39;organisation au protocole a été le développement d&#39;une méthode robuste de suivi des échantillons, en adaptant le logiciel commercial MBioLIMS à la procédure de l&#39;écran ARNi en collaboration avec le ModulBio entreprise. À chaque étape de la procédure, les plaques sont désormais identifiés par un code barre généré dans le LIMS. Comme le LIMS enregistre également l&#39;identité des clones dans chaque puits sur chaque plaque, allant de bien à gène putatif est simple. Et au-delà de cela, les liens LIMS à la base de données WormBase communauté, permettant un accès direct à des données moléculaires et fonctionnelles sur un gène donné. Le LIMS facilite également les analyses à effectuer sur la plaque ou même au niveau des chromosomes et du génome entier. Pour ces analyses, les données sont stockées d&#39;une manière qui est tout à fait compatible avec les outils décrits précédemment comme COPAmulti<sup&gt; 16</sup&gt;.</p&gt;<p class="jove_content"&gt; Un point critique dans la méthode expérimentale est l&#39;ajustement de la quantité de nourriture par rapport au nombre de vers. Pour de nombreux tests, il est essentiel que les vers ne meurent pas de faim à la fin de l&#39;expérience, mais il est également important de ne pas avoir trop de nourriture au début. Nous avons observé qu&#39;une forte densité de nourriture diminue l&#39;induction de fluorescence dans le protocole en utilisant des vers portant le p<em&gt; Nlp-29</em&gt; :: Rapporteur GFP. Cela peut refléter une diminution de l&#39;efficacité d&#39;infection lorsque les vers sont cultivées dans des bactéries trop (BS, résultats non publiés). Dans le même temps, les conditions de culture de la bactérie ARNi est également important, car cela détermine si ARNdb est produite à un niveau optimal. Certains protocoles à grande échelle exigent l&#39;ARNi culture de clones ARNi sur gélose avant l&#39;ensemencement des cultures liquides<sup&gt; 8</sup&gt;. Nous avons obtenu gene silencing robuste en omettant cette étape. Nous avons opté pour un excès de croissance de chaque culture pour assurer des résultats homogènes. Le protocole décrit prévoit en effet suffisamment de matière pour effectuer 2 ou 3 écrans en parallèle. Dans l&#39;intérêt d&#39;obtenir les résultats les plus robustes, nous privilégions, cependant, la réalisation d&#39;écrans en double en utilisant des cultures indépendantes de vers, bactéries et spores fongiques ARNi.</p&gt;<p class="jove_content"&gt; Une autre étape essentielle est liée à l&#39;utilisation de moyennes qui ne soit ni trop humide ni trop sec. Si les plaques ne sont pas assez sèches, la bactérie ARNi ne se dessèche pas correctement et les vers de se baigner pendant l&#39;essai. Cela peut causer des problèmes. Par exemple, les vers ne sont pas efficacement infectées par le<em&gt; D. coniospora</em&gt; Dans un liquide (C Couillault, résultats non publiés). D&#39;autre part, dans des plaques sèches, l&#39;osmolarité du milieu augmente la culture. Cela a des effets directs sur la physiologie ver<sup&gt; 17</sup&gt;, Comprenant l&#39;induction de certains gènes peptides antimicrobiens, tels que<em&gt; 29 nlp</em&gt;<sup&gt; 18</sup&gt;. Le volume utilisé pour distribuer les vers et les spores est essentielle pour permettre un séchage efficace et rapide. Si plus de 10 uL de liquide est ajouté à des plaques fraîches, les puits peuvent prendre plusieurs heures pour sécher. Cela peut signifier qu&#39;il est difficile d&#39;utiliser des méthodes robotiques pour ajouter des vers sur les plaques. Par exemple, chaque ver est distribué par le Biosort dans l&#39;ordre de 1 pl de liquide. Nous distribuons actuellement les vers manuellement que nous permet aussi de surmonter la tendance de vers en solution dans les sédiments.</p&gt;<p class="jove_content"&gt; Nous avons déjà indiqué un ajustement de la Biosort qui permettrait à une plaque de 96 puits à analyser en 36 minutes<sup&gt; 19</sup&gt;. Fabricant de la machine UBI offre maintenant une modification de la Biosort à diminuer encore le temps d&#39;analyse. Avec le &quot;FastReFlex&quot;, les échantillons sont directement acheminé à travers la cellule d&#39;écoulement pour l&#39;analyse, en contournant le filtre à bulles-piège. Il en résulte une réduction significative du temps d&#39;acquisition de données; une plaque entière peut être analysé en 22 min permettant d&#39;environ 20 plaques à traiter par jour. Si les plaques ne sont pas gelés, cependant, il y aurait un intervalle de 8 heures entre les premiers et le dernier. Étant donné la durée relativement courte de l&#39;expérience, avec l&#39;infection qui dure seulement 18 heures, cela pourrait introduire une variable inacceptable dans les plaques. Non seulement les plaques de gel avant l&#39;analyse résoudre ce problème, mais il est également essentiel dans le contexte de l&#39;analyse à haut débit que, avec le protocole standard actuel et de manutention de liquides robots, 2 personnes peuvent facilement traiter jusqu&#39;à 50 plaques par semaine. Actuellement, nous avons l&#39;habitude d&#39;écran 30 plaques par semaine, ce qui permet un écran l&#39;ensemble du génomedoivent avoir lieu en moins de 2 mois.</p&gt;<p class="jove_content"&gt; Une caractéristique intéressante de ce protocole est le fait qu&#39;il utilise la gélose solide, permettant des dosages qui ne peuvent être réalisées en milieu liquide. Comme la physiologie ver diffère selon les conditions de culture<sup&gt; 20,21</sup&gt;, Être capable de réaliser des tests sur milieu solide peut également compléter habituelles à base de liquide écrans. Pour certains phénotypes (par exemple le mouvement), l&#39;analyse peut être effectuée directement avec des plaques de milieu de 96 puits solides. Pour l&#39;écran actuel, cependant, que le Biosort ne pouvons pas échantillonner à partir des plaques de milieu solide, les vers sont d&#39;abord transférés à l&#39;état liquide avant de le congeler. D&#39;autre part, le Biosort COPAS permet une analyse multiparamétrique quantitative compatible avec la mesure de nombreux phénotypes différents. Il est probable que, pour n&#39;importe quel écran donnée ne sont pas tous les paramètres mesurés sera le premier de lecture. Néanmoins, ces mesures peuvent être utiles pour plusieurs raisons. Ils permettent de déterminer si le dosage se sont déroulées correctement. Par exemple, on peut vérifier sur le nombre et la taille des vers dans chaque puits. Ce n&#39;est que lorsque un clone ARNi devrait bloquer la croissance ou la reproduction doit y avoir un écart par rapport aux valeurs attendues. Wells avec moins de 20 vers ou / et des animaux 30% plus petites (TOF) par rapport à un stanstandard de contrôle sans effet sur la croissance (<em&gt; Par exemple GFP (ARNi</em&gt;)) Peuvent être exclus des analyses ultérieures. La fonction de ces clones peuvent être traitées en utilisant un protocole alternatif dans lequel les vers sont exposés à des bactéries ARNi après leur développement est bien avancé<sup&gt; 15</sup&gt;. Les différents paramètres peuvent également être utilisés pour affiner gène candidat (hit) de sélection, par exemple, limiter coups seulement à ceux qui ont une incidence fluorescence verte, mais pas rouge. La méthode choisie pour la sélection précise succès dépendra du type d&#39;écran et de son exacte de lecture. Compte tenu de la complexité des données générées à partir d&#39;un génome quantitative, grand écran, la collaboration avec un statisticien peut être nécessaire.</p&gt;<p class="jove_content"&gt; Le protocole que nous décrivons peut être facilement adaptée à d&#39;autres types de tests. Par exemple, lors de l&#39;étape d&#39;infection<em&gt; D. coniospora</em&gt; Pourrait être remplacé par n&#39;importe quel autre agent pathogène capable d&#39;infecter sur des supports solides. Ce peut être utile comme nous l&#39;avons constaté que bon nombre des facteurs de virulence importants pour l&#39;infection à Serratia marcescens dans une culture liquide ne jouent pas un rôle lors de l&#39;infection en milieu solide (E. Pradel, communication personnelle). Le protocole est également totalement compatible avec les tests de composés chimiques<sup&gt; 22-24</sup&gt;, Ou pour identifier des composés à la bioactivité l&#39;utilisation de bibliothèques différentes de bactéries<sup&gt; 25</sup&gt;, Devrait donc trouver une utilité générale dans le<em&gt; C. elegans</emCommunauté de la recherche&gt;.</p

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> BactoAgar </td><td> BD Diagnostic Systems </td><td> 214010 </td><td></td></tr><tr><td> Ampicilline </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> Tout fournisseur </td><td></td><td></td></tr><tr><td> Film adhésif AeraSeal </td><td> Dutscher </td><td> 760214 </td><td></td></tr><tr><td> Isopropylique β-D-thiogalactopyranoside (IPTG) </td><td> Thermo Scientific </td><td> AB-0481 </td><td></td></tr><tr><td> K 2 HPO 4 </td><td> Tout fournisseur </td><td></td><td></td></tr><tr><td> KH 2 PO 4 </td><td> Tout fournisseur </td><td></td><td></td></tr><tr><td> MgSO 4 </td><td> Tout fournisseur </td><td></td><td></td></tr><tr><td> NaCl </td><td> Tout fournisseur </td><td></td><td></td></tr><tr><td> Na 2 HPO 4 </td><td> Tout fournisseur </td><td></td><td></td></tr><tr><td> NaOH </td><td> Tout fournisseur </td><td></td><td></td></tr><tr><td> Plaque de 96 puits profond </td><td> Thermo Scientific </td><td> AB-0932 </td><td></td></tr><tr><td> Plaque de 96 puits à plat </td><td> FALCON </td><td> 353072 </td><td></td></tr><tr><td> Plaque de 96 puits rond </td><td> FALCON </td><td> 353077 </td><td></td></tr><tr><td> Tétracycline </td><td> Sigma-Aldrich </td><td> T8032 </td><td></td></tr><tr><td> Triton X-100 </td><td> Tout fournisseur </td><td></td><td></td></tr><tr><td> TECAN robots </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> Union 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

L&#39;ARN interférence est une méthode puissante pour comprendre la fonction des gènes, en particulier quand elles sont menées à une échelle du génome entier et dans un contexte quantitatif. Dans C. elegans, la fonction du gène peut être renversé simplement et efficacement en se nourrissant les vers avec des bactéries exprimant un ARN double brin correspondant à un gène spécifique 1. Alors que la création de bibliothèques de clones d&#39;ARNi couvrant la plupart des C. elegans génome 2,3 ouvert la voie à de véritables études génomiques fonctionnelles (voir, par exemple 4-7), la plupart des méthodes établies sont laborieuses. Moy et ses collègues ont mis au point des protocoles semi-automatiques qui facilitent l&#39;échelle du génome écrans 8. L&#39;approche repose sur l&#39;imagerie microscopique et l&#39;analyse d&#39;image. Nous décrivons ici un protocole alternatif pour un haut débit du génome, grand écran, basée sur la manipulation robotique de clones bactériens ARNi, une analyse quantitative à l&#39;aide de la Biosort COPAS (Union Biometrica (UBI)), et unelogiciel intégré: l&#39;MBioLIMS (Laboratory Information Management System de Modul-Bio) une technologie qui offre un débit plus élevé pour la gestion des données et le suivi des échantillons. Procédé permettant écrans à être effectuée sur des plaques de milieu solide. Ceci est particulièrement important pour certaines études, comme celles portant sur ​​les interactions hôte-pathogène dans C. elegans, car certains microbes ne sont pas efficacement infecter des vers dans une culture liquide. Nous montrons comment la méthode peut être utilisée pour quantifier l&#39;importance des gènes dans les anti-fongiques l&#39;immunité innée dans C. elegans. Dans ce cas, l&#39;approche repose sur l&#39;utilisation d&#39;une souche transgénique portant un épiderme infection inductible du gène rapporteur fluorescent, avec la GFP sous le contrôle du promoteur du gène du peptide antimicrobien de la PNL 29 et un journaliste en rouge fluorescent qui est exprimé de façon constitutive dans le épiderme. Celle-ci prévoit un contrôle interne pour l&#39;intégrité fonctionnelle de l&#39;épidermeet non spécifique du transgène taire 9. Lorsque les vers de contrôle sont infectées par le champignon, ils émettent une fluorescence verte. Abattre par ARNi d&#39;un gène requis pour des résultats nlp expression 29 de fluorescence diminuée après l&#39;infection. Actuellement, ce protocole permet à plus de 3000 clones d&#39;ARNi à être testé et analysé par semaine, ouvrant la possibilité de filtrer l&#39;ensemble du génome en moins de 2 mois.

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

Nous décrivons un protocole à l&#39;aide<em&gt; C. elegans</em&gt; Et les bibliothèques d&#39;alimentation ARNi qui permet la mesure automatisée de plusieurs paramètres tels que la fluorescence, la taille et l&#39;opacité de chaque ver dans une population. Nous donnons un exemple d&#39;un écran pour identifier les gènes impliqués dans la lutte contre l&#39;immunité innée dans fongique<em&gt; C. elegans</em&gt;.

Quantitatifs et automatisé à haut débit du génome à l&#39;échelle écrans ARNi dans 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. ...

quantitative-automated-high-throughput-genome-wide-rnai-screens-c

Research

JoVE Journal

Biology

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

17.5K vues.  Université de la Méditerranée. Nous décrivons un protocole à l'aide C. elegans Et les bibliothèques d'alimentation ARNi qui permet la mesure automatisée de plusieurs paramètres tels que la fluorescence, la taille et l'opacité de chaque ver dans une population. Nous donnons un exemple d'un écran pour identifier les gènes impliqués dans la lutte contre l'immunité innée dans fongique C. elegans.

Vidéo: 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.

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.

Développement de l&#39;imagerie automatisée et d&#39;analyse pour les écrans chimiques du poisson zèbre.

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

Recherche

Biologie

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.

Analyse du génome entier en utilisant ChIP d&#39;identifier les gènes cibles spécifiques Isoform

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.

Criblage à haut débit et de biodétection avec fluorescent C. elegans Souches

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.

Dépistage ARNi pour identifier les phénotypes postembryonnaires dans 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.

À haut débit de cartographie physique des chromosomes à l&#39;aide automatisée<em&gt; In situ</em&gt; Hybridation

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.

La délétion de gènes dans le génome entier<em&gt; Streptococcus sanguinis</em&gt; Par PCR à haut débit

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.

Biochimique et High Throughput évaluation microscopique de la masse grasse dans<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.

Haut-débit criblage de modulateurs chimique des gènes post-transcriptionnelle réglementées

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is an alternative approach to chromatin immunoprecipitation (ChIP). An expressed fusion protein consisting of the protein of interest and the E. coli DNA adenine methyltransferase can methylate the adenine base in GATC motifs near the sites of protein-DNA interactions. Adenine-methylated DNA fragments can then be specifically amplified and detected. The original DamID assay detects the genomic locations of methylated DNA fragments by hybridization to DNA microarrays, which is limited by the availability of microarrays and the density of predetermined probes. In this paper, we report the detailed protocol of integrating high throughput DNA sequencing into DamID (DamID-seq). The large number of short reads generated from DamID-seq enables detecting and localizing protein-DNA interactions genome-wide with high precision and sensitivity. We have used the DamID-seq assay to study genome-nuclear lamina (NL) interactions in mammalian cells, and have noticed that DamID-seq provides a high resolution and a wide dynamic range in detecting genome-NL interactions. The DamID-seq approach enables probing NL associations within gene structures and allows comparing genome-NL interaction maps with other functional genomic data, such as ChIP-seq and RNA-seq.

We describe herein an assay by coupling DNA adenine methyltransferase identification (DamID) to high throughput sequencing (DamID-seq). This improved method provides a higher resolution and a wider dynamic range, and allows analyzing DamID-seq data in conjunction with other high throughput sequencing data such as ChIP-seq, RNA-seq, etc.

DamID-seq: L&#39;échelle du génome Cartographie des protéine-ADN Interactions par séquençage à haut débit de fragments d&#39;ADN adénine-méthylé

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is ...