Nous reconnaissons le soutien financier du CPRIT (RP100119) et la Fondation Welch (I-1665).

Protocole complet prend environ 4 jours. 1. Préparation des cellules pour les siRNA et rapporteur transfection Nous allons présenter ici un protocole pour interroger les bibliothèques de siRNA à l&#39;aide d&#39;un petit ensemble de siRNA (384 tableau différent des pools de siRNA en format 96 puits chaque ensemble étant composé de siRNA 4x ciblant un gène unique) à partir d&#39;une base de données siRNA du génome pour des fins d&#39;illustration. Pour cette étude, nous allons exploiter HCT116 cellules qui présentent la signalisation Wnt et Kras déviants, et l&#39;activité de p53. La lignée cellulaire appropriée dans les études visant à interroger d&#39;autres processus cellulaires d&#39;intérêt devront être identifiés et évalués de la même façon. <ol><li> Laver 5 x 10 cm 2 plaques de cellules 80-90% de confluence HCT116 avec du PBS, puis les cellules de la récolte par la trypsine en utilisant 1 ml de solution de trypsine / plaque suivie d&#39;une neutralisation avec 10 ml de milieu de Eagle modifié par Dulbecco (DMEM) contenant 2% de Fetal sérum bovin (FBS) et 1% de pénicilline / streptomycine / plaque. </li><li> Transfert cellules en suspension dans un tube conique de 50 ml et centrifuger à ~ 130 g pendant 5 min, retirer le surnageant et remettre en suspension le culot cellulaire dans 10 ml de DMEM / FBS à 2% / 1% de pénicilline / streptomycine. </li><li> Compter le nombre de cellules avec un compteur de cellules, puis faire une solution de cellules avec 10 5 cellules / ml, garder 150 suspension de cellules ml pour l&#39;étape suivante. Planche 10 4 cellules (100 pi) dans chaque puits d&#39;une plaque de culture cellulaire de 96 puits à l&#39;aide d&#39;un distributeur de liquide automatisé multipoint (désormais multipoint). La suspension cellulaire devrait être suffisante dans ce cas pour le placage 12 x plaques à 96 puits. </li><li> Incuber les boîtes avec des cellules à 37 ° C dans un incubateur avec 5% de CO 2 lors de la préparation du mélange de transfection. </li></ol> 2. Préparation de la Construct Reporter Stock Solution <ol><li> Isoler journaliste de haute qualité construire ADN en utilisant des kits midiprep standard (NucleoBond Xtra Midi produite parClontech) et diluer l&#39;ADN à une concentration finale de 1 mg / ml pour chaque reporter. </li><li> Préparez 100 pi de construction rapporteur solution mère en combinant 30 ul de p53-FL, 30 pl de 8X TCF-RL, 30 pl de Elk1-Gal4, 6 pl UAS-CL et 4 ul de H 2 O pour obtenir un mélange d&#39;ADN avec un ratio final 1:1:1:0.2 des journalistes. Concentration finale de l&#39;ADN de cette solution mère de mélange d&#39;ADN est de 0,3 mg / ml. </li></ol> 3. Préparation du siRNA / ADN transfection Mix Dans cette partie du protocole, nous allons préparer siRNA / ADN mélanges de transfection qui sera suffisant pour la transfection 12 x 96 plaques à puits. Pour cette bibliothèque de test de 4 x 96 puits de siRNA, nous transfecter chaque pool de siRNA en trois exemplaires. Le réactif de transfection, nous allons utiliser est appelé Effectene (Qiagen). Dans nos mains c&#39;est le seul réactif qui travaille pour la livraison simultanée de siRNA et de l&#39;ADN dans des cellules cultivées. <ol><li> Diluer 80 pi de journaliste construction Stock solution dans 8 ml de tampon CE (Kit Effectene) pour obtenir une solution d&#39;ADN avec une concentration finale de 3 pg / ml. En général, préparer suffisamment de mélange de solution d&#39;ADN pour une utilisation immédiate. </li><li> Plate 20 pi de cette solution d&#39;ADN à chaque puits d&#39;une 96 puits / plaque. Le nombre de plaques à 96 puits dépendra de combien de pools de siRNA seront testés. Par exemple, dans ce cas, nous aurons besoin de 4 x plaques à 96 puits pour évaluer 384 piscines de siRNA différents. </li><li> Les siRNA à tester doivent être à une concentration du stock de 5 pm. Sinon, ils peuvent être réplique plaquée et dilué à cette concentration en utilisant le tampon de dilution recommandée associée à chaque bibliothèque. </li><li> Transfert 2 pi de chaque stock de siRNA de 5 uM dans le puits correspondant de la plaque PCR contenant le stock d&#39;ADN dilué avec un Biomek Automated Liquid Handler. Selon l&#39;ampleur de l&#39;étude, une pipette multi-canaux peut suffire. </li><li> Maintenant, nous allons ajouter 1 l de solution Enhancer (kit Effectene) dans chaque puits de l&#39;ADN / siRNA plate. Ce nouveau peut être réalisée soit avec un gestionnaire de liquide automatisé ou pipette multicanaux. </li><li> Laisser la réaction Enhancer se produire à température ambiante pendant 5 min, puis ajouter 3 pi de réactif de transfection Effectene à chaque puits et incuber à température ambiante pendant 10 minutes supplémentaires. </li><li> Pour arrêter la formation d&#39;un complexe de transfection, ajouter 10 ul de DMEM / FBS à 2% / 1% de pénicilline / streptomycine dans chaque puits de la plaque PCR. </li><li> Transférer 10 ul du mélange de transfection pour chaque puits de la plaque de 96 puits contenant les cellules (extrait de l&#39;incubateur de cellules). Comme chaque transfection sera réalisée en trois exemplaires, le même mélange sera appliqué à deux plaques et 96 autres contenant des cellules. </li><li> Incuber les cellules avec des mélanges de transfection ajoutés à 37 ° C dans un environnement humidifié avec 5% de CO2 pendant 36 heures. </li></ol> 4. Déterminer les activités luciférase dans des échantillons cellulaires Après 36 h, les activités luciférase sont prêts pour la mesuredans des cellules transfectées. Le critère doit être déterminée sur une base par expérience. Dans notre étude, un temps d&#39;incubation de 36 heures avait déjà donné un signal suffisamment robuste pour dire la détermination du résultat. Comme cette étude intègre plusieurs journalistes (un sécrété dans le milieu de culture, et deux autres exprimée dans le cytoplasme de la cellule), nous allons obtenir des mesures à la fois de milieu de culture ainsi que d&#39;un lysat cellulaire. <ol><li> Détection de l&#39;activité d&#39;AC dans le milieu de culture: <ol><li> 20 pl de milieu de culture est une réplique en plaqué un opaque 96 puits blanc (soit en utilisant le Liquid Handler Biomek ou pipette multicanaux). </li><li> Ajouter 20 ul de tampon de dosage CL (systèmes de ciblage) dans chaque puits. </li><li> Ajouter 10 ul de Cypridina substrat luciférine (systèmes de ciblage) à chaque puits et détecter l&#39;activité de CL en utilisant un luminomètre (le lecteur de plaque PHERAstar produit par BMG par exemple). </li></ol></li><li> Détection de Floride et de l&#39;activité RL: <ol><li> Lyse le caunes en retirant d&#39;abord le milieu de culture. Ceci peut être accompli rapidement en tournant la plaque à l&#39;envers et en jetant moyen dans un évier. Moyenne restant peut être retiré en tapotant doucement la plaque vers le bas de la tête sur du papier absorbant. Ajouter 30 ul de 1X Passif tampon de lyse (Promega) dans chaque puits de cellules en utilisant le multipoint et le placer sur une bascule de plate-forme (Bellco est une société qui fabrique ces) fixer une vitesse moyenne pendant 5 min à la température ambiante. </li><li> Ajouter 20 ul de réactif dosage de luciférase II (partie de la trousse de luciférase double Promega) en utilisant l&#39;activité FL multipoint et mesurer immédiatement d&#39;utiliser le luminomètre. Ensuite, ajoutez Stop &amp; Glo réactif (Promega) qui va étancher l&#39;activité FL et permettre la mesure de l&#39;activité RL. Remarque: si vous utilisez des mélanges de substrats qui permettent durée de signal étendu (tels que des kits luciférase Dual-Glo de Promega), l&#39;utilisation des kits flash nécessite une mesure rapide lors de l&#39;addition de substrat (moins de 5 min). </li></ol></li></ol>

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	<li>Bronstein, I., Fortin, J., Stanley, P. E., Stewart, G. S., Kricka, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemiluminescent+and+bioluminescent+reporter+gene+assays.">Chemiluminescent and bioluminescent reporter gene assays.</a> Analytical Biochemistry. 219, 169-181 (1994).</li><li>Miraglia, L. J., King, F. J., Damoiseaux, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+the+light:+luminescent+reporter+gene+assays.">Seeing the light: luminescent reporter gene assays.</a> Combinatorial Chemistry &amp; High Throughput Screening. 14, 648-657 (2011).</li><li>McGuire, S. E., Roman, G., Davis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+systems+in+Drosophila:+a+synthesis+of+time+and+space.">Gene expression systems in Drosophila: a synthesis of time and space.</a> Trends in Genetics: TIG. 20, 384-391 (2004).</li><li>Smirnova, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+Neh2-luciferase+reporter+and+its+application+for+high+throughput+screening+and+real-time+monitoring+of+Nrf2+activators.">Development of Neh2-luciferase reporter and its application for high throughput screening and real-time monitoring of Nrf2 activators.</a> Chem. Biol. 18, 752-765 (2011).</li><li>Chen, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule-mediated+disruption+of+Wnt-dependent+signaling+in+tissue+regeneration+and+cancer.">Small molecule-mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer.</a> Nat. Chem. Biol. 5, 100-107 (2009).</li><li>Tang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+RNAi+screen+for+Wnt/beta-catenin+pathway+components+identifies+unexpected+roles+for+TCF+transcription+factors+in+cancer.">A genome-wide RNAi screen for Wnt/beta-catenin pathway components identifies unexpected roles for TCF transcription factors in cancer.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 9697-9702 (2008).</li><li>Lum, L. D., Kulak, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+Luciferase+Reporter+Assay+Systems.">Multiplexed Luciferase Reporter Assay Systems.</a> United States patent. , (2012).</li><li>Funk, W. D., Pak, D. T., Karas, R. H., Wright, W. E., Shay, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transcriptionally+active+DNA-binding+site+for+human+p53+protein+complexes.">A transcriptionally active DNA-binding site for human p53 protein complexes.</a> Mol. Cell Biol. 12, 2866-2871 (1992).</li><li>DasGupta, R., Kaykas, A., Moon, R. 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Genet. 19, 379-383 (1998).</li><li>Korinek, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+members+of+the+Tcf+family+implicated+in+Wnt/beta-catenin+signaling+during+embryogenesis+in+the+mouse.">Two members of the Tcf family implicated in Wnt/beta-catenin signaling during embryogenesis in the mouse.</a> Mol. Cell Biol. 18, 1248-1256 (1998).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+molecular+characterization+of+human+colon+and+rectal+cancer.">Comprehensive molecular characterization of human colon and rectal cancer.</a> Nature. 487, 330-337 (2012).</li><li>Koboldt, D. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrative+analysis+of+genome-wide+RNA+interference+screens.">Integrative analysis of genome-wide RNA interference screens.</a> Science Signaling. 2, pt4 (2009).</li><li>Falschlehner, C., Steinbrink, S., Erdmann, G., Boutros, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+RNAi+screening+to+dissect+cellular+pathways:+a+how-to+guide.">High-throughput RNAi screening to dissect cellular pathways: a how-to guide.</a> Biotechnology Journal. 5, 368-376 (2010).</li><li>Boutros, M., Bras, L. P., Huber, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+cell-based+RNAi+screens.">Analysis of cell-based RNAi screens.</a> Genome Biology. 7, R66 (2006).</li><li>Gilbert, D. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+multiplex+cell+viability+assay+for+high-throughput+RNAi+screening.">A novel multiplex cell viability assay for high-throughput RNAi screening.</a> PLoS ONE. 6, e28338 (2011).</li></ol>

OK et LL sont des auteurs sur un brevet en instance relatives à la technologie multiplex luciférase.

Il existe plusieurs fournisseurs de systèmes rapporteurs luciférase (telles que le Promega, systèmes de ciblage, New England Biolabs, et Thermo Fisher) qui fournissent des ressources en ligne utiles pour ceux divertir un écran à haut débit pour la première fois. En outre, un certain nombre d&#39;excellentes critiques concernant l&#39;optimisation de l&#39;utilisation d&#39;écrans à haut débit pour la découverte de gènes et comment l&#39;ARNi basée sur les résultats du dépistage peuvent être intégrés au...

Une variété de journalistes luciférase basés pour surveiller un large éventail de processus biologiques cellulaires sont disponibles commercialement. La majorité de ces constructions d&#39;ADN codant pour des protéines luciférase qui sont amenés à exprimer par des stimuli spécifiques de cellules ou de perturbations. Les plupart des journalistes transcription base robuste placent l&#39;expression d&#39;une enzyme luciférase sous le contrôle des éléments activateurs synthétiques ou régions du promoteur du gène bien établies pour leur fiabilité en rapporter un événement 1,2 transcriptionnelle physiologiquement pertinents. Il existe également des systèmes trans-rapporteur de la luciférase qui utilisent GAL4 UAS pour conduire l&#39;expression de la protéine luciférase. Gal4 de levure est un activateur de la transcription et de l&#39;UAS est un activateur de Gal4 qui se lie spécifiquement à activer la transcription des séquences de gènes placés en aval de celui-ci 3. Ces types de systèmes luciférase sont généralement pris en charge par deux plasmides d&#39;ADN - on encode la protéine GAL4 fusionné à la regulatory partie d&#39;une protéine d&#39;intérêt, et le deuxième code pour une protéine luciférase placé sous le contrôle d&#39;une ou plusieurs séquences d&#39;UAS. Ainsi, le signal luciférase cellulaire reflète le niveau d&#39;activité de la protéine d&#39;intérêt. Une autre utilisation courante de journalistes luciférase à base est de surveiller la stabilité des protéines par lequel une protéine d&#39;intérêt est fusionnée à une enzyme luciférase 4. Quel que soit le type de journaliste, un caveat emptor est à noter ici que l&#39;on ne peut assumer la spécificité de tout journaliste aurait été bien interrogé. Ainsi, la diligence raisonnable est nécessaire dans l&#39;intégration des nouvelles constructions rapporteurs dans toute stratégie de recherche. Le choix de l&#39;enzyme luciférase lecture peut être aussi important que la fiabilité des éléments d&#39;ADN qui contrôlent son expression. Alors que la luciférase de luciole (FL) est l&#39;enzyme la plus couramment utilisée dans les constructions disponibles dans le commerce, l&#39;avènement de nouveaux systèmes de rapporteur de la luciférase qui ne nécessitent pas l&#39;ATP (comme dans le casdes réactions FL-based) ou qui intègrent des enzymes plus stables qui émettent des signaux forts promettre d&#39;améliorer l&#39;efficacité et la fiabilité de cette plate-forme de recherche. Une remarque importante concernant la sélection des journalistes luciférase dans des études chimiques est la sensibilité des FL à l&#39;inhibition chimique qui pourrait donner lieu à de faux rapports. Dans notre expérience, d&#39;autres enzymes telles que Renilla ou Gaussia luciférase (RL et GL, respectivement) qui utilisent coelentérazine comme substrat sont moins facilement inhibé par des petites molécules 5. Le format le plus courant pour le multiplexage luciférase read-out implique l&#39;utilisation de FL et RL largement donné la disponibilité de fonctions faciles à utiliser kits avec la capacité à mesurer de manière séquentielle des activités enzymatiques à partir d&#39;un seul échantillon. Dans la plupart des ensembles, les échantillons sont d&#39;abord exposées à la luciférine pour révéler les niveaux d&#39;activité FL suivis d&#39;un réactif d&#39;extinction qui est simultanément déposés avec coelentérazine pour donner une secondesignal de RL Dary. Avec l&#39;ajout d&#39;autres luciferases qui peuvent être sécrétés comme GL ou que l&#39;utilisation encore un autre substrat comme Cypridina luciférase (CL), un plus grand nombre de possibilités pour générer des données à haute teneur en utilisant luciferases ont émergé. Un exemple d&#39;un écran ARNi du génome entier en utilisant cette technique peut être trouvée ici 6. Ces avancées technologiques ont à leur tour stimulé le développement de mécanismes spécifiques pour étancher chaque type d&#39;enzyme luciférase pour limiter la diaphonie 7. Dans nos mains, nous constatons une plus grande fréquence des «effets de bord» (tendances inexplicables dans l&#39;activité cellulaire associée au bord de plaques de culture à titre élevé), avec luciferases sécrétées telles que GL ou CL. D&#39;autre part, ces luciferases sécrétées sont utiles pour les essais cinétiques, car elles permettent d&#39;échantillonnage du signal sans dénaturer la viabilité cellulaire. Pour notre étude présentée ici, nous allons surveiller simultanément l&#39;activité de trois cancer pertinentesprocessus cellulaires: la p53, Kras, et les voies de transduction du signal Wnt. Les journalistes intégrés dans notre étude sont le FL journaliste PP53-TA-Luc de Clontech (ci-après dénommé le journaliste p53-FL) 8, un système de journaliste Elk1-GAL4/UAS-CL (ci-Elk-1 journaliste, Agilent), et le 8X TCF RL journaliste qui intègre de multiples éléments activateurs synthétiques reconnus par la voie Wnt régulateurs de transcription connus comme des facteurs de lymphocytes T (ou FCT) 9-11.

<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>PathDetect Elk1 Trans-Reporting System</td>
			<td>Agilent Technologies Inc.</td>
			<td>219005</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pathway Profiling Luciferase System 4 pp53-TA-Luc Vector</td>
			<td>Clontech Lab Inc.</td>
			<td>631914</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Effectene Transfection Reagent</td>
			<td>Qiagen Inc.</td>
			<td>301427</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dual-Luciferase Reporter Assay System</td>
			<td>Promega Corp.</td>
			<td>E1960</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cypridina Luciferase Assay reagent</td>
			<td>Targeting Systems</td>
			<td>VLAR-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HCT116 cells</td>
			<td>ATCC</td>
			<td>CCL-247</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CytoTox-Fluo Cytotoxicity Assay</td>
			<td>Promega Corp.</td>
			<td>G9260</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>MultiFlo Microplate Dispenser</td>
			<td>Labsystems Inc.</td>
			<td>Model 832</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Biomek FXP Laboratory Automation Workstation</td>
			<td>Beckman Coulter Inc.</td>
			<td>A31842</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PHERAstar FS-multi-mode HTS microplate reader</td>
			<td>BMG Labtech Inc.</td>
			<td>0471-101A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bright-Line Reichert Hemacytometers</td>
			<td>Hausser Scientific Company</td>
			<td>1490</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

a multiplexed luciferase-based screening platform for interrogating cancer-associated signal transduction in cultured cells

Interrogation échelle du génome de la fonction des gènes en utilisant l&#39;interférence ARN (ARNi) s&#39;annonce très prometteuse pour l&#39;identification rapide des vulnérabilités de cellules cancéreuses chimiquement traitables. Limiter le potentiel de cette technologie est l&#39;incapacité à définir rapidement la base mécaniste des résultats phénotypiques et ainsi informer le développement de stratégies thérapeutiques moléculaires ciblées. Nous décrivons ici les méthodes de déconstruire phénotypes cellulaires induits par le gène ARNi à médiation visant l&#39;utilisation des systèmes multiplexés journaliste qui permettent le suivi du cancer touche les processus associés à la cellule. Cette méthode de criblage à haut contenu est polyvalent et peut être facilement adapté pour le dépistage d&#39;autres types de grandes bibliothèques moléculaires.

Malgré les progrès de la cartographie du paysage mutationnel de divers cancers à l&#39;aide des efforts de séquençage du génome massives 12-14, nous n&#39;avons pas encore mis en place une approche systématique pour la traduction de ces observations dans les stratégies d&#39;intervention. Dans le cancer colorectal (CRC), les mutations qui sont prévus pour affecter les composants de trois processus cellulaires - Wnt / β-caténine, p53, et la signalisation Kras - se retrouvent dans 99% de toutes les t...

Watch this Scientific Journal Video about A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells at JoVE.com

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Atteindre un niveau compréhension des systèmes de processus cellulaires est un objectif de la biologie cellulaire moderne. Nous décrivons ici les stratégies pour les journalistes luciférase multiplexage de diverses fonctions cellulaires points d&#39;extrémité pour interroger la fonction des gènes en utilisant l&#39;échelle du génome bibliothèques ARNi.

Une plateforme de criblage à base de luciférase multiplexé pour interroger associée au cancer transduction du signal dans des cellules cultivées

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

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11.0K vues.  UT Southwestern Medical Center. Atteindre un niveau compréhension des systèmes de processus cellulaires est un objectif de la biologie cellulaire moderne. Nous décrivons ici les stratégies pour les journalistes luciférase multiplexage de diverses fonctions cellulaires points d'extrémité pour interroger la fonction des gènes en utilisant l'échelle du génome bibliothèques ARNi.

Vidéo: A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

How to Measure Cortical Folding from MR Images: a Step-by-Step Tutorial to Compute Local Gyrification Index

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will be identifiable at any age. As recently reviewed by Mangin and colleagues2, several methods exist to quantify different characteristics of gyrification. For instance, sulcal morphometry can be used to measure shape descriptors such as the depth, length or indices of inter-hemispheric asymmetry3. These geometrical properties have the advantage of being easy to interpret. However, sulcal morphometry tightly relies on the accurate identification of a given set of sulci and hence provides a fragmented description of gyrification. A more fine-grained quantification of gyrification can be achieved with curvature-based measurements, where smoothed absolute mean curvature is typically computed at thousands of points over the cortical surface4. The curvature is however not straightforward to comprehend, as it remains unclear if there is any direct relationship between the curvedness and a biologically meaningful correlate such as cortical volume or surface. To address the diverse issues raised by the measurement of cortical folding, we previously developed an algorithm to quantify local gyrification with an exquisite spatial resolution and of simple interpretation. Our method is inspired of the Gyrification Index5, a method originally used in comparative neuroanatomy to evaluate the cortical folding differences across species. In our implementation, which we name local Gyrification Index (lGI1), we measure the amount of cortex buried within the sulcal folds as compared with the amount of visible cortex in circular regions of interest. Given that the cortex grows primarily through radial expansion6, our method was specifically designed to identify early defects of cortical development.
In this article, we detail the computation of local Gyrification Index, which is now freely distributed as a part of the FreeSurfer Software (<a href="http://surfer.nmr.mgh.harvard.edu/">http://surfer.nmr.mgh.harvard.edu/</a>, Martinos Center for Biomedical Imaging, Massachusetts General Hospital). FreeSurfer provides a set of automated reconstruction tools of the brain's cortical surface from structural MRI data. The cortical surface extracted in the native space of the images with sub-millimeter accuracy is then further used for the creation of an outer surface, which will serve as a basis for the lGI calculation. A circular region of interest is then delineated on the outer surface, and its corresponding region of interest on the cortical surface is identified using a matching algorithm as described in our validation study1. This process is repeatedly iterated with largely overlapping regions of interest, resulting in cortical maps of gyrification for subsequent statistical comparisons (Fig. 1). Of note, another measurement of local gyrification with a similar inspiration was proposed by Toro and colleagues7, where the folding index at each point is computed as the ratio of the cortical area contained in a sphere divided by the area of a disc with the same radius. The two implementations differ in that the one by Toro et al. is based on Euclidian distances and thus considers discontinuous patches of cortical area, whereas ours uses a strict geodesic algorithm and include only the continuous patch of cortical area opening at the brain surface in a circular region of interest.

Measuring gyrification (cortical folding) at any age represents a window into early brain development. Hence, we previously developed an algorithm to measure local gyrification at thousands of points over the hemisphere1. In this paper, we detail the computation of this local gyrification index.

Comment mesurer pliant corticale à partir d&#39;images IRM: un tutoriel pas-à-pas à calculer l&#39;indice gyrification locale

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will ...

Recherche

Médecine

High Content Screening in Neurodegenerative Diseases

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be addressed as a matter of great urgency1-4. Multiple complementary 'omics' approaches have provided clues as to the genetic risk factors and pathogenic mechanisms underlying numerous neurodegenerative diseases, but most findings still require functional validation5. For example, a recent genome wide association study for Parkinson's Disease (PD), identified many new loci as risk factors for the disease, but the underlying causative variant(s) or pathogenic mechanism is not known6, 7. As each associated region can contain several genes, the functional evaluation of each of the genes on phenotypes associated with the disease, using traditional cell biology techniques would take too long. 
	There is also a need to understand the molecular networks that link genetic mutations to the phenotypes they cause. It is expected that disease phenotypes are the result of multiple interactions that have been disrupted. Reconstruction of these networks using traditional molecular methods would be time consuming. Moreover, network predictions from independent studies of individual components, the reductionism approach, will probably underestimate the network complexity8. This underestimation could, in part, explain the low success rate of drug approval due to undesirable or toxic side effects. Gaining a network perspective of disease related pathways using HT/HC cellular screening approaches, and identifying key nodes within these pathways, could lead to the identification of targets that are more suited for therapeutic intervention.
	High-throughput screening (HTS) is an ideal methodology to address these issues9-12. but traditional methods were one dimensional whole-well cell assays, that used simplistic readouts for complex biological processes. They were unable to simultaneously quantify the many phenotypes observed in neurodegenerative diseases such as axonal transport deficits or alterations in morphology properties13, 14. This approach could not be used to investigate the dynamic nature of cellular processes or pathogenic events that occur in a subset of cells. To quantify such features one has to move to multi-dimensional phenotypes termed high-content screening (HCS)4, 15-17. HCS is the cell-based quantification of several processes simultaneously, which provides a more detailed representation of the cellular response to various perturbations compared to HTS. 
	HCS has many advantages over HTS18, 19, but conducting a high-throughput (HT)-high-content (HC) screen in neuronal models is problematic due to high cost, environmental variation and human error. In order to detect cellular responses on a 'phenomics' scale using HC imaging one has to reduce variation and error, while increasing sensitivity and reproducibility. 
	Herein we describe a method to accurately and reliably conduct shRNA screens using automated cell culturing20 and HC imaging in neuronal cellular models. We describe how we have used this methodology to identify modulators for one particular protein, DJ1, which when mutated causes autosomal recessive parkinsonism21. 
	Combining the versatility of HC imaging with HT methods, it is possible to accurately quantify a plethora of phenotypes. This could subsequently be utilized to advance our understanding of the genome, the pathways involved in disease pathogenesis as well as identify potential therapeutic targets.

We describe a methodology combining automated cell culturing with high-content imaging to visualize and quantify multiple cellular processes and structures, in a high-throughput manner. Such methods can aid in the further functional annotation of genomes as well as identify disease gene networks and potential drug targets.

Dépistage du contenu haute sur les maladies neurodégénératives

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be ...

The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

The lower urinary tract (LUT) functions as a dynamic reservoir that is able to store urine and to efficiently expel it at a convenient time. While storing urine, however, the bladder is exposed for prolonged periods to waste products. By acting as a tight barrier, the epithelial lining of the LUT, the urothelium, avoids re-absorption of harmful substances. Moreover, noxious chemicals stimulate the bladder's nociceptive innervation and initiate voiding contractions that expel the bladder's contents. Interestingly, the bladder's sensitivity to noxious chemicals has been used successfully in clinical practice, by intravesically infusing the TRPV1 agonist capsaicin to treat neurogenic bladder overactivity1. This underscores the advantage of viewing the bladder as a chemosensory organ and prompts for further clinical research. However, ethical issues severely limit the possibilities to perform, in human subjects, the invasive measurements that are necessary to unravel the molecular bases of LUT clinical pharmacology. A way to overcome this limitation is the use of several animal models2. Here we describe the implementation of cystometry in mice and rats, a technique that allows measuring the intravesical pressure in conditions of controlled bladder perfusion.
	After laparotomy, a catheter is implanted in the bladder dome and tunneled subcutaneously to the interscapular region. Then the bladder can be filled at a controlled rate, while the urethra is left free for micturition. During the repetitive cycles of filling and voiding, intravesical pressure can be measured via the implanted catheter. As such, the pressure changes can be quantified and analyzed. Moreover, simultaneous measurement of the voided volume allows distinguishing voiding contractions from non-voiding contractions3.
	Importantly, due to the differences in micturition control between rodents and humans, cystometric measurements in these animals have only limited translational value4. Nevertheless, they are quite instrumental in the study of bladder pathophysiology and pharmacology in experimental pre-clinical settings. Recent research using this technique has revealed the key role of novel molecular players in the mechano- and chemo-sensory properties of the bladder.

Cystometry is an efficient technique to measure bladder function of small animals in vivo. The bladder is continuously infused at rates controlled through an intravesical catheter, whereas the urethra is left free for micturition. This allows for repetitive filling and emptying of the bladder, while intravesical pressure and voided volume are recorded.

L&#39;utilisation de la cystomanométrie chez les petits rongeurs: une étude de la vessie Chemosensation

	The lower urinary tract (LUT) functions as a  dynamic reservoir that is able to store urine and to efficiently expel it at a  convenient time. While ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

Dépistage du mélanome Modificateurs l&#39;aide d&#39;un modèle de tumeur Zebrafish Autochtone

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Etudier le cancer du pancréas Stem Cell Caractéristiques pour le développement de nouvelles stratégies de traitement

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

A High-content In Vitro Pancreatic Islet &amp;#946;-cell Replication Discovery Platform

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

Un haut contenu<em&gt; In vitro</em&gt; Pancreatic Islet β-cellulaire Plateforme de découverte de la réplication

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have been limited compounds available so far to attenuate TLR signaling and there have been no clinically approved TLR inhibitors (except the anti-malarial drug hydroxychloroquine) in clinical use. In light of rapid advances in nanotechnology, manipulation of immune responsiveness using nano-devices may provide a new strategy to treat these diseases. Herein, we present a high throughput screening method for quickly identifying novel bioactive nanoparticles that inhibit TLR signaling in phagocytic immune cells. This screening platform is built on THP-1 cell-based reporter cells with colorimetric and luciferase assays. The reporter cells are engineered from the human THP-1 monocytic cell line by stable integration of two inducible reporter constructs. One expresses a secreted embryonic alkaline phosphatase (SEAP) gene under the control of a promoter inducible by the transcription factors NF-&#954;B and AP-1, and the other expresses a secreted luciferase reporter gene under the control of promoters inducible by interferon regulatory factors (IRFs).Upon TLR stimulation, the reporter cells activate transcription factors and subsequently produce SEAP and/or luciferase, which can be detected using their corresponding substrate reagents. Using a library of peptide-gold nanoparticle (GNP) hybrids established in our previous studies as an example, we identified one peptide-GNP hybrid that could effectively inhibit the two arms of TLR4 signaling cascade triggered by its prototypical ligand, lipopolysaccharide (LPS). The findings were validated by standard biochemical techniques including immunoblotting. Further analysis established that this lead hybrid had a broad inhibitory spectrum, acting on multiple TLR pathways, including TLR2, 3, 4, and 5. This experimental approach allows a rapid assessment of whether a nanoparticle (or other therapeutic compounds) can modulate specific TLR signaling in phagocytic immune cells.

Toll-like receptor (TLR) signaling plays an important role in the pathophysiology of many human inflammatory diseases, and regulating TLR responses by bioactive nanoparticles is anticipated to be beneficial in many inflammatory conditions. THP-1 cell-based reporter cells provide a versatile and robust screening platform for identifying novel inhibitors of TLR signaling.

Criblage de nanoparticules bioactives dans des cellules immunitaires phagocytaires pour les inhibiteurs de la signalisation des récepteurs de type péage

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have ...

Routine Screening Method for Microparticles in Platelet Transfusions

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood banks. Cells fragment off microparticles (MP) when they are stressed. Blood and blood components may contain cellular fragments from a variety of cells, most notably from activated platelets. When performing their roles as innate immune cells and major players in coagulation and hemostasis, platelets change shape and generate microparticles. With dynamic light scattering (DLS)-based microparticle detection, it is possible to differentiate activated (high microparticle) from non-activated (low microparticle) platelets in transfusions, and optimize the use of this scarce blood product. Previous research suggests that providing non-activated platelets for prophylactic use in hematology-oncology patients could reduce their risk of becoming refractory and improve patient care. The goal of this screening method is to routinely differentiate activated from non-activated platelets. The method described here outlines the steps to be performed for routine platelet inventory management in a hospital blood bank: obtaining a sample from a platelet transfusion, loading the sample into the capillary for DLS measurement, performing the DLS test to identify microparticles, and using the reported microparticle content to identify activated platelets.

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative in hospital blood banks. The goal is to differentiate activated from non-activated platelets to optimize the use of platelets. Providing non-activated platelets to hematology-oncology patients might reduce their high risk to become refractory.

Routine de procédé de criblage de microparticules dans les Transfusions de plaquettes

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood ...

Hydra, a Computer-Based Platform for Aiding Clinicians in Cardiovascular Analysis and Diagnosis

Cardiovascular diseases (CVDs) are the leading cause of death throughout the world. The total risk of developing CVD is determined by the combined effect of different cardiovascular risk factors (e.g., diabetes, raised blood pressure, unhealthy diet, tobacco use, stress, etc.) that commonly coexist and act multiplicatively. Most CVDs can be prevented by an early identification of the highest risk factors and an appropriate treatment. The stratification of cardiovascular risk factors involves a wide range of parameters and tests that specialists use in their clinical practice. In addition to cardiovascular (CV) risk stratification, ambulatory blood pressure monitoring (ABPM) also provides relevant information for diagnostic and treatment purposes. This work presents a list of protocols based on the Hydra platform, a web-based system for clinical decision support which incorporates a set of functionalities and services that are required for complete cardiovascular analysis, risk assessment, early diagnosis, treatment and monitoring of patients over time. The program includes tools for inputting and managing comprehensive patient data, organized into different checkups to track the evolution over time. It also has a risk stratification tool to compute a CV risk factor based upon several risk stratification tables of reference. Additionally, the program includes a tool that incorporates ABPM analysis and allows the extraction of valuable information by monitoring blood pressure over a specific period of time. Finally, the reporting service summarizes the most relevant information in a set of reports that aid clinicians in their clinical decision-making process.

This article presents a protocol based on Hydra &#8212; a web-based system for clinical decision support that integrates a full and detailed set of functionalities and services required by physicians for complete cardiovascular analysis, risk assessment, early diagnosis, treatment, and monitoring over time.

Hydra, une plateforme informatique pour aider les cliniciens dans le diagnostic et d’analyse cardiovasculaire

Cardiovascular diseases (CVDs) are the leading cause of death throughout the world. The total risk of developing CVD is determined by the combined effect ...

In Vitro Method to Control Concentrations of Halogenated Gases in Cultured Alveolar Epithelial Cells

Acute respiratory distress syndrome (ARDS) is a syndrome of diffuse alveolar injury with impaired alveolar fluid clearance and severe inflammation. The use of halogenated agents, such as sevoflurane or isoflurane, for the sedation of intensive care unit (ICU) patients can improve gas exchange, reduce alveolar edema, and attenuate inflammation during ARDS. However, data on the use of inhaled agents for continuous sedation in the ICU to treat or prevent lung damage is lacking. To study the effects of halogenated agents on alveolar epithelial cells under "physiologic" conditions, we describe an easy system to culture cells at the air-liquid interface and expose them to halogenated agents to provide precise controlled "air" fractions and "medium" concentrations for these agents. We developed a sealed air-tight chamber in which plates with human alveolar epithelial immortalized cells could be exposed to a precise, controlled fraction of sevoflurane or isoflurane using a continuous gas flow provided by an anesthetic machine circuit. Cells were exposed to 4% of sevoflurane and 1% of isoflurane for 24 hours. Gas mass spectrometry was performed to determine the concentration of halogenated agents dissolved in the medium. After the first hour, the concentrations of sevoflurane and isoflurane in the medium were 251 mg/L and 25 mg/L, respectively. The curves representing the concentrations of both sevoflurane and isoflurane dissolved in the medium showed similar courses over time, with a plateau reached at one hour after exposure. 
This protocol was specifically designed to reach precise and controlled concentrations of sevoflurane or isoflurane in vitro to improve our understanding of mechanisms involved in epithelial lung injury during ARDS and to test novel therapies for the syndrome.

In Vitro Method to Control Concentrations of Halogenated Gases in Cultured Alveolar Epithelial Cells

We describe an easy protocol specifically designed to reach precise and controlled concentrations of sevoflurane or isoflurane in vitro in order to improve our understanding of mechanisms involved in the epithelial lung injury and to test novel therapies for acute respiratory distress syndrome.

In Vitro Méthode de contrôle des Concentrations de gaz halogénés en cellules épithéliales alvéolaires

Acute respiratory distress syndrome (ARDS) is a syndrome of diffuse alveolar injury with impaired alveolar fluid clearance and severe inflammation. The ...