C. elegans transgènes ont été fournis par le Centre de Génétique Caenorhabditis (Université du Minnesota, Minneapolis, MN). Ce travail a été soutenu par le NIH R21 octroi NS0667678-01 à KS. Tous les auteurs ont participé à la collecte, l&#39;analyse et l&#39;interprétation des données. CKL, KS, et KPC participé à la rédaction et la révision du manuscrit.

1. Préparation de la nourriture vers bactérienne Jour 1 <ol><li> Ajouter 5 ml d&#39;eau saturée E. coli OP50 bouillon de culture bactérienne ml à 500 Terrific complété avec 50 pg / ml de streptomycine et de grandir dans un incubateur agitateur (225 rpm) pendant la nuit à 37 ° C. </li></ol> Jour 2 <ol start="2"><li> Séparer la culture de la nuit bactérienne en dix tubes de 50 ml et les bactéries par centrifugation dans une centrifugeuse réfrigérée à 2.500 rcf pendant 20 minutes. </li><li> Décanter bouillon LB et resuspendre chaque culot bactérien dans 10 ml de liquide milieux de croissance des nématodes (NGM). Secouer horizontalement dans un shaker de plancher pendant 15 minutes pour remettre en suspension les bactéries. Pour faire tampon NGM, ajouter 3 g de NaCl à 1 L d&#39;eau déminéralisée et autoclave. Laisser refroidir à 55 ° C et ajouter dans l&#39;ordre les solutions suivantes stérile: 1 ml de 1 M CaCl 2, 1 ml de 1 M MgSO 4, et 25 ml de phosphate de potassium 1 M, pH 6,0. </li><li> Centrifuger la culture bactérienne dans une centrifugeuse réfrigérée à 2.500 rcf pendant 20 minutes. </li><li> Décanter tampon NGM et peser le culot bactérien. </li><li> Ajouter un volume égal de tampon NGM pour remettre les boulettes, partie aliquote de 3 ml de bactéries se concentrer dans des tubes de 15 ml et conserver à -20 ° C. </li></ol> 2. À grande échelle C. elegans culture liquide Nous utilisons une lignée transgénique VP596 (dvIs19 [pAF15 (TPS-4:: GFP:: SNA)]; vsIs33 [dop-3: DP]) transportant deux constructions fluorescentes: Pgst -4:: GFP 14 pour surveiller SKN-1 activité et PDOP-3: DP 17 pour servir de standard pour la normalisation des numéros ver. Jour 1 <ol><li> Mélanger 150 ml de tampon NGM, 1,5 ml de bouillon LB, 150 pi de 5 mg / ml de cholestérol (dans l&#39;éthanol), et 75 ul de 100 mg / ml de streptomycine. Filtre-stériliser le mélange et ajouter à un ballon de 1 litre stérile. REMARQUE: l&#39;ajout de bouillon LB 1% de tampon NGM aide à prévenir les vers de coller à verre et en plastique, mais ce montant n&#39;est pas suffisant pour soutenir la croissance des bactéries. </li><li> Synchroniser les vers à la procédure standard de l&#39;hypochlorite de 18 ans. Jusqu&#39;à 0,5 ml de vers gravides peuvent être traitées dans un seul tube de 15 ml avec 5 ml de solution d&#39;hypochlorite (3,75 ml d&#39;eau stérile, 1 ml de javel, et 250 pi NaOH 10 N). Lavez les oeufs libérés à l&#39;eau stérile et les remettre en suspension dans 10 ml de tampon NGM. </li><li> Diluez un échantillon d&#39;œufs 100 fois NGM tampon (par exemple, 100 ul dans 10 ml) et de compter le nombre d&#39;oeufs séparés en trois aliquotes de 5 pi. Multiples, le nombre moyen de 200.000 (100 x facteur de dilution 2000) pour estimer le nombre total d&#39;œufs. </li><li> Ajouter 200 000 à 2 millions d&#39;œufs à la fiole avec un tampon de NGM et secouer la culture à 100 rpm, à 20 ° C. </li></ol> Jour 2 <ol start="5"><li> Au moins 16 h après l&#39;ajout d&#39;oeufs dans le ballon, utiliser une pipette stérile sérologique pour enlever environ 0,5 ml de la culture ver suspendu. Pipeter 5 gouttes trois ul sur le couvercle stérile d&#39;une boîte de Pétri. Placez le couvercle dans un congélateur à -20 ° C pendant 1-2 minutes pour paralyser les vers. Comptez le nombre moyen de vers vivants éclos par 5 ul puis plusieurs par 30 000 (150 ml / 5 pl) pour estimer le nombre total de vers éclos. </li><li> Dégeler un tube de gelée OP50 culture bactérienne (protocole 1.6) et ajouter 3 ml de 50% dans la culture OP50 ver par 500 000 vers éclos. Agiter le flacon à 100 rpm à 20 ° C. Worms se développer en larves L4 et les jeunes adultes dans environ 51 heures. La DO600 de bactéries devrait être d&#39;environ 2.200 au début. Surveiller la quantité de bactéries par absorption et d&#39;ajouter de plus si la DO 600 tombe en dessous de 0,900. </li></ol> 3. La collecte et la distribution de Worm Jour 4 <ol><li> Utiliser une pipette stérile sérologique au transfert d&#39;environ 0,5 ml de la culture ver suspendu à une plaque de gélose NGM standard. Afficher les vers avec un stéréomicroscope pour s&#39;assurer que la plupart sont des larves L4 ou de jeunes adultes. Larves L4 aura un spot ventrale claire dans le milieu du corps. Les jeunes adultes seront légèrement plus grand et ne sera pas une tache claire. </li><li> Verser la culture ver dans des tubes de 50 ml stériles et placer les tubes dans un portoir sur la paillasse. Autoriser les vers de se contenter de 10 minutes environ. Eliminer le surnageant par aspiration ou pipetage. REMARQUE: Cette étape peut être important d&#39;enlever les œufs non éclos ou des vers qui ne se développent pas car ils se déposent plus lentement que les larves L4 et les jeunes adultes. </li><li> Ramassez tous les vers dans un seul tube de 50 ml et laver avec un tampon de NGM avec du bouillon LB 1% (NGM + LB) 3-4 fois pour éliminer les bactéries. NOTE: Ceci est fait de mieux dans une centrifugeuse en collectant des vers à 500 fcr pendant 30 sec et en remplaçant le surnageant avec le NGM frais + tampon LB. </li><li> Après le lavage, remplir le tube avec le NGM + tampon LB à 50 ml et versez dans un flacon à vis sans fin personnalisée de distribution. Ajouter un barreau de petites et remuer pour garder les vers suspendu. Comptez èmee nombre de vers en trois 5 gouttes ul comme indiqué plus haut. Diluer avec le NGM + LB jusqu&#39;à ce que vous obtenez 12 à 15 vers par 5 ul baisse (de 2.5 à 3.0 ~ vers / ul). REMARQUE: Un minimum d&#39;environ 20 ml (60.000 vers) est nécessaire pour combler l&#39;espace mort du flacon de distribution et d&#39;une cassette distributeur. Chaque plaque 384 puits nécessite environ 35 000 vers ou 11,5 ml de vers suspendu. </li><li> Chargez une cassette de 10 ul de distribution et stériliser par amorçage avec de l&#39;éthanol à 70%. Rincer l&#39;éthanol par amorçage à l&#39;eau stérile. </li><li> Insérez l&#39;extrémité de la cassette de distribution dans le ballon de sorte qu&#39;il est juste au-dessus de la barre remuez sans perturber son mouvement. Assurez-vous que les vers restent en suspension dans le flacon entier. Programme du distributeur de se passer à basse vitesse avec 2 pré-impulsions. Premier et exécuter au moins 10 ml de vers suspendu. Remplissez plaques comme nécessaire rapidement pour empêcher les vers de s&#39;installer dans le tube. </li><li> Sceller les plaques avec du ruban adhésif respirant. </li><li> Placez les plaques sur une table d&#39;agitation dans un incubateur à la température appropriée pour votre analyse. Ne pas empiler les assiettes. </li></ol> 4. L&#39;analyse par fluorescence <ol><li> Placer la plaque cible dans un lecteur de microplaques pour mesurer l&#39;intensité de fluorescence de chaque puits avec la longueur d&#39;onde appropriée d&#39;émission et d&#39;excitation (Filtres pour notre analyse: la GFP 485/20ex 528/20em; DP 540/25ex 590/35em). </li><li> Calculer le rapport de la GFP / RFP et normaliser les lectures des puits témoin (sans composé activant) pour déterminer la différence exacte pli de l&#39;intensité de fluorescence provenant des traitements individuels. </li></ol> 5. Les résultats représentatifs: Notre SKN-1 essai utilise une souche journaliste de chromosomes double intégrée (VP596). Comme le montre la figure 1, le nombre de vers est bien corrélé au volume distribué. Comme le montrent les figures 2A et 2B, le total des GFP et DP de fluorescence par puits est hautement reproductible d&#39;un puits à travers une plaque de 384 puits. Comme le montre la figure 2C, l&#39;ONU-induite Pgst-4:: fluorescence de la GFP est linéairement corrélée à PDOP-3: DP dans 384 puits. Lorsqu&#39;il est exprimé comme un rapport de la GFP / RFP, la fluorescence devient hautement reproductibles d&#39;un puits à travers une plaque de 384 puits (comparer le coefficient de variation de la figure 2D pour 2A) démontrant la capacité de la PDOP-3:: Un journaliste demande de propositions pour réduire la variabilité. Comme le montre la figure 3, l&#39;induction de la GFP relatives à la DP avec un SKN-1 activation des xénobiotiques (38 juglone uM) est robuste et hautement reproductible à travers une plaque de 384 puits. <img src="/files/ftp_upload/2745/2745fig1.jpg" alt="Figure 1"/> Figure 1. Nombre de vers en fonction du volume distribué. Worms ont été dilués à environ 2/μl et distribués dans une plaque à 24 puits pour le comptage manuel avec un stéréomicroscope. N = 8 puits par volume. <img src="/files/ftp_upload/2745/2745fig2.jpg" alt="Figure 2"/> Figure 2. Fluorescence totale de vers distribuée dans une plaque 384 puits est reproductible. Worms ont été dilués à environ ul 2.5/μl et 30 a été distribué dans chaque puits d&#39;une plaque de 384 puits. GFP (A) et DP (B) de fluorescence de tous les puits ont un coefficient de variation inférieur à 9%. (C) fluorescence de la GFP est fortement corrélé à la DP fluorescence. (D) Calcul du ratio de la GFP à la DP a réduit le coefficient de variation au-dessous de 6% (A, B et D) Les lignes pleines indiquent les moyens et les lignes brisées indiquent trois déviations standard au-dessus ou en dessous de la moyenne. <img src="/files/ftp_upload/2745/2745fig3.jpg" alt="Figure 3"/> . Figure 3 Activation de Pgst-4:: GFP est robuste et cohérente. Environ 75 L4 vers ont été distribués dans tous les puits d&#39;une plaque de 384 puits et 38 uM juglone a été ajouté dans chaque colonne. GFP et DP fluorescence a été mesurée après 21 h d&#39;incubation. La relative moyenne des ratios de fluorescence de tout contrôle (1,0) et la juglone puits (8,9) sont marqués par des lignes pleines. Trois déviations standard au-dessus du contrôle de la moyenne et inférieure à la moyenne juglone sont marqués par des lignes brisées.

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Nous présentons une méthode pour la culture et la distribution de grands nombres de nématodes transgéniques. L&#39;équipement utilisé pour les vers de la culture est la norme pour les laboratoires effectuant le clonage moléculaire et la manipulation de liquides et de l&#39;équipement de fluorescence est standard pour les laboratoires de traitement de grands nombres de microplaques. D&#39;autres méthodes de distribution grand nombre de vivre C. elegans nécessitent des particules onéreux équipement de...

<table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th><th> Commentaires (optionnel) </th></tr><tr><td> Bouillon LB </td><td> Recherche International Products Corp </td><td> L24066 </td><td></td></tr><tr><td> Terrific Bouillon </td><td> Recherche International Products Corp </td><td> T15100 </td><td></td></tr><tr><td> Synergy ™ HT multi-mode lecteur de microplaques </td><td> BioTek </td><td></td><td> Filtres: GFP 485/20ex 528/20em; DP 540/25ex 590/35em </td></tr><tr><td> MicroFlo Select Distributeur </td><td> BioTek </td><td></td><td></td></tr><tr><td> Worm flacon de distribution </td><td> Sud Scientific Inc, Micanopy, Floride </td><td></td><td> Personnalisé assemblés (352) 284-2531 </td></tr><tr><td> 384 microplaques </td><td> Greiner Bio-One </td><td> 5678-1209 </td><td></td></tr><tr><td> Ruban adhésif respirant </td><td> Nunc </td><td> 241205 </td><td></td></tr><tr><td> (5-hydroxy-p-naphthoqinone) Juglone </td><td> ACROS </td><td> 121640010 </td><td> Juglone est dissoute dans du DMSO </td></tr><tr><td> DMSO </td><td> Sigma-Aldrich </td><td> D-5879 </td><td></td></tr><tr><td> C. elegans transgéniques souche </td><td> Auteur du laboratoire </td><td> VP596 </td><td> Pgst-4:: GFP et PDOP-3: DP </td></tr></tbody></table>

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

Criblage à haut débit (HTS) est une approche puissante pour identifier des modulateurs chimiques des processus biologiques. Toutefois, de nombreux composés identifiés dans les écrans en utilisant des modèles de culture cellulaire sont souvent considérés comme toxiques ou pharmacologiquement inactive in vivo 1-2. Dépistage dans les modèles animaux entiers peuvent aider à éviter ces écueils et de rationaliser la voie du développement de médicaments. C. elegans est un organisme modèle multicellulaire bien adapté pour HTS. Il est de petite taille (&lt;1 mm) et peut être économiquement cultivé et distribué dans les liquides. C. elegans est également l&#39;un des modèles animaux les plus dociles expérimentale permettant une identification rapide et détaillée de la drogue mode d&#39;action 3. Nous décrivons un protocole pour la culture et la distribution des souches fluorescentes de C. elegans pour criblage à haut débit de chimiothèques ou la détection de contaminants environnementaux qui modifient l&#39;expression d&#39;un gène spécifique. Un grand nombre de vers de développement synchronisé sont cultivées en milieu liquide, récoltées, lavées, et suspendu à une densité définie. Worms sont ensuite ajoutés au noir, fond plat, plaques 384 puits en utilisant un distributeur péristaltique liquide. Les petites molécules à partir d&#39;une bibliothèque chimique ou échantillons de test (par exemple, eau, nourriture, ou le sol) peuvent être ajoutés aux puits avec des vers. In vivo, en temps réel l&#39;intensité de fluorescence est mesurée avec un lecteur de microplaques à fluorescence. Cette méthode peut être adapté à n&#39;importe quel gène inductible en C. elegans pour lequel un journaliste convenable est disponible. Beaucoup de stress inductible et de développement des voies de transcription sont bien définis dans C. elegans et les souches transgéniques GFP journaliste existent déjà pour beaucoup d&#39;entre eux 4. Lorsqu&#39;il est combiné avec les journalistes appropriée transgéniques, notre méthode peut être utilisée pour dépister les modulateurs voie ou à développer de solides analyses biocapteur pour les contaminants environnementaux. Nous démontrons notre C. elegans culture et de distribution de protocole avec un test HTS nous avons développé pour surveiller le C. &#39;n&#39; elegans bouchon facteur de transcription cols SKN-1. SKN-1 et son homologue de Nrf2 activer des gènes de mammifères cytoprotecteur durant le stress oxydatif et des xénobiotiques 5-10. Nrf2 protège les mammifères de nombreuses liée à l&#39;âge des troubles tels que le cancer, la neurodégénérescence, et de l&#39;inflammation chronique et est devenue une cible majeure chimiothérapeutiques 11-13. Notre analyse est basée sur une GFP journaliste transgéniques pour le SKN-1 gène cible -4 TPS 14, codant pour une glutathion-S 6 transférase. La TPS est également journaliste de -4 un biocapteur pour les produits chimiques xénobiotiques et oxydatif qui activent SKN-1 et peut être utilisé pour détecter de faibles niveaux de contaminants tels que l&#39;acrylamide et le méthyl-mercure, 15-16.

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

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

Une procédure de liquides à base de culture et de la distribution de<em&gt; C. elegans</em&gt; Souches exprimant des protéines rapporteur fluorescent est décrite qui ne nécessite pas d&#39;équipement de tri coûteux. Cette approche peut être appliquée à de nombreux inductible<em&gt; C. elegans</em&gt; Des gènes pour la découverte de médicaments ou de biocapteurs de 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 ...

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

17.8K vues.  University of Florida. Une procédure de liquides à base de culture et de la distribution de C. elegans Souches exprimant des protéines rapporteur fluorescent est décrite qui ne nécessite pas d'équipement de tri coûteux. Cette approche peut être appliquée à de nombreux inductible C. elegans Des gènes pour la découverte de médicaments ou de biocapteurs de contaminants.

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

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

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

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

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

Criblage à haut débit activité endoglucanase fongique dans les Escherichia coli</em

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

Recherche

Biologie

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 ...

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

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

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

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

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. ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

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

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

À haut débit Affinity Purification de protéines recombinantes-étiquette

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

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

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

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

Criblage à haut débit pour les petites molécules modulateurs de canaux potassiques entrants Rectifier

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

C. elegans Chemotaxis Assay

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

C. elegans Chemotaxis Assay

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

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

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

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

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

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

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 Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

Criblage à haut débit fonctionnelle utilisant une maison double lueur de dosage de luciférase

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

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 ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

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

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

Identification de paires Kinase-substrat en utilisant criblage à haut débit


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