Ce travail a été soutenu par un financement du National Institutes of Health des subventions 1R21NS073097-01 et 1R01DK082884 (JSD) et la Fondation pour la PIER11VCTR instituts nationaux de subvention.

<ol>
	<li>Ehrlich, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inward+rectifier+potassium+currents+as+a+target+for+atrial+fibrillation+therapy.">Inward rectifier potassium currents as a target for atrial fibrillation therapy.</a> J. Cardiovasc. Pharmacol. 52 (2), 129 (2008).</li><li>Bhave, G., Lonergan, D., Chauder, B. A., Denton, J. S. <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+modulators+of+inward+rectifier+K++channels:+recent+advances+and+future+possibilities.">Small-molecule modulators of inward rectifier K+ channels: recent advances and future possibilities.</a> Future Med. Chem. 2 (5), 757 (2010).</li><li>Swartz, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tarantula+toxins+interacting+with+voltage+sensors+in+potassium+channels.">Tarantula toxins interacting with voltage sensors in potassium channels.</a> Toxicon. 49 (2), 213 (2007).</li><li>Jin, W., Lu, Z. <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+high-affinity+inhibitor+for+inward-rectifier+K++channels.">A novel high-affinity inhibitor for inward-rectifier K+ channels.</a> Biochemistry. 37 (38), 13291 (1998).</li><li>Jin, W., Lu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+a+stable+form+of+tertiapin:+a+high-affinity+inhibitor+for+inward-rectifier+K++channels.">Synthesis of a stable form of tertiapin: a high-affinity inhibitor for inward-rectifier K+ channels.</a> Biochemistry. 38 (43), 14286 (1999).</li><li>Roy, A., McDonald, P. R., Sittampalam, S., Chaguturu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open+access+high+throughput+drug+discovery+in+the+public+domain:+a+Mount+Everest+in+the+making.">Open access high throughput drug discovery in the public domain: a Mount Everest in the making.</a> Curr. Pharm. Biotechnol. 11 (7), 764 (2010).</li><li>Weaver, C. D., 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+thallium-sensitive,+fluorescence-based+assay+for+detecting+and+characterizing+potassium+channel+modulators+in+mammalian+cells.">A thallium-sensitive, fluorescence-based assay for detecting and characterizing potassium channel modulators in mammalian cells.</a> J. Biomol. Screen. 9 (8), 671 (2004).</li><li>Lewis, L. M., 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=High-throughput+screening+reveals+a+small-molecule+inhibitor+of+the+renal+outer+medullary+potassium+channel+and.">High-throughput screening reveals a small-molecule inhibitor of the renal outer medullary potassium channel and.</a> 76 (5), 1094 (2009).</li><li>Bhave, G., 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+a+selective+small-molecule+inhibitor+of+Kir1.1,+the+renal+outer+medullary+potassium+channel.">Development of a selective small-molecule inhibitor of Kir1.1, the renal outer medullary potassium channel.</a> Mol. Pharmacol. 79 (1), 42 (2011).</li><li>Raphemot, R., 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=Discovery,+characterization,+and+structure-activity+relationships+of+an+inhibitor+of+inward+rectifier+potassium+(Kir)+channels+with+preference+for+Kir2.3,+Kir3.x,+and+Kir7.1.">Discovery, characterization, and structure-activity relationships of an inhibitor of inward rectifier potassium (Kir) channels with preference for Kir2.3, Kir3.x, and Kir7.1.</a> Front Pharmacol. 2, 75 (2012).</li><li>Niswender, C. M., 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+assay+of+Gi/o-linked+G+protein-coupled+receptor+coupling+to+potassium+channels+provides+new+insights+into+the+pharmacology+of+the+group+III+metabotropic+glutamate+receptors.">A novel assay of Gi/o-linked G protein-coupled receptor coupling to potassium channels provides new insights into the pharmacology of the group III metabotropic glutamate receptors.</a> Mol. Pharmacol. 73 (4), 1213 (2008).</li></ol>

Aucun conflit d&#39;intérêt déclaré.

Traitement des données: Une fois les données recueillies, une étape commune dans l&#39;analyse consiste à normaliser réponse de fluorescence de chaque puits, F, à sa valeur initiale au début de l&#39;expérience, F 0. Ceci est communément appelé le &quot;rapport statique» et symbolisé &quot;F / F 0&quot;. Dans le cas où F 0 est dominé par le colorant indicateur du fonctionnement rapport statique pour corriger sensiblement nombreux facteurs tels qu...

An erratum was issued for: <a href="https://www.jove.com/video/4209/high-throughput-screening-for-small-molecule-modulators-inward">High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels</a>. The Protocol section has been updated.
The formula in step 7.1 of the Protocol has been updated from:
Z prime = 1- (3SDp + 3SDn)/|meanp + meann |
to:
Z prime = 1- (3SDp + 3SDn)/|meanp - meann |

An erratum was issued for: <a href="https://www.jove.com/video/4209/high-throughput-screening-for-small-molecule-modulators-inward">High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels</a>. The Protocol section has been updated.

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

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> pcDNA5/TO </td><td> Invitrogen </td><td> V1033-20 </td><td> Vecteur d&#39;expression inductible par la tétracycline </td></tr><tr><td> T-rex-cellules HEK293 </td><td> Invitrogen </td><td> R71007 </td><td> Lignée cellulaire inductible par la tétracycline </td></tr><tr><td> Lipofectamine LTX / Plus réactif </td><td> Invitrogen </td><td> 15338100 </td><td> Réactif de transfection </td></tr><tr><td> FBS </td><td> ATLANTA Biologicals </td><td> S11550 </td><td> Milieux de culture cellulaire </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 11965 </td><td> Milieux de culture cellulaire </td></tr><tr><td> Hygromycine B </td><td> Invitrogen </td><td> 10687-010 </td><td> Milieux de culture cellulaire </td></tr><tr><td> Blasticidine S </td><td> Invitrogen </td&gt; <td> R210-01 </td><td> Milieux de culture cellulaire </td></tr><tr><td> Pénicilline / Streptomycine </td><td> Invitrogen </td><td> 15140 </td><td> Milieux de culture cellulaire </td></tr><tr><td> HBSS-divalent gratuit </td><td> Mediatech </td><td> 21022CV </td><td> Lavage des cellules </td></tr><tr><td> Trypsine 0,25% </td><td> Mediatech </td><td> 25053CI </td><td> Dissociation cellulaire </td></tr><tr><td> Tétracycline-HCl </td><td> Sigma </td><td> T9823 </td><td> Réactif à induction </td></tr><tr><td> FBS dialyse </td><td> ATLANTA Biologicals </td><td> S12650 </td><td> Géloses </td></tr><tr><td> FluoZin-2 </td><td> Invitrogen </td><td> F24189 </td><td> Colorant fluorescent </td></tr><tr><td> Pluronic F-127 </td><td> Invitrogen </td><td> P-3000MP </td><td> Colorant de chargement </td></tr><tr><td> HBSS </td><td> Invitrogen </td><td> 14175 </td><td> Tampon de dosage </td></tr><tr><td> HEPES </td><td> Invitrogen </td><td> 15630 </td><td> Tampon de dosage </td></tr><tr><td> NaHCO 3 </td><td> Sigma </td><td> S6297 </td><td> Tl + relance tampon </td></tr><tr><td> MgSO 4 </td><td> Sigma </td><td> M2643 </td><td> Tl + relance tampon </td></tr><tr><td> CaSO 4 • 2H 2 O </td><td> Sigma </td><td> C3771 </td><td> Tl + relance tampon </td></tr><tr><td> Le D-glucose </td><td> Sigma </td><td> G7528 </td><td> Tl + relance tampon </td></tr><tr><td> Sulfate de thallium </td><td> Aldrich </td><td> 204625 </td><td> Tl + relance tampon </td></tr><tr><td> HEPES </td><td> Sigma </td><td> H4034 </td><td> Tl + relance tampon </td></tr><tr><td> DMSO </td><td> Sigma </td><td> D4540 </td><td> Solvant </td></tr><tr><td> Huit canaux pipette électronique </td><td> Biohit </td><td> E300 </td><td> Placage cellule en 384 -plaques à puits </td></tr><tr><td> BD PureCoat amine recouvertes de plaques 384 puits </td><td> BD Biosciences </td><td> 356719 </td><td> Microplaques de dosage </td></tr><tr><td> Echo qualifié 384-Eh bien polypropylène microplaque (384pp) </td><td> Labcyte </td><td> P-05525 </td><td> Microplaques sources composés </td></tr><tr><td> Microplaques en polypropylène de 384 puits </td><td> Greiner Bio-One </td><td> 781280 </td><td></td></tr><tr><td> Multidrop Combi distributeur de réactif </td><td> Thermo Scientific </td><td> 5840300 </td><td></td></tr><tr><td> Laveur de microplaques ELx405 </td><td> BioTek </td><td> ELx405HT </td><td> Lavage de cellules automatisé </td></tr><tr><td> Echo manipulateur de liquide </td><td> Labcyte </td><td> Labcyte Echo 550 </td><td></td></tr><tr><td> Bravo automatisée plate-forme de manipulation des liquides </td><td> Agilent Technologies </td><td> Modèle standard </td><td></td></tr><tr><td> Hamamatsu FDSS 6000 </td><td> Hamamatsu </td><td></td><td> Kinetic lecteur de plaque d&#39;imagerie </td></tr></tbody></table> Tableau 1. Liste des Matériels et réactifs.

high-throughput screening for small-molecule modulators of inward rectifier potassium channels

Certains membres de la famille redresseur canal potassique vers l&#39;intérieur (Kir) sont postulés cibles thérapeutiques pour une variété de troubles, y compris l&#39;hypertension, la fibrillation auriculaire, et de 1,2 douleur. Pour la plupart, toutefois, les progrès vers la compréhension de leur potentiel thérapeutique, voire fonctions physiologiques a été ralentie par le manque de bons outils pharmacologiques. En effet, la pharmacologie moléculaire de la famille redresseur entrant est restée loin derrière celle de la superfamille S4 de potassium voltage-dépendants (Kv), les canaux pour lesquels un certain nombre d&#39;affinité nanomolaire et très modulateurs sélectifs des toxines peptidiques ont été découverts 3. La toxine du venin d&#39;abeille tertiapin et ses dérivés sont de puissants inhibiteurs de Kir1.1 et Kir3 canaux 4,5, mais peptides sont d&#39;une utilité limitée thérapeutique ainsi que expérimentalement en raison de leurs propriétés antigéniques et la biodisponibilité médiocre, la stabilité métabolique et la pénétrance des tissus. Le développement de puissantset sélectifs de petites molécules sondes avec des propriétés pharmacologiques améliorées sera un élément clé pour bien comprendre la physiologie et le potentiel thérapeutique des canaux Kir. La moléculaire bibliothèques Sondes de production Network Center (MLPCN) supportée par le National Institutes of Health (NIH) Fonds commun a créé des opportunités pour les scientifiques académiques pour initier des campagnes de sonde de découverte de cibles moléculaires et les voies de signalisation dans le besoin d&#39;une meilleure pharmacologie 6. L&#39;offre aux chercheurs l&#39;accès MLPCN aux centres de dépistage l&#39;échelle industrielle et la chimie médicinale et de l&#39;informatique en charge de développer de petites molécules sondes pour élucider la fonction des gènes et des réseaux de gènes. L&#39;étape critique pour gagner l&#39;entrée à l&#39;MLPCN est le développement d&#39;un test robuste cible ou une voie spécifique qui est susceptible de criblage à haut débit (HTS). Ici, nous décrivons comment développer une base de fluorescence thallium (Tl +) flux assaY sur Kir canal de fonction pour le criblage à haut débit composé 7,8,9,10. L&#39;essai est basé sur la perméabilité de la chaîne K + pores de la Tl + K + congénère. Un disponibles dans le commerce fluorescent Tl + colorant reporter est utilisé pour détecter le flux transmembranaire de Tl + à travers le pore. Il ya au moins trois colorants disponibles dans le commerce qui sont appropriés pour Tl + tests de flux: BTC, FluoZin-2, 7,8 et Fluxor. Ce protocole décrit le développement de tests à l&#39;aide FluoZin-2. Bien qu&#39;à l&#39;origine développés et commercialisés comme un indicateur de zinc, FluoZin-2 présente une augmentation forte et dose-dépendante de l&#39;émission de fluorescence lors de Tl + contraignant. Nous avons commencé à travailler avec FluoZin-2 avant Fluxor était disponible 7,8 et ont continué à le faire 9,10. Cependant, les étapes de développement de tests sont essentiellement identiques pour les trois colorants, et les utilisateurs devraient déterminer quels colorant est le plus approprié pour leur propre needs. Nous discutons également des critères de performance de l&#39;analyse qui doit être atteint à prendre en considération pour l&#39;entrée à l&#39;MLPCN. Depuis Tl + imprègne facilement la plupart des canaux K +, le dosage doit être adapté à la plupart des objectifs de canaux K +.

L&#39;utilisation d&#39;un système d&#39;expression inductible par la tétracycline permet un contrôle pratique interne pour distinguer Tl + flux à travers les voies endogènes et le canal Kir d&#39;intérêt. Figure 1 montre quelques exemples de cartes de placage de cellules utilisées dans différents types d&#39;expériences. Les positions des puits contenant les cellules non induites ou induite par la tétracycline sont indiquées par des couleurs différentes. Figure 2A</stron...

Watch this Scientific Journal Video about High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels at JoVE.com

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

Méthodes d&#39;élaboration et de validation d&#39;un dosage quantitatif de fluorescence pour mesurer l&#39;activité de potassium redresseur actif (Kir) des canaux pour le criblage de composés à haut débit est présenté.

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, ...

high-throughput-screening-for-small-molecule-modulators-inward

Research

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Biology

14.9K vues.  Vanderbilt University School of Medicine. Méthodes d'élaboration et de validation d'un dosage quantitatif de fluorescence pour mesurer l'activité de potassium redresseur actif (Kir) des canaux pour le criblage de composés à haut débit est présenté.

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

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

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

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

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

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

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

Recherche

Biologie

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 ...

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue1,2. GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (Gi and Go) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K+ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate.
GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders3. However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations3.
Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K+ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K+ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry4 or radiotracer analysis5, the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.

A real-time screening procedure for identifying drugs that interact with G protein-gated inward rectifier K+ (GIRK) channels is described. The assay utilizes membrane potential-sensitive fluorescent dyes to measure GIRK channel activity. This technique is adaptable for use on a number of cell lines.

Un test de dépistage fluorescent pour identifier des modulateurs de canaux Girk

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...

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 ...

A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

The Drosophila embryo has long been a powerful laboratory model for elucidating molecular and genetic mechanisms that control development. The ease of genetic manipulations with this model has supplanted pharmacological approaches that are commonplace in other animal models and cell-based assays. Here we describe recent advances in a protocol that enables application of small molecules to the developing fruit fly embryo. The method details steps to overcome the impermeability of the eggshell while maintaining embryo viability. Eggshell permeabilization across a broad range of developmental stages is achieved by application of a previously described d-limonene embryo permeabilization solvent (EPS1) and by aging embryos at reduced temperature (18 &#176;C) prior to treatments. In addition, use of a far-red dye (CY5) as a permeabilization indicator is described, which is compatible with downstream applications involving standard red and green fluorescent dyes in live and fixed preparations. This protocol is applicable to studies using bioactive compounds to probe developmental mechanisms as well as for studies aimed at evaluating teratogenic or pharmacologic activity of uncharacterized small molecules.

A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

Introduction of small molecules to the developing Drosophila embryo offers great potential for characterizing biological activity of novel compounds, drugs, and toxins as well as for probing fundamental developmental pathways. Methods described herein outline steps that overcome natural barriers to this approach, expanding the utility of the Drosophila embryo model.

Une méthode de perméabilisation de<em&gt; Drosophila</em&gt; Embryons pour dosages de petites molécules activité

The Drosophila embryo has long been a powerful laboratory model for elucidating molecular and genetic mechanisms that control development. The ease of ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

Un petit écran Molecule Manuel approchant à haut débit utilisant embryons de poissons zèbres

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Recouvrement des adultes coeurs de poisson zèbre à haut débit pour des applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

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 ...

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge flexibility in order to identify various EPS.

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.