C. elegans Transgene wurden von den Caenorhabditis Genetics Center (University of Minnesota, Minneapolis, MN) zur Verfügung gestellt. Diese Arbeit wurde vom NIH R21 gewähren NS0667678-01 bis KS unterstützt. Alle Autoren nahmen an der Erhebung, Analyse und Interpretation der Daten. CKL, KS, und KPC nahmen schriftlich und Überarbeitung des Manuskripts.

1. Vorbereitung der bakteriellen Wurm Lebensmittel Tag 1 <ol><li> 5 ml gesättigte E. coli OP50 Bakterienkultur zu 500 ml Brühe Terrific mit 50 ug / ml Streptomycin ergänzt und wachsen in einem Schüttelinkubator (225 rpm) über Nacht bei 37 ° C. </li></ol> Tag 2 <ol start="2"><li> Split der Nacht Bakterienkultur in zehn 50 ml-und Zentrifugenröhrchen Bakterien in einer Kühlzentrifuge bei 2.500 rcf für 20 Minuten. </li><li> Abdekantieren LB Brühe und resuspendieren jeder Bakterienpellet in 10 ml Flüssigkeit Nematoden Nährmedien (NGM). Schütteln Sie horizontal in einem Stockwerk Schüttler für 15 Minuten, um die Bakterien zu resuspendieren. Um NGM-Puffer, 3 g NaCl auf 1 L entionisiertem Wasser und Autoklaven. Abkühlen auf 55 ° C und in Ordnung fügen Sie die folgende sterile Lösungen: 1 ml 1 M CaCl 2, 1 ml 1 M MgSO 4, und 25 ml 1 M Kaliumphosphat, pH 6,0. </li><li> Centrifuge die Bakterienkultur in einer Kühlzentrifuge bei 2.500 rcf für 20 Minuten. </li><li> Abdekantieren NGM-Puffer und wiegen das Bakterienpellet. </li><li> Fügen Sie dem gleichen Volumen NGM-Puffer zu den Pellets, Aliquot 3 ml Bakterien Konzentrat in 15 ml Tuben, und bei -20 ° C resuspendieren </li></ol> 2. Großflächige C. elegans Flüssigkultur Wir verwenden eine transgene Linie VP596 (dvIs19 [pAF15 (gst-4:: GFP:: NLS)]; vsIs33 [dop-3:: RFP]) mit zwei fluoreszierenden Konstrukte: Pgst -4:: GFP 14 bis SKN-1 Monitor Aktivität und PDOP-3:: RFP 17 bis als Standard für die Wurm-Nummer Normalisierung dienen. Tag 1 <ol><li> Mix 150 ml NGM-Puffer, 1,5 ml LB-Medium, 150 ul von 5 mg / ml Cholesterin (in Ethanol) und 75 ul 100 mg / ml Streptomycin. Filter-sterilisiert die Mischung und fügen Sie einen 1-Liter sterile Flasche. HINWEIS: Bei Zugabe von 1% LB Brühe auf NGM Puffer hilft, Würmer Anhaften von Glas-und Kunststoffprodukte zu verhindern, aber dieser Betrag nicht ausreicht, um das Wachstum von Bakterien zu unterstützen. </li><li> Synchronisieren Würmer mit dem Standard-Hypochlorit Verfahren 18. Bis zu 0,5 ml gravid Würmer können in einem einzigen 15 ml Tube mit 5 ml Natriumhypochlorit-Lösung (3,75 ml sterilem Wasser, 1 ml Bleiche, und 250 ul 10 N NaOH) verarbeitet werden. Waschen Sie die Freigabe Eier mit sterilem Wasser und resuspendieren Sie in 10 ml NGM Puffer. </li><li> Verdünnen Sie eine Probe der Eier 100-fach in NGM Puffer (z. B. 100 ul in 10 ml) und zählen die Anzahl der Eier in drei separate 5 ul Aliquots. Mehrere der durchschnittlichen Anzahl von 200.000 (100 Verdünnungsfaktor x 2.000) zur Schätzung der Gesamtzahl der Eier. </li><li> Add 200.000 bis 2 Millionen Eier in den Kolben mit NGM-Puffer und schütteln Sie die Kultur bei 100 Umdrehungen pro Minute bei 20 ° C. </li></ol> Tag 2 <ol start="5"><li> Mindestens 16 h nach der Zugabe Eier in den Kolben, eine sterile serologische Pipette ca. 0,5 ml des suspendierten Wurm Kultur zu entfernen. Pipet drei 5 ul Tropfen auf die sterile Deckel einer Petrischale. Setzen Sie den Deckel in einer -20 ° C Gefrierschrank für 1-2 Minuten, um Würmer zu lähmen. Zählen Sie die durchschnittliche Anzahl von lebenden geschlüpften Würmer pro 5 ul und dann mehrere von 30.000 (150 ml / 5 pl) auf die Gesamtzahl der geschlüpften Würmer zu schätzen. </li><li> Tauwetter ein Rohr aus gefrorenem OP50 Bakterienkultur (Protocol 1,6) und 3 ml 50% OP50 in die Wurm Kultur pro 500.000 geschlüpften Würmer. Schütteln Sie die Flasche mit 100 UpM bei 20 ° C. Worms wird in L4-Larven und jungen Erwachsenen in etwa 51 Stunden zu entwickeln. Die OD600 von Bakterien sollte etwa 2.200 an der Anfang sein. Überwachen Sie die Menge der Bakterien durch Absorption und fügen Sie mehr, wenn die OD 600 fällt unter 0,900. </li></ol> 3. Worm Sammlung und Abgabe Tag 4 <ol><li> Verwenden Sie eine sterile serologische Pipette ca. 0,5 ml des suspendierten Wurm Kultur zu einem Standard NGM-Agar-Platte übertragen. Sehen Sie sich die Würmer mit einem Stereomikroskop, um sicherzustellen, dass die meisten L4-Larven oder junge Erwachsene sind. L4-Larven haben einen ventralen hellen Fleck in der Mitte des Körpers. Junge Erwachsene werden etwas größer und haben keine klare Ort. </li><li> Gießen Sie den Wurm Kultur in sterile 50 ml Röhrchen und setzen Sie die Rohre in einem Reagenzglas Rack auf dem Labortisch. Lassen Sie die Würmer etwa 10 Minuten absetzen. Entfernen Sie den Überstand durch Absaugen oder Pipettieren. HINWEIS: Dieser Schritt kann es wichtig sein, unhatched Eier oder Würmer, die sich nicht entwickeln sich zu entfernen, weil sie langsamer als L4-Larven und junge Erwachsene werden zu begleichen. </li><li> Sammeln Sie alle Würmer in einem einzigen 50 ml Tube und wäscht mit NGM-Puffer mit 1% LB-Bouillon (NGM + LB) 3-4 mal, um Bakterien zu entfernen. Hinweis: Dies geschieht am besten in einem Ausschwingrotor zentrifugiert durch das Sammeln von Würmern bei 500 rcf für 30 sec und Austausch der Überstand mit frischen NGM + LB-Puffer. </li><li> Nach dem Waschen, füllen Sie den Schlauch mit NGM + LB-Puffer auf 50 ml und gießen Sie in ein benutzerdefiniertes Wurm Verzicht Kolben. Fügen Sie einen kleinen Rührstab und rühren, damit die Würmer ausgesetzt. Graf the Anzahl von Würmern in drei 5-ul Tropfen wie oben angegeben. Verdünnen mit NGM + LB, bis Sie 12-15 Würmer pro 5 ul Tropfen (~ 2,5-3,0 Würmer / ul) zu bekommen. HINWEIS: Ein Minimum von etwa 20 ml (60.000 Würmer) wird benötigt, um den Totraum der Abgabe Kolben und Spender Kassette zu füllen. Jeder 384-Well-Platte benötigt etwa 35.000 Würmer oder 11,5 ml suspendiert Würmer. </li><li> Legen Sie eine 10 pl Dispensierkassette und sterilisieren durch Grundierung mit 70% Ethanol. Spülen Sie das Ethanol durch Grundierung mit sterilem Wasser. </li><li> Führen Sie das Ende des Dispensierkassette in den Kolben, so dass es direkt über dem Rührstab ohne Unterbrechung der Bewegung ist. Stellen Sie sicher, dass die Würmer das ganze Kolben suspendiert bleiben. Programm der Spender bei niedriger Drehzahl mit 2 pre-Impulse zu verzichten. Prime und führen mindestens 10 ml suspendiert Würmer. Füllen Platten so schnell um die Würmer aus, sich in den Schlauch zu verhindern benötigt. </li><li> Seal die Platten mit atmungsaktivem Band. </li><li> Legen Sie die Platten auf einem Schütteltisch in einem Brutschrank bei der geeigneten Temperatur für Ihren Test. Stapeln Sie nicht die Platten. </li></ol> 4. Fluoreszenz-Analyse <ol><li> Legen Sie die Zieltafel in einem Mikroplatten-Reader, um die Fluoreszenz-Intensität der einzelnen Vertiefungen mit den entsprechenden Emissions-und Anregungswellenlänge (Filter für unseren Test: GFP 485/20ex 528/20em; RFP 540/25ex 590/35em) zu messen. </li><li> Berechnen Sie das Verhältnis von GFP / RFP und normalisieren mit den Messwerten der Kontroll-Wells (ohne aktivierende Verbindung), um die genaue fache Differenz der Fluoreszenzintensität von einzelnen Behandlungen abgeleitet bestimmen. </li></ol> 5. Repräsentative Ergebnisse: Unsere SKN-1-Test verwendet eine chromosomal integrierte Dual-Reporter-Stamm (VP596). Wie in Abbildung 1 gezeigt, ist die Zahl der Würmer auch, um die Lautstärke verzichtet korreliert. Wie in 2A und 2B, insgesamt GFP und RFP-Fluoreszenz pro Vertiefung dargestellt ist in hohem Maße reproduzierbar von Brunnen zu Brunnen über eine 384-Well-Platte. : GFP-Fluoreszenz ist linear auf PDOP-3 korreliert::: RFP über 384 Wells, wie in Abbildung 2C, un-induzierte Pgst-4 gezeigt. Wenn sie als ein Verhältnis von GFP / RFP ausgedrückt, wird Fluoreszenz hoch reproduzierbare von Brunnen zu Brunnen über eine 384-Well-Platte (vgl. Variationskoeffizient aus Abbildung 2D zu 2A) demonstriert die Fähigkeit der PDOP-3:: RFP Reporter Variabilität zu reduzieren. Wie in Abbildung 3, die Induktion der GFP gegenüber RFP mit A dargestellt SKN-1-aktivierenden Xenobiotika (38 uM Juglon) ist robust und sehr gut reproduzierbare über eine 384-Well-Platte. <img src="/files/ftp_upload/2745/2745fig1.jpg" alt="Abbildung 1"/> Abbildung 1. Anzahl der Würmer zu Volumen verzichtet. Worms wurden auf etwa 2/μl verdünnt und in eine 24-Well-Platte für die manuelle Zählung mit einem Stereomikroskop. N = 8 Vertiefungen pro Volumen. <img src="/files/ftp_upload/2745/2745fig2.jpg" alt="Abbildung 2"/> Abbildung 2. Summe Fluoreszenz von Würmern in einer 384-Well-Platte verzichtet reproduzierbar ist. Worms wurden auf etwa 2.5/μl und 30 ul verdünnt wurde in jede Vertiefung einer 384-Well-Platte verzichtet. GFP (A) und RFP (B) Fluoreszenz von allen Wells hatte einen Variationskoeffizienten unter 9%. (C) GFP-Fluoreszenz ist stark an RFP Fluoreszenz korreliert. (D) Berechnung des Verhältnisses der GFP zu RFP reduziert der Variationskoeffizient unter 6% (A, B und D) durchgezogenen Linien zeigen die Mittel und gestrichelten Linien zeigen drei Standardabweichungen über oder unter dem Mittelwert. <img src="/files/ftp_upload/2745/2745fig3.jpg" alt="Abbildung 3"/> . Abbildung 3 Aktivierung Pgst-4:: GFP ist robust und konsistent. Etwa 75 L4 Würmer wurden in alle Wells einer 384-Well-Platte und 38 uM Juglon verzichtet war in jeder anderen Spalte hinzugefügt. GFP und RFP Fluoreszenz wurde nach 21 h Inkubation gemessen. Die mittlere relative Fluoreszenz-Verhältnisse aller Steuer (1,0) und Juglon Brunnen (8,9) sind mit durchgezogenen Linien markiert. Drei Standardabweichungen über der Kontrolle bedeuten, und unter dem Juglon meine, sind mit gestrichelten Linien markiert.

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Wir stellen eine Methode zur Kultivierung und Abgabe einer großen Anzahl von transgenen Nematoden. Die Ausrüstung für die Kultivierung von Würmern verwendet wird Standard für Labors, die molekulare Klonierung und der Liquid-Handling-und Fluoreszenz-Ausstattung ist Standard für Labors Verarbeitung einer großen Anzahl von Mikroplatten. Andere Methoden der Abgabe eine große Anzahl von Live-C elegans teure Partikel Sortieranlagen 19. Die Pgst-4:: GFP-Assay kann zum Screening niedermoleku...

<table border="1"><tbody><tr><th> Name des Reagenzes </th><th> Firma </th><th> Katalog-Nummer </th><th> Kommentare (optional) </th></tr><tr><td> LB-Medium </td><td> Research Products International Corp </td><td> L24066 </td><td></td></tr><tr><td> Terrific Bouillon </td><td> Research Products International Corp </td><td> T15100 </td><td></td></tr><tr><td> Synergy ™ HT Multi-Mode Mikroplatten-Reader </td><td> BioTek </td><td></td><td> Filter: GFP 485/20ex 528/20em; RFP 540/25ex 590/35em </td></tr><tr><td> MicroFlo Select Dispenser </td><td> BioTek </td><td></td><td></td></tr><tr><td> Worm Verzicht Kolben </td><td> Süd Scientific Inc., Micanopy, FL </td><td></td><td> Benutzerdefinierte montiert (352) 284-2531 </td></tr><tr><td> 384 Mikrotiterplatten </td><td> Greiner Bio-One </td><td> 5678-1209 </td><td></td></tr><tr><td> Atmungsaktive Dichtband </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> Juglon ist in DMSO gelösten </td></tr><tr><td> DMSO </td><td> Sigma-Aldrich </td><td> D-5879 </td><td></td></tr><tr><td> C. elegans transgenen Stammes </td><td> Autor Labor </td><td> VP596 </td><td> Pgst-4:: GFP und PDOP-3:: RFP </td></tr></tbody></table>

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

High-Throughput-Screening (HTS) ist ein leistungsstarkes Konzept zur Identifizierung von chemischen Modulatoren der biologischen Prozessen. Allerdings sind viele Verbindungen in Bildschirmen mit Zellkultur-Modellen identifiziert oft, dass sie giftig oder pharmakologisch inaktiv in vivo 1-2. Screening in ganz Tiermodelle können zur Vermeidung dieser Gefahren und optimieren den Weg zur Entwicklung von Medikamenten. C. elegans ist ein vielzelligen Organismus-Modell auch für HTS geeignet. Es ist klein (&lt;1 mm) und kann wirtschaftlich gezüchtet werden und abgefüllt in Flüssigkeiten. C. elegans ist auch einer der experimentell handhabbar Tiermodelle erlauben eine schnelle und detaillierte Identifizierung von Wirkstoffkandidaten mode-of-Aktion 3. Wir beschreiben ein Protokoll für die Kultivierung und Abgabe fluoreszierender Stämme von C. elegans für das Hochdurchsatz-Screening von chemischen Bibliotheken oder den Nachweis von Umweltschadstoffen, dass die Expression eines bestimmten Gens zu verändern. Eine große Anzahl von entwicklungspolitisch synchronisiert Würmer sind in flüssiger Kultur angebaut, geerntet, gewaschen und aufgehängt an einer definierten Dichte. Würmer sind dann schwarz, mit flachem Boden 384-well-Platten mit einer peristaltischen Flüssigkeitsspender aufgenommen. Kleine Moleküle aus einer chemischen Bibliothek oder Proben (z. B. Wasser, Nahrung oder Boden) kann in die Vertiefungen mit Würmern hinzugefügt werden. In vivo Echtzeit-Fluoreszenz-Intensität mit einem Fluoreszenz-Mikroplatten-Reader gemessen. Diese Methode kann zu jeder induzierbaren Gens in C. angepasst werden elegans, für die eine geeignete Reporter zur Verfügung steht. Viele induzierbare Stress-und Entwicklungsbiologie transkriptionelle Wege sind gut in C definiert elegans und GFP transgenen Reporter-Stämme bereits für viele von ihnen 4 existieren. Wenn mit der entsprechenden transgenen Reporter kombiniert, können unsere Methode verwendet, um für die Bahn-Modulatoren Bildschirm oder robust Biosensor-Assays für Umweltschadstoffe zu entwickeln. Wir zeigen unseren C. elegans Kultur und Abgabe-Protokoll mit einem HTS-Assay wir entwickelt, um die C-Monitor elegans cap &#39;n&#39; Kragen Transkriptionsfaktor SKN-1. SKN-1 und seine Säuger Homolog Nrf2 aktivieren zytoprotektive Gene während der oxidative Stress und xenobiotische 5-10. Nrf2 schützt Säugetiere aus zahlreichen altersbedingten Erkrankungen wie Krebs, Neurodegeneration und chronischen Entzündungen und ist zu einem wichtigen chemotherapeutischen Ziel 11-13. Unser Test auf einem GFP transgenen Reporter für die SKN-1 Zielgen gst -4 14 basiert, die kodiert für ein Glutathion-S-Transferase 6. Die gst -4 Reporter ist auch ein Biosensor für Fremdstoff-und oxidative Chemikalien, die SKN-1 zu aktivieren und können verwendet werden, um niedrige Konzentrationen von Kontaminanten wie Acrylamid und Methyl-Quecksilber 15-16 zu erkennen.

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

Ein Verfahren zur Flüssig-Kultivierung und die Abgabe von<em&gt; C. elegans</em&gt; Stämme, fluoreszierenden Reporter-Proteinen wird beschrieben, dass keine teuren Sortieranlagen. Dieser Ansatz kann auf zahlreiche induzierbaren angewendet werden<em&gt; C. elegans</em&gt; Gene für die Wirkstoffforschung oder Biosensorik von Schadstoffen.

High-Throughput-Screening und Biosensorik mit Fluorescent C. elegans Die Stämme

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 Aufrufe.  University of Florida. Ein Verfahren zur Flüssig-Kultivierung und die Abgabe von C. elegans Stämme, fluoreszierenden Reporter-Proteinen wird beschrieben, dass keine teuren Sortieranlagen. Dieser Ansatz kann auf zahlreiche induzierbaren angewendet werden C. elegans Gene für die Wirkstoffforschung oder Biosensorik von Schadstoffen.

Serie: 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.

High Throughput Screening von Fungal Endoglucanaseaktivität in Escherichia coli</em

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

Forschung

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.

RNAi-Screening auf postembryonalen Phänotypen in Identifizieren 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.

Quantitative und automatisierte Hochdurchsatz-genomweiten RNAi-Screens in 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.

High-throughput Reinigung von Affinity-markierter rekombinanter Proteine

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.

High-throughput Screening niedermolekularer Modulatoren von Inward Gleichrichter-Kaliumkanäle

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.

C. elegans Chemotaxis-Assay

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.

Biochemische und High Throughput Mikroskopische Beurteilung der Fettmasse in<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.

Hochdurchsatz-Screening mit einem Funktionshomemade Dual-Luciferase Assay-Schein

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.

Hochdurchsatz-Screening für Chemische Modulatoren der Post-transkriptionell regulierten Genen

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.

Identifikation von Kinase-Substrat-Pairs Mit 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 ...