Diese Arbeit wurde vom US Public Health Service Award NS-071530 gefördert.

1. Herstellung von Zellen <ol><li> Wachsen Hypophyse AtT20-Zellen in Dulbeccos modifiziertem Eagle-Medium (DMEM) mit 10% Pferdeserum und aufrechtzuerhalten Kulturen bei 37 ° C in einer befeuchteten Atmosphäre von 5% CO 2 ergänzt. </li><li> Pflegen Sie die Kultur durch Subkultivierung der Zellen alle 5 bis 7 Tage mit einem Standard-Verfahren Trypsinierung. </li><li> Die Vertiefungen von schwarzen, klaren unten, 96-well Platten mit 50 ul von Poly-L-Lysin. Die Vertiefungen für 30 Minuten in den Inkubator zu trocknen. </li><li> Tafel die Zellen in den Platten mit 96 Vertiefungen, enthaltend 200 ul Medium bei einer Dichte von 30.000 Zellen pro Vertiefung und Lagerung der Zellen in dem Inkubator für 3-4 Tage. </li><li> Verwenden eine Aspiration Setup, bestehend aus einem verschlossenen 4-Liter-Flasche mit einer Vakuumquelle an einem Ende und einem 200 ul Pipettenspitze an dem anderen Ende, langsam absaugen Kulturmedium aus der Zellmonolayer. </li><li> Waschen Sie die Zellen mit 200 ul normalen Pufferlösung (132 mM NaCl, 5 mM KCl, 1 mM CaCl 2, 1 mM MgCl 2, 5 mM Dextrose, 5 mM HEPES, pH 7,4 mit NaOH). </li><li> Geben Sie 200 ul Puffer Lösung, die das Membranpotential-sensitiven Farbstoffs (MPD) DIBAC 4 (3) oder HLB 021-152 (5 pM) in jede Vertiefung und inkubieren Sie die Zellen für 40 min bei 37 ° C </li></ol> 2. Vorbereitung der Testverbindungen und Cell Treatment <ol><li> Bereiten Sie eine serielle Verdünnung der Testverbindungen (Standard = 10 mM in DMSO) aus einer Verbindung Bibliothek (in 96-Well-Format) mit einer Versette (ThermoFisher) automatisierte Liquid-Handling-System. </li><li> Entfernen Sie die 96-Well Platte, welche die AtT20 Zellen aus dem Inkubator und damit die Platte an die Raumtemperatur anpassen. Absaugen der alte Puffer und durch frisches Pufferlösung, die die MPD an die Zellen. </li><li> Mit der Liquid-Handling-System anwenden verschiedenen Konzentrationen (10 nM bis 10 pM) der Testverbindungen (2 bis 20 ul) und Control-Lösungen (0,1% DMSO) (in Pufferlösung containing der MPD) an die 96-Well-Platte mit den Zellen. </li><li> Inkubieren der Zellen für 5 min mit Testverbindungen vor der Fluoreszenz-Messungen. </li></ol> 3. Die Aktivierung von GIRK Kanäle, Neonlicht Messung und Analyse <ol><li> Legen Sie die 96-Well-Platte in eine Biotek Synergy2 fluoreszierenden Platte Leser und erhalten Sie die fluoreszierenden Hintergrund Signal an Anregungs-und Emissionswellenlänge von 520 und 560 nm auf. </li><li> Anwenden der GPCR-Liganden (Somatostatin = 200 nM oder Carbachol = 10 pM) oder Kontroll-Lösung (Puffer allein) (20 mL) in die Vertiefungen (Gesamtvolumen = 220 mL), um die Kanäle mit den GIRK Synergy2 Injektor zu aktivieren. Daten sammeln Punkte bei 10 s Intervallen über einen 300 s Abtastperiode bei Anregungs-und Emissionswellenlänge von 520 und 560 nm auf. </li><li> Besorgen Sie den zeitlichen Verlauf und Spitze-Amplitude des fluoreszierenden Änderung über Biotek Gen5 Software und exportieren Sie die Daten nach Excel zur weiteren Analyse. </li&gt; <li> Berechnen Sie die Z&#39;-Faktor, um die Zuverlässigkeit des Tests zu bestimmen. Platten für Z&#39;-Faktor Bestimmung verwendet wurde, enthielt zwei Reihen von jeweils positiv (GPCR-Liganden) und negativen (Puffer allein) Kontrollen. Die Z&#39;-Faktor ist definiert als: Z &#39;= 1 - (P + 3σ 3σ N) / | μ P - μ N |, wobei μ μ P &amp; N sind das Mittel der positive und negative Steuersignale und &amp;sgr; P &amp; · N sind die Standardabweichungen der positive und negative Steuersignale, bzw. 6. Z&#39;-Faktoren im Bereich von 0,5 bis 1,0 zeigen, dass die Qualität des Assays ausgezeichnete 6 ist. Platten mit Z&#39;-Faktoren unter 0,5 werden aus der Analyse ausgeschlossen. </li><li> Verbindungen, die die Fluoreszenz-Signal modulieren, werden in Gegenwart von Ba 2 + oder tertiapin-Q erneut getestet, um Einwärtsgleichrichter K +-Kanal Spezifität zu bestätigen. </li></ol> 4. Repräsentativer Ergebnisse Ein Beispiel des Fluoreszenzsignals unter Verwendung des GIRK Kanal Assay wird in 2 gezeigt. Die Zugabe des GPCR-Liganden Somatostatin (200 nM), um den AtT20-Zellen verursachte eine schnelle, zeitabhängige Abnahme der HLB 021-152 Fluoreszenzsignal (2). Im Gegensatz dazu erzeugte Zugabe von Kontrolllösung eine kleine schnelle Anstieg in der Fluoreszenz, die mit der Zeit auf die Grundlinie zurück (Abb. 2). Vergleich der Spitzenwert Fluoreszenzwerte in 96-Well-Platten mit Steuerung und Somatostatin-Lösungen injiziert hat eine Z&#39;-Faktoren im Bereich von 0,5 bis 0,7. Für weitere Quantifizierung wurde die Kontrolle Datensatz aus der Somatostatin-Rekord subtrahiert und der resultierende Somatostatin-und Kleinschreibung Signal analysiert (Abbildung 2). Der Test stellt ein Verfahren zur schnellen Identifizierung Medikamente, die GIRK Kanäle modulieren. Zum Beispiel Medikamente, die Hemmung der GIRK channel sollte GPCR-Liganden-vermittelte Abnahme der Fluoreszenz, indem verhindert wird K +-Ausstrom aus den Zellen zu reduzieren. Wie in 3, der Behandlung der Zellen mit tertiapin-Q, ein Toxin gezeigt, dass Blöcke GIRK Kanäle 7, die eine Hemmung der Somatostatin-vermittelte Fluoreszenz Änderung hergestellt. Propafenon, ein Antiarrhythmikum, dass die Blöcke GIRK Kanäle im Herzen 8, inhibierte auch den fluoreszierenden Änderung (Abbildung 4). Der Assay kann auch nützlich sein zur Identifizierung von Aktivatoren der GIRK Kanal. Die Anwendung dieser Ethanol (100 &amp; 200 mM), verursacht ein Aktivator des Kanals GIRK 2,3, keine signifikante Veränderung des Fluoreszenzsignals im Vergleich zur Lösung (p&gt; 0,5) zu steuern. <img src="/files/ftp_upload/3850/3850fig1.jpg" alt="1"/> Abbildung 1. Experimentelle Gestaltung des GIRK Kanal Fluoreszenz-Assay. AtT20-Zellen werden in einem Puffer mit einer Membran inkubierte Potential Fluoreszenzfarbstoff (D). Die Farbstoffmoleküle geben Sie in die Zellen und werden nach der Bindung an intrazelluläre Proteine ​​(oben) fluoreszierend. Die Bindung von Somatostatin (SOM) in seine GPCR stimuliert die inhibitorische Protein G (G i) bewirkt die Aktivierung des GIRK Kanal (unten). Die anschließende Efflux von K + aus der Zellen bewirkt, dass das Membranpotential weiter in den negativen und die Farbstoffmoleküle, um die Zellen zu verlassen. Als Ergebnis können die Fluoreszenzsignal abnimmt (unten). <img src="/files/ftp_upload/3850/3850fig2.jpg" alt="2"/> Abbildung 2. Vertreter HLB 021-152 Fluoreszenzsignal im Laufe der Zeit in den AtT20 Zellen während der Zugabe von Somatostatin-oder Kontroll-Lösung gemessen. Das Verhältnis der Fluoreszenzintensität (F / F O) wurde durch Teilen des Signals in Gegenwart (F) von Somatostatin (oder Kontroll-Lösung) von der Grundlinie Signal vor (F O) gemessen wird, berechnet addition von Somatostatin (oder Control-Lösung). Jeder Punkt stellt den Mittelwert ± SE in 5-6 Vertiefungen erhalten. Somatostatin oder Kontrolle wurde zum Zeitpunkt Null (↓) zugegeben. Die Zugabe des Kontrolllösung führte zu einer geringen vorübergehender Anstieg des Fluoreszenzsignals. Dies resultiert aus einer Temperaturänderung durch Injektion der Lösung verursacht. <img src="/files/ftp_upload/3850/3850fig3.jpg" alt="Abbildung 3"/> 3. Behandlung der Zellen mit AtT20 tertiapin-Q (500 nM) hemmt die Somatostatin-vermittelte Abnahme der Fluoreszenz-Signal durch Bindung an und blockiert die GIRK Kanäle. Jeder Punkt stellt den Mittelwert ± SE in 4-6 Brunnen erhalten. Somatostatin wurde zum Zeitpunkt Null (↓) zugegeben. <img src="/files/ftp_upload/3850/3850fig4.jpg" alt="Abbildung 4"/> Abbildung 4. Die Behandlung der Zellen mit AtT20 Propafenon (20 uM) hemmt auch die Somatostatin-vermittelten Abnahme derdas Fluoreszenzsignal. Jeder Punkt stellt den Mittelwert ± SE in 5-6 Vertiefungen erhalten. Somatostatin wurde zum Zeitpunkt Null (↓) zugegeben.

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Während Membranpotential Fluoreszenzfarbstoffe verwendet wurden, um Medikamente, die Ionenkanäle modulieren 9,10 zu identifizieren, ist dies der erste Bericht über ihre Anwendung für die neuronale GIRK Channel Drug Discovery. Die GIRK Kanal Fluoreszenz-Assay hier vorgestellten bietet eine schnelle, zuverlässige und Echtzeit-Methode für das Screening von Liganden-gesteuerte K +-Kanäle. Der Assay kann zur Verwendung mit einer Vielzahl von Zellen, einschließlich immortalisierten Zelllinien (HE...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> DMEM </td><td> CellGro </td><td> 10-013 </td></tr><tr><td> Pferdeserum </td><td> Invitrogen </td><td> 16050-114 </td></tr><tr><td> 96-Well-Platten </td><td> Corning </td><td> 3603 </td></tr><tr><td> Poly-L-Lysin </td><td> Sigma-Aldrich </td><td> P4707 </td></tr><tr><td> Somatostatin </td><td> Sigma-Aldrich </td><td> S9129 </td></tr><tr><td> Carbachol </td><td> Sigma-Aldrich </td><td> C4382 </td></tr><tr><td> HLB 021-152 </td><td> AnaSpec </td><td> 89300 </td></tr><tr><td> Versette automatisierten Liquid Handler </td><td> ThermoFisher </td><td> 650-01 </td></tr><tr><td> Synergy2 fluoreszierenden Platte Leser </td><td> Biotek </td><td></td></tr><tr><td> Gen5 Analyse software </td><td> Biotek </td><td></td></tr></tbody></table> Tabelle 1. Tabelle der spezifischen Reagenzien und Geräte.

a fluorescent screening assay for identifying modulators of girk channels

G-Protein-gesteuerten K + Einwärtsgleichrichter (GIRK) Kanäle fungieren als zelluläre Mediatoren einer Vielzahl von Hormonen und Neurotransmittern und im Gehirn, Herz, Skelettmuskel und endokrines Gewebe 1,2 ausgedrückt. GIRK Kanäle aktiviert werden nach der Bindung von Liganden (Neurotransmitter, Hormone, Arzneimittel, etc.), um die Plasma-Membran-gebundenen G-Protein-gekoppelte Rezeptoren (GPCRs). Diese Bindung bewirkt, dass die Stimulation des G-Proteine ​​(G i und G o), die anschließend zu binden und aktivieren Sie die GIRK Kanal. Nach dem Öffnen der GIRK Kanal erlaubt die Bewegung von K + aus der Zelle verursacht das Ruhemembranpotential zu mehr negativ. Als Folge davon sinkt GIRK Kanal-Aktivierung in Neuronen spontane Aktionspotential Bildung und hemmt die Freisetzung von exzitatorischen Neurotransmittern. Im Herzen, hemmt die Aktivierung des GIRK Kanal Schrittmacher-Aktivität dadurch eine Verlangsamung der Herzfrequenz. <pclass = &quot;jove_content&quot;&gt; GIRK Kanäle neuartigen Ansatzpunkt für die Entwicklung neuer Therapeutika für die Behandlung neuropathischer Schmerzen, Drogensucht, Herzrhythmusstörungen und anderen Erkrankungen 3. Allerdings bleibt die Pharmakologie dieser Kanäle weitgehend unerforscht. Obwohl eine Reihe von Medikamenten, einschließlich Antiarrhythmika, Antipsychotika und Antidepressiva die GIRK Kanal blockieren, ist diese Hemmung nicht selektiv und bei relativ hohen Wirkstoffkonzentrationen 3. Hier beschreiben wir eine Echtzeit-Screening-Test zur Identifizierung neuer Modulatoren von GIRK Kanäle. In diesem Assay werden neuronalen AtT20-Zellen, die GIRK Kanäle mit Membranpotential Fluoreszenzfarbstoffe, wie Bis-(1,3-dibutylbarbituric Säure) geladen Trimethin Oxonol [Dibac 4 (3)] oder HLB 021-152 (1 ). Die Farbstoffmoleküle werden stark fluoreszierende folgende Aufnahme in die Zellen (Abbildung 1). Behandlungder Zellen mit GPCR-Liganden stimuliert die GIRK Kanäle zu öffnen. Das resultierende K +-Ausstrom aus der Zelle bewirkt, dass das Membranpotential zu werden negativ und das Fluoreszenzsignal zu verringern (Abbildung 1). So können Medikamente, die K +-Ausstrom modulieren durch den Kanal GIRK untersucht mit Hilfe eines Fluoreszenz-Plattenlesegerät werden. Im Gegensatz zu anderen Ionenkanal-Screening-Assays, wie Atomabsorptionsspektrometrie 4 oder Radiotracer Analyse 5 stellt die GIRK Kanal Fluoreszenz-Assay einen schnellen, in Echtzeit und kostengünstige Screening-Verfahren.

Watch this Scientific Journal Video about A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels at JoVE.com

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

Ein Echtzeit-Screening-Verfahren zur Identifizierung von Medikamenten, die mit G-Protein-gated Einwärtsgleichrichter K interagieren<sup&gt; +</sup&gt; (GIRK) Kanälen beschrieben. Der Test basiert Membranpotential Fluoreszenzfarbstoffe zu GIRK Kanal-Aktivität zu messen. Diese Technik eignet sich sowohl für den Einsatz auf einer Reihe von Zelllinien.

Ein fluoreszierendes Screening Assay zur Identifizierung von Modulatoren der GIRK Kanäle

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

a-fluorescent-screening-assay-for-identifying-modulators-girk

Research

JoVE Journal

Biology

14.3K Aufrufe.  University of South Carolina, School of Medicine. Ein Echtzeit-Screening-Verfahren zur Identifizierung von Medikamenten, die mit G-Protein-gated Einwärtsgleichrichter K interagieren + (GIRK) Kanälen beschrieben. Der Test basiert Membranpotential Fluoreszenzfarbstoffe zu GIRK Kanal-Aktivität zu messen. Diese Technik eignet sich sowohl für den Einsatz auf einer Reihe von Zelllinien.

Serie: A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

Mutagenese und funktionale Analyse von Ionenkanälen heterolog in Säugerzellen exprimierten

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Forschung

Biologie

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.

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 ...

Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life science research and clinical diagnostics. However, ELISA has limitations. It is typically performed in a 96-well microplate, and the wells are coated with capture antibody, requiring a relatively large amount of sample to capture an antigen of interest . The large surface area of the wells and the hydrophobic binding of capture antibody can also lead to non-specific binding and increased background. Additionally, most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity. Such amplification is not always linear and can thus skew results.
In the past 15 years, a new technology has emerged that offers the benefits of the ELISA, but also enables higher throughput, increased flexibility, reduced sample volume, and lower cost, with a similar workflow 1, 2. Luminex xMAP Technology is a microsphere (bead) array platform enabling both monoplex and multiplex assays that can be applied to both protein and nucleic acid applications 3-5. The beads have the capture antibody covalently immobilized on a smaller surface area, requiring less capture antibody and smaller sample volumes, compared to ELISA, and non-specific binding is significantly reduced. Smaller sample volumes are important when working with limiting samples such as cerebrospinal fluid, synovial fluid, etc. 6. Multiplexing the assay further reduces sample volume requirements, enabling multiple results from a single sample.
Recent improvements by Luminex include: the new MAGPIX system, a smaller, less expensive, easier-to-use analyzer; Low-Concentration Magnetic MagPlex Microspheres which eliminate the need for expensive filter plates and come in a working concentration better suited for assay development and low-throughput applications; and the xMAP Antibody Coupling (AbC) Kit, which includes a protocol, reagents, and consumables necessary for coupling beads to the capture antibody of interest. (See Materials section for a detailed list of kit contents.)
In this experiment, we convert a pre-optimized ELISA assay for TNF-alpha cytokine to the xMAP platform and compare the performance of the two methods 7-11. TNF-alpha is a biomarker used in the measurement of inflammatory responses in patients with autoimmune disorders.
We begin by coupling four candidate capture antibodies to four different microsphere sets or regions. When mixed together, these four sets allow for the simultaneous testing of all four candidates with four separate detection antibodies to determine the best antibody pair, saving reagents, sample and time. Two xMAP assays are then constructed with the two most optimal antibody pairs and their performance is compared to that of the original ELISA assay in regards to signal strength, dynamic range, and sensitivity.

An ELISA can be easily converted to a Luminex xMAP assay and, through the benefits of multiplexing, several antibodies can be screened simultaneously to identify an optimum antibody pair, resulting in increased sensitivity and dynamic range, while reducing assay cost.

Umbau eines Capture-ELISA zu einem Luminex xMAP Assay unter Verwendung eines Multiplex-Antikörper-Screening-Methode

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life ...

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, ...

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 ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

Green Fluorescent Protein-basierte Screening Expression von Membranproteinen in<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as from eukaryotes. This increase has been largely driven by the successful push to obtain structural information on membrane proteins. However, the ability to functionally interrogate these proteins has not advanced at the same rate and is often limited to qualitative assays of limited quantitative value, thereby limiting the mechanistic insights that they can provide. An assay to quantitatively investigate the transport activity of reconstituted Cl- channels or transporters is described. The assay is based on the measure of the efflux rate of Cl- from proteoliposomes following the addition of the K+ ionophore valinomycin to shunt the membrane potential. An ion sensitive electrode is used to follow the time-course of ion efflux from proteoliposomes reconstituted with the desired protein. The method is highly suited for mechanistic studies, as it allows for the quantitative determination of key properties of the reconstituted protein, such as its unitary transport rate, the fraction of active protein and the molecular mass of the functional unit. The assay can also be utilized to determine the effect of small molecule compounds that directly inhibit/activate the reconstituted protein, as well as to test the modulatory effects of the membrane composition or lipid-modifying reagents. Where possible, direct comparison between results obtained using this method were found to be in good agreement with those obtained using electrophysiological approaches. The technique is illustrated using CLC-ec1, a CLC-type H+/Cl- exchanger, as a model system. The efflux assay can be utilized to study any Cl- conducting channel/transporter and, with minimal changes, can be adapted to study any ion-transporting protein.

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

Proteoliposomes are used to study purified channels and transporters reconstituted in a well-defined biochemical environment. An experimental procedure to measure efflux mediated by these proteins is illustrated. The steps to prepare proteoliposomes, perform the recordings, and analyze data to quantitatively determine the functional properties of the reconstituted protein are described.

Ein Proteoliposom Basierte Efflux-Assay auf Einzelmoleküleigenschaften von Cl Bestimmen<sup&gt; -</sup&gt; Kanäle und Transporter

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as ...

Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery of fluorescent proteins and their genes have enabled the engineering of protein fusions for localization, the analysis of transcriptional activation and translation of proteins of interest, or the general tracking of individual cells and cell populations. The use of fluorescent protein genes in combination with retroviral technology has further allowed the expression of these proteins in mammalian cells in a stable and reliable manner. Shown here is how one can utilize these genes to give cells within a population of cells their own biosignature. As the biosignature is achieved with retroviral technology, cells are barcoded &#180;indefinitely&#180;. As such, they can be individually tracked within a mixture of barcoded cells and utilized in more complex biological applications. The tracking of distinct populations in a mixture of cells is ideal for multiplexed applications such as discovery of drugs against a multitude of targets or the activation profile of different promoters. The protocol describes how to elegantly develop and amplify barcoded mammalian cells with distinct genetic fluorescent markers, and how to use several markers at once or one marker at different intensities. Finally, the protocol describes how the cells can be further utilized in combination with cell-based assays to increase the power of analysis through multiplexing.

Since the discovery of the green fluorescent protein gene, fluorescent proteins have impacted molecular cell biology. This protocol describes how expression of distinct fluorescent proteins through genetic engineering is used for barcoding individual cells. The procedure enables tracking distinct populations in a cell mixture, which is ideal for multiplexed applications.

Genetische Barcoding mit Fluoreszierende Proteine ​​für Multiplex-Anwendungen

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery ...

Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish kidney develops and functions in almost the same way as humans. A major difference in the construction of the human kidney is the presence of millions of nephrons compared to the zebrafish that has only two. However, simplifying such a complex system into basic functional units has aided our understanding of how the kidney develops and operates. In zebrafish, the midline located glomerulus is responsible for the initial blood filtration into two pronephric tubules that diverge to run bilaterally down the embryonic axis before fusing to each other at the cloaca. The pronephric tubules are heavily populated by motile cilia that facilitate the movement of filtrate along the segmented tubule, allowing the exchange of various solutes before finally exiting via the cloaca2-4. Many genes responsible for CKD, including those related to ciliogenesis, have been studied in zebrafish5. However, a major draw back has been the difficulty in evaluating zebrafish kidney function after genetic manipulation. Traditional assays to measure kidney dysfunction in humans have proved non translational to zebrafish, mainly due to their aquatic environment and small size. For example, it is not physically possible to extract blood from embryonic staged fish for analysis of urea and creatinine content, as they are too small. In addition, zebrafish do not produce enough urine for testing on a simple proteinuria &#8216;dipstick&#8217;, which is often performed during initial patient examinations. We describe a fluorescent assay that utilizes the optical transparency of the zebrafish to quantitatively monitor the clearance of a fluorescent dye, over time, from the vasculature and out through the kidney, to give a read out of renal function1,6-9.

The zebrafish is a popular tool to model chronic kidney disease (CKD). However, their small size makes it impossible to evaluate renal function using traditional methods. We describe a fluorescent dye kidney clearance assay1 that allows quantitative analysis of zebrafish kidney function in CKD.

Bewertung der Zebrafisch Nierenfunktion unter Verwendung einer fluoreszierenden Räumungs Assay

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish ...

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 ...