Questo lavoro è stato supportato da premio statunitense Public Health Service NS-071530.

1. Preparazione delle cellule <ol><li> Grow pituitari AtT20 cellule in terreno Dulbecco modificato Eagle (DMEM) supplementato con siero di cavallo 10% e mantenere colture a 37 ° C in atmosfera umidificata al 5% CO 2. </li><li> Mantenere la cultura Trapianto delle cellule ogni 5 a 7 giorni utilizzando una procedura standard tripsinizzazione. </li><li> Coat i pozzi di nero, fondo chiaro, piastre a 96 pozzetti con 50 pl di poli-L-lisina. Lasciare asciugare i pozzetti per 30 minuti in incubatrice. </li><li> Piastra le cellule nelle piastre a 96 pozzetti contenenti 200 pl mezzi a densità di 30.000 cellule per pozzetto e conservare le cellule in incubatore per 3-4 giorni. </li><li> Utilizzare una configurazione aspirazione, costituito da un tappo 4 barattolo l collegato ad un vuoto su una estremità e una punta di pipetta 200 pl all&#39;altra estremità, per aspirare lentamente il terreno di coltura del monostrato cellulare. </li><li> Lavare le cellule con 200 pl di soluzione tampone normale (132 mM NaCl, 5 mM KCl, 1 mM CaCl 2, 1 mM MgCl 2, destrosio 5 mM, HEPES 5 mM, pH 7,4 con NaOH). </li><li> Aggiungere 200 pl di soluzione tampone contenente il potenziale-colorante sensibile membrana (MPD) DiBAC 4 (3) o HLB 021-152 (5 pM) a ciascun pozzetto ed incubare le cellule per 40 min a 37 ° C. </li></ol> 2. Preparazione dei composti in esame e il trattamento con cellule <ol><li> Preparare una diluizione seriale di composti di prova (fotografia = 10 mM in DMSO) da una libreria composto (in formato a 96 pozzetti) utilizzando un (ThermoFisher) Versette sistema automatizzato di gestione dei liquidi. </li><li> Rimuovere la piastra a 96 pozzetti, contenente le cellule AtT20, dall&#39;incubatore e permettono la piastra di ritornare a temperatura ambiente. Aspirare il buffer sconto vecchio e applicare la soluzione tampone fresco contenente il MPD alle cellule. </li><li> Utilizzando il sistema di manipolazione di liquido applica varie concentrazioni (10 nM a 10 pM) dei composti di prova da 2 a 20 pl) e soluzioni di controllo (0,1% DMSO) (in soluzione tampone containing il MPD) alla piastra a 96 pozzetti contenenti le cellule. </li><li> Incubare le cellule per 5 min con composti di prova prima delle misurazioni fluorescenti. </li></ol> 3. Attivazione di canali GIRK, misurazione e analisi fluorescente <ol><li> Inserire la piastra a 96 pozzetti in un lettore di piastre Synergy2 Biotek fluorescente e ottenere il segnale fluorescente di fondo a lunghezze d&#39;onda di eccitazione e di emissione di 520 e 560 nm, rispettivamente. </li><li> Applicare i ligandi GPCR (somatostatina = 200 nM o carbacolo = 10 pM) o la soluzione di controllo (solo tampone) (20 pl) ad ogni pozzetto (volume totale = 220 mL) per attivare i canali GIRK utilizzando il sistema Synergy2 iniettore. Raccogliere punti dati a intervalli di 10 s 300 s per un periodo di campionamento a lunghezze d&#39;onda di eccitazione e di emissione di 520 e 560 nm, rispettivamente. </li><li> Ottenere il decorso e l&#39;ampiezza di picco del cambiamento fluorescente utilizzando Biotek Gen5 software ed esportare i dati in Excel per ulteriori analisi. </li&gt; <li> Calcolare il Z&#39;-fattore per determinare l&#39;affidabilità del saggio. Lastre utilizzate per la determinazione Z&#39;-factor contenuta 2 righe ciascuno di positivo (ligando GPCR) e negativi (solo tampone) controlli. Il Z&#39;-fattore è definito come: Z &#39;= 1 - (3σ P + 3σ N) / | P μ - N μ |, dove μ P &amp; μ N sono mezzi di controllo positivo e segnali di controllo negativi, e σ P &amp; N σ sono le deviazioni standard del controllo positivo e segnali di controllo negativo, rispettivamente 6. Z&#39;fattori nell&#39;intervallo da 0.5 a 1.0 indicare che la qualità del saggio è eccellente 6. Piastre con Z&#39;-fattori inferiori a 0,5 sono esclusi dall&#39;analisi. </li><li> Composti che modulano il segnale fluorescente sono riesaminati in presenza di Ba 2 + o tertiapin-Q per confermare attivo raddrizzatore K + specificità canale. </li></ol> 4. RRisultati epresentative Un esempio del segnale di fluorescenza misurata usando il saggio canale GIRK è mostrato in Figura 2. L&#39;aggiunta della somatostatina ligando GPCR (200 nM) per le cellule AtT20 causato una rapida, dipendente dal tempo diminuzione del segnale fluorescente 021-152 HLB (Figura 2). Al contrario, l&#39;aggiunta di soluzione di controllo prodotto un piccolo aumento istantaneo della fluorescenza che con il tempo restituito al basale (Figura 2). Confronto tra i valori di picco fluorescenti in piastre da 96 pozzetti iniettati con controllo e soluzioni somatostatina dato un Z&#39;fattori nell&#39;intervallo da 0,5 a 0,7. Per ulteriore quantificazione, il record di controllo è stato sottratto dal record somatostatina e la risultante somatostatina sensibile segnale analizzato (Figura 2). Il saggio fornisce un metodo per identificare rapidamente farmaci che modulano i canali GIRK. Ad esempio, i farmaci che inibiscono la cha GIRKnnel dovrebbe ridurre GPCR ligando-mediate diminuzioni della fluorescenza impedendo K + efflusso dalle cellule. Come mostrato in Figura 3, il trattamento delle cellule con tertiapin-Q, una tossina che blocca i canali GIRK 7, prodotto una inibizione del cambio somatostatina-mediata fluorescente. Propafenone, un farmaco antiaritmico che blocca i canali GIRK nel cuore 8, anche inibito il cambiamento fluorescente (Figura 4). Il dosaggio può anche essere utile per identificare attivatori del canale GIRK. Tuttavia, l&#39;applicazione di etanolo (100 e 200 mM), un attivatore del canale GIRK 2,3, causato alcun cambiamento significativo nel segnale fluorescente rispetto ai controlli soluzione (p&gt; 0,5). <img src="/files/ftp_upload/3850/3850fig1.jpg" alt="Figura 1"/> Figura 1. Disegno sperimentale del test GIRK canale fluorescente. AtT20 cellule sono incubate in tampone contenente un Membrane potenziale sensibile colorante fluorescente (D). Le molecole di colorante entrare nelle cellule e diventano fluorescenti sul legame alle proteine ​​intracellulari (pannello superiore). Il legame di somatostatina (Som) alla sua GPCR stimola la proteina G inibitoria (G i) provocando l&#39;attivazione del canale GIRK (pannello inferiore). L&#39;efflusso successiva K + dalle cellule provoca il potenziale di membrana a diventare più negative e le molecole di colorante per uscire le cellule. Come risultato diminuisce segnale fluorescente (pannello inferiore). <img src="/files/ftp_upload/3850/3850fig2.jpg" alt="Figura 2"/> Figura 2. Rappresentante HLB 021-152 segnale fluorescente misurato nel tempo nei AtT20 cellule durante l&#39;aggiunta di somatostatina o soluzione di controllo. Il rapporto tra l&#39;intensità della fluorescenza (F / F O) è stata calcolata dividendo il segnale in presenza (F) di somatostatina (o soluzione di controllo) dal segnale basale misurata prima (F O) annunciodizione della somatostatina (o soluzione di controllo). Ogni punto rappresenta la media ± SE ottenuta in 5-6 pozzetti. La somatostatina o soluzione di controllo è stata aggiunta al tempo zero (↓). Aggiunta della soluzione di controllo ha provocato un piccolo aumento transitorio del segnale fluorescente. Questo risultato di una variazione di temperatura causato dalla iniezione della soluzione. <img src="/files/ftp_upload/3850/3850fig3.jpg" alt="Figura 3"/> Figura 3. Trattamento delle cellule con AtT20 tertiapin-Q (500 nM) inibisce la somatostatina-mediata diminuzione del segnale fluorescente legandosi e bloccando i canali GIRK. Ogni punto rappresenta la media ± SE ottenuta in 4-6 pozzetti. La somatostatina è stato aggiunto al tempo zero (↓). <img src="/files/ftp_upload/3850/3850fig4.jpg" alt="Figura 4"/> Figura 4. Trattamento delle cellule con AtT20 propafenone (20 pM) inibisce anche la somatostatina-mediata diminuzioneil segnale fluorescente. Ogni punto rappresenta la media ± SE ottenuta in 5-6 pozzetti. La somatostatina è stato aggiunto al tempo zero (↓).

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Mentre il potenziale di membrana coloranti fluorescenti sensibili sono stati utilizzati per identificare i farmaci che modulano i canali ionici 9,10, questo è il primo rapporto della loro applicazione per la scoperta di farmaci neuronale GIRK canale. Il saggio GIRK canale fluorescente qui presentato fornisce un metodo rapido, affidabile e in tempo reale per lo screening del ligando-dipendenti canali del K +. Il dosaggio può essere modificato per l&#39;utilizzo con un&#39;ampia gamma di cellule, c...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> DMEM </td><td> Cellgro </td><td> 10-013 </td></tr><tr><td> Siero di cavallo </td><td> Invitrogen </td><td> 16050-114 </td></tr><tr><td> 96-pozzetti </td><td> Corning </td><td> 3603 </td></tr><tr><td> Poly-L-lisina </td><td> Sigma-Aldrich </td><td> P4707 </td></tr><tr><td> Somatostatina </td><td> Sigma-Aldrich </td><td> S9129 </td></tr><tr><td> Carbacolo </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 gestore automatizzata dei liquidi </td><td> ThermoFisher </td><td> 650-01 </td></tr><tr><td> Synergy2 lettore di piastre a fluorescenza </td><td> Biotek </td><td></td></tr><tr><td> Gen5 analisi software </td><td> Biotek </td><td></td></tr></tbody></table> Tabella 1. Tabella di reagenti e attrezzature specifiche.

a fluorescent screening assay for identifying modulators of girk channels

Proteina G-dipendenti verso l&#39;interno del raddrizzatore K + (GIRK) canali di agire come mediatori cellulari di una vasta gamma di ormoni e neurotrasmettitori e sono espressi nel cervello, cuore, muscolo scheletrico e del tessuto endocrino 1,2. GIRK canali si attivano in seguito il legame di ligandi (ormoni, neurotrasmettitori, farmaci, ecc) per la loro membrana plasmatica-bound, accoppiati a proteine ​​G recettori (GPCR). Tale legame determina la stimolazione delle proteine ​​G (G e G o i) che legano e successivamente attivare il canale GIRK. Una volta aperto il canale GIRK consente il movimento di K + fuori dalla cellula causando la membrana riposo potenziale di diventare più negativo. Di conseguenza, canale attivazione GIRK in neuroni diminuisce formazione spontanea potenziale di azione e inibisce il rilascio di neurotrasmettitori eccitatori. Nel cuore, l&#39;attivazione del canale GIRK inibisce l&#39;attività del pacemaker così rallentando la frequenza cardiaca. <pclass = &quot;&quot;&gt; jove_content canali GIRK rappresentano nuovi bersagli per lo sviluppo di nuovi agenti terapeutici per il trattamento del dolore neuropatico, tossicodipendenza, aritmie cardiache e altri disturbi 3. Tuttavia, la farmacologia di questi canali rimane in gran parte inesplorato. Sebbene un certo numero di farmaci anti-aritmici tra cui agenti, i farmaci antipsicotici e antidepressivi bloccare il canale GIRK, questa inibizione non è selettiva e si verifica a concentrazioni relativamente elevate di droga 3. Qui, noi descriviamo un tempo reale saggio di screening per identificare modulatori di nuovi canali GIRK. In questo saggio, cellule neuronali AtT20, che esprimono canali GIRK, vengono caricati con membrana coloranti fluorescenti sensibili potenziale come bis-(1,3-dibutylbarbituric acido) trimethine oxonol [DiBAC 4 (3)] o HLB 021-152 (Figura 1 ). Le molecole di colorante fluorescente diventano fortemente seguente captazione nelle cellule (Figura 1). Trattamentodelle cellule con leganti GPCR stimola i canali GIRK per aprire. La risultante K + efflusso fuori dalla cellula causa del potenziale di membrana a diventare più negativo e il segnale fluorescente per diminuire (Figura 1). Pertanto, i farmaci che modulano K + efflusso attraverso il canale GIRK può essere saggiata usando un lettore di piastra fluorescente. A differenza di altri saggi di screening dei canali ionici, quali spettrometria di assorbimento atomico o 4 radiotracciante analisi 5, il saggio GIRK canale fluorescente fornisce una procedura di screening rapido, in tempo reale e poco costoso.

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

A real-time procedura di screening per identificare i farmaci che interagiscono con la proteina G-gated verso l&#39;interno raddrizzatore K<sup&gt; +</sup&gt; (GIRK) canali viene descritto. Il test utilizza coloranti fluorescenti potenziale di membrana sensibili per misurare l&#39;attività dei canali GIRK. Questa tecnica è adattabile per l&#39;uso su un certo numero di linee cellulari.

Un test di screening a fluorescenza per l&#39;identificazione di modulatori Canali 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 ...

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Research

JoVE Journal

Biology

14.3K Views.  University of South Carolina, School of Medicine. A real-time procedura di screening per identificare i farmaci che interagiscono con la proteina G-gated verso l'interno raddrizzatore K + (GIRK) canali viene descritto. Il test utilizza coloranti fluorescenti potenziale di membrana sensibili per misurare l'attività dei canali GIRK. Questa tecnica è adattabile per l'uso su un certo numero di linee cellulari.

Video: 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.

Mutagenesi e Analisi funzionale dei canali ionici Heterologously Espresso in cellule di mammifero

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 ...

Ricerca

Biologia

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 e biopercezione con fluorescente C. elegans Ceppi

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.

Conversione di un ELISA Capture ad un saggio Luminex xMAP utilizzando un anticorpo Multiplex metodo di screening

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 per la piccola molecola modulatori di canali di potassio Inward Rectifier

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.

High-throughput screening funzionale utilizzando una casalinga 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 ...

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-base Espressione Proiezione di proteine ​​di membrana 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.

A Proteoliposome-Based Efflux Assay per determinare le proprietà singola molecola di Cl<sup&gt; -</sup&gt; Canali e trasportatori

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.

Barcoding genetica con proteine ​​fluorescenti per applicazioni multiplexati

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.

Valutazione del rene Zebrafish Funzione Con un test Clearance fluorescente

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.

Screening ad alto rendimento per i modulatori chimici di Genes post-trascrizionale regolamentati

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...