Questo lavoro è stato finanziato dal NIH assegni di ricerca K01AR053210, R01AR061484 e una borsa di National Psoriasis Foundation.

1. Informazioni di base Il sistema del doppio ibrido di lievito è una potente tecnica genetica utilizzata per scoprire interazioni proteina-proteina 16, 17. Diversi tipi di 2-sistemi ibridi, come i sistemi basati su lexa, il sistema di reclutamento Sos, e batteri o cellule di mammifero a base di 2-ibridi, sono commerciali disponibili, questo articolo si concentra in particolare sulle modifiche dei più comunemente usati GAL4 base lievito 2-sistema ibrido. In breve, il metodo si basa sulle proprietà della proteina GAL4 lievito che si compone di domini separabili responsabile per l&#39;attivazione del DNA-binding e trascrizionale. La proteina esca è espresso come una fusione a DNA-binding GAL4 dominio (DNA-BD), mentre le proteine ​​prede sono espressi come fusioni al dominio GAL4 attivazione (AD). L&#39;interazione tra esca e proteine ​​di fusione preda porta alla attivazione trascrizionale di GAL4 siti di legame contenente geni reporter che sono integrati nella g di lievitoenome. Il principio di Y2H è illustrato nella fig. 1 e la procedura sperimentale è sintetizzato nella fig. 2. 2. Materiali necessari e le soluzioni <ol><li> La crescita media YPD (una miscela di peptone, estratto di lievito, destrosio e in proporzioni ottimali per la coltivazione maggior parte dei ceppi Saccharomyces cerevisiae). </li><li> Minime basi SD (Minimal sintetico definito (SD) basi includono una base di azoto lievito, solfato di ammonio, e una fonte di carbonio, destrosio. Dropout (DO), gli integratori possono essere aggiunti alla base minimale SD per fare una sintesi, un terreno specifico manca specificato nutrienti). </li><li> Leu / Trp-Dropout (DO) supplemento (che contiene ogni amminoacido essenziale ad eccezione di leucina e triptofano) </li><li> Dropout -His/-Leu/-Trp/-Ura (DO) Supplemento (contenente ogni amminoacido essenziale tranne che per la leucina, triptofano, istidina, e uracile) </li><li> Luria brodo (LB) (Triptone 10 g / L, estratto di lievito 5 g / l, NaCl 5 g / L) </li><li> X-Gal (5-bromo-4-cloro-3-indolil-β-D-galattopiranoside) soluzione: pronta come una dose di 20 mg / ml di soluzione concentrata in N, N-Dimetilformammide </li><li> TE 10X (100 mM Tris-HCl (pH 7,5), 10 mM EDTA, autoclavato) </li><li> Herring sonicato o sperma DNA di salmone, bolliti (10 mg / ml) </li><li> LIAC 10X (1 acetato di litio M, autoclavato) </li><li> 50% PEG-3350 la soluzione, sterilizzata per filtrazione </li><li> 3-Amino-1, 2, 4-triazolo (3AT) </li><li> Kanamicina </li><li> Ampicillina </li><li> ELECTROMAX DH5α cellule </li><li> Z buffer: 16,1 g Na2HPO4 7H2O (o 8,52 g anidro), 5,5 g NaH2PO4 H2O (pari al 4,8 ganhydrous), 0,75 g di KCl, 0,246 g MgSO4 7H2O (o 0,12 g anidro), sciolto in 1 L autoclave, acqua distillata e regolato pH 7,0. </li></ol> 3. Bait generazione (pDBLeu-PGRN) Un frammento di cDNA PGRN codifica manca peptide segnale (aa21-588) è stato clonato direzionale in I-Sal non ho siti del vettore pDBLeu (ProQuest il sistema del doppio ibrido, Invitrogen),mantenendo lo stesso frame di lettura traduzione come dominio di GAL4 DNA Binding per generare pDBLeu-PGRN. <ol><li> Amplificare il frammento di cDNA PGRN sopra descritti mediante PCR utilizzando primer oligonucleotidi progettati per contenere siti di restrizione (Sal io al 5&#39;end e non io a 3&#39;end) per consentire in-frame di fusione. </li><li> Gel purificare il prodotto di PCR, digestione con endonucleasi di restrizione Not I e Sal I. Preparare il DB vettore pDBLeu dalla digestione di restrizione doppio con enzimi di restrizione stessa. </li><li> Legare il frammento PGRN restrizione nel pDBLeu vettore linearizzato e trasformare in DH5α con selezione per LB + kanamicina a 25 mcg / ml. </li><li> Verificare la correzione del costrutto di sequenziamento del DNA. </li></ol> 4. Piccola scala di trasformazione di esche plasmide <ol><li> Inoculare 5 ml di YPD con una colonia di Mav203 (Invitrogen), agitare una notte a 30 ° C. </li><li> Diluire la cultura durante la notte in 50 ml di YPD. Crescere un additional 2-4 ore. </li><li> Agglomerare le cellule a 3000 rpm per 5 minuti a temperatura ambiente. Risospendere il pellet in 40 ml di autoclave, acqua distillata. </li><li> Re-agglomerare le cellule. Risospendere in 2 ml di soluzione di I, incubare a RT 10 min. Soluzione I: 10xLiAc 0,5 ml, 0,5 ml 10xTE, 4 ml di H 2 O </li><li> Dispensare il DNA per tubi: 2-3 ml di pDBLeu-PGRN (0.25μg/μl) e 10 ml di denaturato, il DNA degli spermatozoi di salmone tranciati (10μg/μl). Aggiungere 100μl cellule di lievito, mescolare bene. </li><li> Aggiungere 700μl soluzione II, mescolare bene. Incubare a 30 ° C per 30 min. Soluzione II: 0,2 ml 10xLiAc, 10xTE 0,2 ml, 1,6 ml, 50% PEG3350. </li><li> Shock termico a 42 ° C per 15 min. </li><li> Pellet per 2 min. Scartare il surnatante. Risospendere il pellet in 200 l autoclave, acqua distillata. </li><li> Piatto la sospensione su SD-Leu piatti con diluizioni seriali. Incubare la piastra a 30 ° C per 2-3 giorni. </li></ol> 5. Bait convalida Prima di eseguire sì° schermo doppio ibrido, test pDBLeu-PGRN per l&#39;auto-attivazione e determinare i livelli basali di espressione del gene HIS3 reporter. Questo test determina se le esche attivare la trascrizione e se l&#39;auto-attivazione può essere neutralizzato da inibitori. 3-Amino-1, 2, 4-triazolo (3-AT) è un inibitore competitivo del HIS3 gene-prodotto e può essere utilizzato per titolare il livello minimo di espressione HIS3 necessari per la crescita di istidina carenti di mezzi di comunicazione. <ol><li> Trasformare il pDBLeu-PGRN in Mav203 ceppo di lievito, la piastra di trasformazione su SD-Leu piastre e incubare per 48-72 ore a 30 ° C. </li><li> Colonie patch di ogni trasformazione con stuzzicadenti autoclave su SD-Leu-His piastre contenenti 3-IN a concentrazioni di 10 mm, 25 mm, 50 mm, 75 mm e 100 mm. 3-AT è un inibitore competitivo dell&#39;enzima HIS3 ed esche solo che mostrano auto-attivazione sarà in grado di crescere in presenza di 3-AT. </li><li> Ceppi esca che crescono sulle piastre containing 100 mM 3-AT non sono adatti per l&#39;utilizzo in doppio ibrido schermo. Per le esche che possono essere utilizzati, utilizzare la più bassa concentrazione di 3-A che inibisce la crescita cellulare. In molti casi, 25 mm di 3-AT è usato per lo screening. </li></ol> 6. biblioteca di screening di cDNA Il plasmide pDBLeu-PGRN viene introdotto in MaV203 utilizzando una piccola trasformazione come descritto sopra. Per introdurre pPC86-library (Invitrogen) in MaV203 (pDBLeu-PGRN), la procedura descritta di seguito dà tipicamente ~ 4 x 10 4 colonie con 0,5 mg di DNA plasmidico biblioteca. Quindi, 2,5 x 10 6 trasformanti lievito richiederà ~ 30,0 mcg pPC86-cDNA DNA biblioteca plasmide, 25 trasformazioni, e cinquanta piastre da 10 cm (SD-Leu-Trp-His-Ura 3 AT). <ol><li> Preparare il numero appropriato di 10 cm di SD-Leu-Trp-His-Ura +3 al piatti (50 per la procedura di seguito). Anche preparare almeno quattro di 10 cm SD-Leu-Trp piastre per stimare il numero di trasformanti. </li><li> Trasformare la libreria inil MaV203 contenente pDBLeu-PGRN secondo la procedura descritta di seguito. <ol><li> Sospendere diverse colonie isolate di MaV203 (pDBLeu-PGRN) in ~ 100μl autoclave, acqua distillata e diffonderle su una di 10 cm SD-Leu piatto. Ripetere la procedura per un secondo SD-Leu piatto. Incubare entrambe le piastre per 18-24 ore a 30 ° C. </li><li> Raschiare e sospendere completamente le cellule in 10 ml in autoclave, acqua distillata. Aggiungere un volume sufficiente di sospensione cellulare a 500 ml di liquido di media YPD in un pallone per dare un OD 600 del ~ 0.1. </li><li> Verificare che l&#39;OD è ~ 0.1 dopo l&#39;inoculazione. </li><li> Agitare a 30 ° C fino a quando il diametro esterno raggiunge i 600 ~ 0.4. </li><li> Preparare freschi: <ol type="a"><li> 110 ml 1X TE / LIAC unendo 11 ml 10X TE, 11 LIAC ml 10X, e 88 ml di acqua in autoclave. </li><li> 16 ml PEG / LIAC combinando 1,6 ml 10X TE, 1,6 ml LIAC 10X, e 12,8 ml al 50% PEG-3350. </li></ol></li><li> Agglomerare le cellule a 3000g per 5 minuti a temperatura ambiente. </li><li> Scartare tha surnatanti e delicatamente risospendere il pellet pipettando su e giù in 100 ml in autoclave, acqua distillata a temperatura ambiente. </li><li> Centrifugare a 3000 g per 5 minuti a temperatura ambiente. </li><li> Eliminare il surnatante delle cellule centrifugato e risospendere il pellet cellulare in 50 ml 1X TE / LIAC soluzione. </li><li> Centrifugare a 3000 g per 5 minuti a temperatura ambiente. </li><li> Rimuovere il surnatante e risospendere il pellet in un volume finale di 2,5 ml 1X TE / LIAC soluzione. </li><li> Eseguire 25 trasformazioni. Unire celle 2,5 ml, 125 microlitri (10μg/μl) denaturati, tranciate DNA dello sperma di salmone, e 30 mcg biblioteca di cDNA. Mescolare delicatamente pipettando su e giù. Aggiungere 15 ml di PEG / LIAC soluzione e mescolare delicatamente. Aliquota in 25 autoclave da 1,5 ml tubi microcentrifuga di 700 microlitri ciascuno. </li><li> Incubare per 30 minuti in un bagno d&#39;acqua 30 ° C. </li><li> Shock termico per 15 minuti in un bagno d&#39;acqua ° 42 C. </li><li> Centrifugare a 6.000 g per 1 minuto a temperatura ambiente. Rimuovere con attenzione il supersupernatante. Risospendere delicatamente ciascun pellet in 400 microlitri in autoclave, acqua distillata pipettando su e giù. </li></ol></li><li> Piastra da 200 microlitri miscela trasformazione da ogni trasformazione sul singolo di 10 cm SD-Leu-Trp-His-Ura 3 AT (3AT concentrazione: 25 mm) con piastre di una barra di diffusione, quindi 25 in autoclave da 1,5 ml, tubi microcentrifuga può fare 50 piastre (SD-Leu-Trp-His-Ura 3 AT). </li><li> Incubare le piastre per 5-10 giorni a 30 ° C. </li><li> Per stimare l&#39;efficienza della trasformazione della reazione, diluizioni seriali di una reazione (1:40, 1:400 e 1:4000) sono placcati su SD-Leu-Trp piatto. Dopo incubazione per 3 giorni a 30 ° C, il numero di colonie viene contato e il numero totale di trasformanti calcolato. </li></ol> 7. X-gal assay <ol><li> Trasferimento colonie alla piastra YPD; incubare a 30 ° C durante la notte. </li><li> Luogo arrotondati membrana di nitrocellulosa sul piatto YPD, assicurarsi che non vi siano bolle tra la membrana e la piastra YPD. Dopo 1-2 minuti, macerto ke tutte le colonie vengono trasferite su membrane (carta di nitrocellulosa). </li><li> Posizionare il lato colonia membrana in barca fatta di carta foglio di alluminio. </li><li> Mettere la membrana in azoto liquido, attendere 20 secondi, e sommergere in azoto liquido per un paio di minuti. </li><li> Estrarre membrana a scongelare. </li><li> Nel frattempo mescolare il reagente seguenti: 1,5 ml di buffer Z 20μl X-gal (20 mg / ml) </li><li> Caduta Z buffer / X-gal in una capsula di Petri, luogo di carta Whatman per di più. </li><li> Con attenzione la carta di nitrocellulosa posto sopra la carta Whatman. </li><li> Incubare a 37 ° fino a quando le colonie blu appaiono. </li></ol> 8. Ritrasformazione test Proteine ​​di fusione Prey (AD-Y) isolato da uno screening della libreria dovrebbe mantenere l&#39;interazione con proteine ​​di fusione esca (DB-X) per indurre i geni relazione e la ritrasformazione di cloni prede ed esche costruire nel lievito può inoltre eliminare i falsi positivi e facilitare aggiungereanalisi itional. <ol><li> Isolare il DNA plasmidico da ceppi di lievito che possono contenere proteine ​​che interagiscono. </li><li> Trasforma DNA in E. coli DH5α cellule, piastra le trasformazioni su LB 100 mcg / ml piastre ampicillina per isolare selettivamente i pPC86-cDNA biblioteca, e incubare una notte a 37 ° C. </li><li> Raccogliere numerose colonie dalla piastra di mettere in LB 100 mcg / ml brodo ampicillina. </li><li> Preparare miniprep DNA ed esaminare l&#39;analisi di restrizione doppia con Sal io e non io </li><li> Co-trasformare la preda plasmide e esca plasmide in MaV203 la targhetta del miscele di trasformazione su SC-Leu-Trp piastre e incubare per 2-3 giorni a 30 ° C. </li><li> Scegli tre colonie di diverso da SC-Leu-Trp piastra, eseguire X-gal saggio. </li></ol> 9. Sequenziamento e l&#39;analisi bioinformatica Sequenza del DNA plasmide che è stato isolato da probabile cloni veri positivi, confrontare queste sequenze a quelle del GenBank utilizzando il BLAST programma, e identificare i due cloni ibridi che corrispondono a geni noti. Dati di sequenziamento ha dimostrato che due dei 12 positivi ottenuti in precedenza erano superficie cellulare TNFR2 (TNFRSF1B/CD120b; adesione # NM_130426). Inoltre, l&#39;interazione tra PGRN e TNFR è stata verificata utilizzando varie proteina-proteina saggi di interazione, anche in vitro in fase solida test vincolante, Co-immunoprecipitazione, Surface Plasmon analisi di risonanza, e citometria a flusso saggio 14. 10. Rappresentante Risultati Il diagramma di flusso dello screening è indicato nella fig. 3. Tipicamente 50-100 candidati clone positivo si ottiene in questa fase. Inizialmente abbiamo isolato 54 candidati clone positivo tra 2,5 milioni trasformanti screening con l&#39;esca PGRN. Candidati clone positivi sono stati poi verificati eseguendo X-gal test. Tipicamente, circa il 50% dei cloni falsi positivi vengono rimossi tramite X-gal test. Abbiamo ottenuto 23 clone positivo candida<html> Ates a questo passaggio per l&#39;esca PGRN (Fig. 4). Ritrasformazione dei cloni preda e costruire esche in lieviti ulteriormente eliminare i falsi positivi e cloni che ancora attivare i geni giornalista probabilmente rappresentano l&#39;veri positivi. Tipicamente, circa il 50% dei candidati clone positivo verrà rimosso. Abbiamo finalmente isolati 12 cloni positivi che interagiscono con PGRN nel lievito. <img alt="Figura 1" src="/files/ftp_upload/3562/3562fig1.jpg" /> Figura 1. Principio di lievito sistema di doppio ibrido <img alt="Figura 2" src="/files/ftp_upload/3562/3562fig2.jpg" /> Figura 2. Pipeline di identificazione di partner di legame con le proteine ​​utilizzando lievito sistema del doppio ibrido <img alt="Figura 3" src="/files/ftp_upload/3562/3562fig3.jpg" /> Figura 3. Diagramma di flusso di lievito di screening a due ibridi biblioteca.rge.jpg &quot;target =&quot; _blank &quot;&gt; Clicca qui per vedere una versione full-size di questa immagine. <img alt="Figura 4" src="/files/ftp_upload/3562/3562fig4.jpg" /> Figura 4. Beta-galattosidasi saggio di candidati clone positivo. A, cloni positivi ottenuti dallo schermo della libreria sono stati trasferiti alla piastra YPD e incubato a 30 ° C durante la notte; B, tutte le colonie su piastra YPD sono stati trasferiti su membrane di nitrocellulosa e beta-galattosidasi test eseguiti.

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Doppio ibrido di lievito di screening ha dimostrato di essere uno strumento efficace per identificare interazioni proteina 16, 17. Rispetto ad altri approcci per identificare le proteine ​​di legame partner, come la biochimica di co-purificazione e proteine ​​chip, lievito sistema del doppio ibrido è un approccio sensibile genetico che può essere utilizzato per lo screening numero molto elevato di sequenze codificanti in un esperimento relativamente semplice, in Inoltre, rileva l&#39;interazione in ...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> ProQuest sistema del doppio ibrido </td><td> Invitrogen </td><td> 10835 </td></tr><tr><td> YPD crescita media </td><td> Clontech </td><td> 630409 </td></tr><tr><td> YPD AGAR </td><td> Clontech </td><td> 630410 </td></tr><tr><td> Minimo SD agar Base </td><td> Clontech </td><td> 630412 </td></tr><tr><td> Leu-DO Supplemento </td><td> Clontech </td><td> 630414 </td></tr><tr><td> -Leu/-Trp DO Supplemento </td><td> Clontech </td><td> 630417 </td></tr><tr><td> -His/-Leu/-Trp/-Ura DO Supplemento </td><td> Clontech </td><td> 630425 </td></tr><tr><td> 3-Amino-1 ,2,4-triazolo (3AT) </td><td> Sigma-Aldrich </td><td> A8056 </td></tr><tr><td> Luria brodo (LB) </td><td> Sigma-Aldrich </td><td> L3022 </td></tr><tr><td> X-Gal </td><td> Invitrogen </td><td> 15520-034 </td></tr><tr><td> Sonicato Salmon Sperm DNA </td><td> Stratagene </td><td> 201190 </td></tr><tr><td> Ampicillina </td><td> AMRESCO </td><td> 0339 </td></tr><tr><td> Kanamicina solfato </td><td> Invitrogen </td><td> 11815-024 </td></tr><tr><td> Efficienza subcloning ™ DH5α ™ cellule competenti </td><td> Invitrogen </td><td> 18265-017 </td></tr><tr><td> Trizma ® di base </td><td> Sigma-Aldrich </td><td> T6066 </td></tr><tr><td> Acetato di litio </td><td> Sigma-Aldrich </td><td> L4158 </td></tr><tr><td> Etilendiammintetraacetato </td><td> Sigma-Aldrich </td><td> ED </td></tr><tr><td> Membrana di nitrocellulosa </td><td> Bio-Rad </td><td> 162-0115 </td></tr><tr><td> 10-cm scatola di Petri </td><td> ITI scientifico </td><td> CT-903 </td></tr><tr><td> Incubatore (30 ° C) </td><td> ATR (Ecotron) </td><td></td></tr></table>

modified yeast-two-hybrid system to identify proteins interacting with the growth factor progranulin

Progranulina (PGRN), noto anche come precursore granulin epithelin (GEP), è un fattore 593-amino-acido crescita autocrino. PGRN è nota per svolgere un ruolo critico in una varietà di processi fisiologici e malattie, tra cui l&#39;embriogenesi precoce, la guarigione della ferita 1, infiammazioni 2, 3, e ospitare difesa 4. PGRN funziona anche come un fattore neurotrofico 5, e le mutazioni nel gene PGRN con conseguente perdita parziale della proteina causano demenza frontotemporale PGRN 6, 7. I nostri studi recenti hanno portato all&#39;isolamento di PGRN come un importante regolatore di sviluppo della cartilagine e del degrado 8-11. Anche se PGRN, scoperto quasi due decenni fa, gioca un ruolo cruciale in molteplici condizioni fisiologiche e patologiche, gli sforzi per sfruttare le azioni di PGRN e comprendere i meccanismi coinvolti sono stati significativamente ostacolata dalla nostra incapacità di identificare il suo legame al recettore (s). Per risolvere questo problema, abbiamo sviluppato un modicato doppio ibrido di lievito (MY2H) approccio basato sulla più comunemente utilizzati GAL4 base 2-sistema ibrido. Rispetto al tradizionale doppio ibrido di lievito schermo, MY2H riduce drasticamente il processo di schermo e riduce il numero di cloni falsi positivi. Inoltre, questo approccio è riproducibile e affidabile, e abbiamo impiegato con successo questo sistema per isolare le proteine ​​leganti di esche, tra cui ioni di canale 12, proteine ​​della matrice extracellulare 10, 13, e del fattore di crescita 14. In questo lavoro, si descrive questa procedura MY2H sperimentale in dettaglio utilizzando PGRN come un esempio che ha portato alla identificazione di TNFR2 come la prima nota PGRN associata recettore 14, 15.

Watch this Scientific Journal Video about Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin at JoVE.com

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Abbiamo modificato il lievito tradizionale doppio ibrido di screening, un efficace strumento genetico per identificare interazioni proteiche. Questa modifica riduce notevolmente il processo, riduce il carico di lavoro e, soprattutto, riduce il numero di falsi positivi. Inoltre, questo approccio è riproducibile ed affidabile.

Modificato lievito-sistema del doppio ibrido per identificare proteine ​​che interagiscono con il fattore di crescita progranulina

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

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28.6K Views.  NYU Hospital for Joint Diseases. Abbiamo modificato il lievito tradizionale doppio ibrido di screening, un efficace strumento genetico per identificare interazioni proteiche. Questa modifica riduce notevolmente il processo, riduce il carico di lavoro e, soprattutto, riduce il numero di falsi positivi. Inoltre, questo approccio è riproducibile ed affidabile.

Video: Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Growth Factor-Coated Bead Placement on Dorsal Forebrain Explants

This video demonstrates two methods for preparing and placing beads, which have been coated with growth factor, on explants of the developing cerebral cortex. These beads can be used to induce spatially restricted gene expression on developing neural tissue such as forebrain explants. Methods are given for using both Affi-Gel beads and heparin acryllic beads.

Fattore-Coated crescita Placement Bead su Espianti Forebrain dorsali

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Biologia

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

The neural tube expresses many proteins in specific spatiotemporal patterns during development. These proteins have been shown to be critical for cell fate determination, cell migration, and formation of neural circuits. Neuronal induction and patterning involve bone morphogenetic protein (BMP), sonic hedgehog (SHH), fibroblast growth factor (FGF), among others. In particular, the expression pattern of Fgf8 is in close proximity to regions expressing BMP4 and SHH. This expression pattern is consistent with developmental interactions that facilitate patterning in the telencephalon.

Here we provide a visual demonstration of a method in which an in ovo preparation can be used to test the effects of Fgfs in the formation of the forebrain. Beads are coated with protein and placed in the developing neural tube to provide sustained exposure. Because the procedure uses small, carefully placed beads, it is minimally invasive and allows several beads to be placed within a single neural tube. Moreover, the method allows for continued development so that embryos can be analyzed at a more mature stage to detect changes in anatomy and in neural patterning. This simple but useful protocol allows for real time imaging. It provides a means to make spatially and temporally limited changes to endogenous protein levels.

A variety of growth factors and proteins interact to induce cells to take on different cell fates during development. Here we demonstrate the use of an in ovo preparation to address possible interactions between different proteins in development by placing beads on E2.5 chick embryos.

Posizionamento Growth Factor-Coated Perline su embrioni di pollo Stadio

The neural tube expresses many proteins in specific spatiotemporal patterns during development.   These proteins have been shown to be critical for cell ...

Preparing T Cell Growth Factor from Rat Splenocytes

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion 1,2. Addition of interleukin 2 to the culture media during the expansion phase is necessary to prevent cell death and sufficient to maintain short-term T cell lines but has been shown to increase Th1 polarization 3. Replacement of interleukin 2 by T cell growth factor (TCGF) which contains a mix of cytokines is more effective than interleukin 2 in maintaining long-term T cell lines in vitro 3. Moreover, TCGF can easily be prepared in large amounts in the laboratory and is much cheaper than recombinant interleukin 2. 
Here, we show how to prepare TCGF from rat splenocyte culture supernatants. For this procedure, we harvest spleens from naive Lewis rats euthanized for thymus and blood collection. We prepare single-cell suspensions from the spleens, lyze the red blood cells by osmotic shock, and seed the splenocytes in culture medium. The cells are stimulated with concanavalin A, a mitogen that non-selectively activates all rat T lymphocytes, inducing the production of cytokines. The culture supernantant is collected 48 hours later andexcess concanavalin A is bound to alpha methyl mannoside to prevent it from activating T cell lines to which TCGF will be added. The TCGF is then sterile-filtered, aliquoted, and stored at -20&deg;C.

We describe the preparation of T cell growth factor used for the in vitro expansion of antigen-specific rat T lymphocyte lines.

Preparazione T fattore di crescita cellulare da splenociti Rat

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion 1,2. ...

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys loop, the glutamate-gated and the P2X receptor channels2. In each case binding of transmitter leads to the opening of a pore through which ions flow down their electrochemical gradients. Many ligand-gated channels are also permeable to calcium ions3, 4, which have downstream signaling roles5 (e.g. gene regulation) that may exceed the duration of channel opening. Thus ligand-gated channels can signal over broad time scales ranging from a few milliseconds to days. Given these important roles it is necessary to understand how ligand-gated ion channels themselves are regulated by proteins, and how these proteins may tune signaling. Recent studies suggest that many, if not all, channels may be part of protein signaling complexes6. In this article we explain how to identify the proteins that bind to the C-terminal aspects of the P2X2 receptor cytosolic domain.
P2X receptors are ATP-gated cation channels and consist of seven subunits (P2X1-P2X7). P2X receptors are widely expressed in the brain, where they mediate excitatory synaptic transmission and presynaptic facilitation of neurotransmitter release7. P2X receptors are found in excitable and non-excitable cells and mediate key roles in neuronal signaling, inflammation and cardiovascular function8. P2X2 receptors are abundant in the nervous system9 and are the focus of this study. Each P2X subunit is thought to possess two membrane spanning segments (TM1 &amp; TM2) separated by an extracellular region7 and intracellular N and C termini (Fig 1a)7. P2X subunits10 (P2X1-P2X7) show 30-50% sequence homology at the amino acid level11. P2X receptors contain only three subunits, which is the simplest stoichiometry among ionotropic receptors. The P2X2 C-terminus consists of 120 amino acids (Fig 1b) and contains several protein docking consensus sites, supporting the hypothesis that P2X2 receptor may be part of signaling complexes. However, although several functions have been attributed to the C-terminus of P2X2 receptors9 no study has described the molecular partners that couple to the intracellular side of this protein via the full length C-terminus. In this methods paper we describe a proteomic approach to identify the proteins which interact with the full length C-terminus of P2X2 receptors.

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

Proteomica per identificare proteine ​​che interagiscono con P2X2 Ligand-Gated canali di comunicazione

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

Split-Ubiquitina membrana a base di lievito Due-Hybrid (MITO) Sistema: un potente strumento per identificare interazioni proteina-proteina

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR-Clip - un metodo per identificare trascrittoma livello i siti di legame delle proteine ​​RNA binding

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

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 di screening per identificare fenotipi postembrionale in 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 ...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking metabolism and inflammation (e.g.&#160;hepatic steatosis)1,2. There has been much progress in the identification of agonist ligands for PXR, however, there are limited descriptions of drug-like antagonists and their binding sites on PXR3,4,5. A critical barrier has been the inability to efficiently purify full-length protein for structural studies with antagonists despite the fact that PXR was cloned and characterized in 1998. Our laboratory developed a novel high throughput yeast based two-hybrid assay to define an antagonist, ketoconazole&#39;s, binding residues on PXR6. Our method involves creating mutational libraries that would rescue the effect of single mutations on the AF-2 surface of PXR expected to interact with ketoconazole. Rescue or &#34;gain-of-function&#34; second mutations can be made such that conclusions regarding the genetic interaction of ketoconazole and the surface residue(s) on PXR are feasible. Thus, we developed a high throughput two-hybrid yeast screen of PXR mutants interacting with its coactivator, SRC-1. Using this approach, in which the yeast was modified to accommodate the study of the antifungal drug, ketoconazole, we could demonstrate specific mutations on PXR enriched in clones unable to bind to ketoconazole. By reverse logic, we conclude that the original residues are direct interaction residues with ketoconazole. This assay represents a novel, tractable genetic assay to screen for antagonist binding sites on nuclear receptor surfaces. This assay could be applied to any drug regardless of its cytotoxic potential to yeast as well as to cellular protein(s) that cannot be studied using standard structural biology or proteomic based methods. Potential pitfalls include interpretation of data (complementary methods useful), reliance on single Y2H method, expertise in handling yeast or performing yeast two-hybrid assays, and assay optimization.

Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.

Reverse lievito sistema Two-ibrida per identificare mammiferi Nuclear Receptor residui che interagiscono con leganti e / o antagonisti

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

Capture spettrometria di massa Compound - un potente strumento per identificare nuovi c-di-GMP effettori Proteine

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

Xenopus laevis come un modello per individuare Traduzione Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...

Modificato lievito-sistema del doppio ibrido per identificare proteine ​​che interagiscono con il fattore di crescita progranulina

Modificato lievito-sistema del doppio ibrido per identificare proteine che interagiscono con il fattore di crescita progranulina