Ringraziamo Fiona Foley per il sostegno e la cura eccellente laboratorio zebrafish. Questo lavoro è stato finanziato una borsa di studio predoctoral AHA a GW (11PRE5730027) e le sovvenzioni a HJY NHLBI (R01HL66292) e JDA (R01HL095690).

Panoramica delle iniezioni zebrafish Stage-specifici embrioni Oligonucleotidi antisenso morfolino (MO), che si legano ad un mRNA mirata e disturbare espressione proteica da tale trascrizione, sono ampiamente utilizzati in knockdown gene (perdita di funzione) Studi in zebrafish 13,14. Strumenti di Gene, LLC offre MO che sono contrassegnati con uno carbossifluoresceina (emette fluorescenza verde) o lissamina (emette fluorescenza rossa) per rilevare MO negli embrioni iniettati usando la microscopia a fluorescenza. Iniettando MO nella cella tuorlo a diversi stadi di sviluppo zebrafish, è possibile fornire il MO a specifici compartimenti dell&#39;embrione (Figure 1A-F). MO iniettato nel tuorlo tra le fasi 1-4 cellule (0-1 hpf) entra tutte le cellule embrionali (figure 1A, D, D &#39;) tramite connessioni con il tuorlo che persistono fino a 32 cellule stadio 15 per facilitare knockdown globale. MO iniettato nel tuorlo durante midblastfasi ula (2,5-3 hpf) possono inserire i progenitori dei DFCS (1B figure, E, E &#39;), che possano attraverso ponti citoplasmatici 8 e funzione del gene atterramento in particolare nel DFC / KV linea cellulare 12, senza entrare maggior parte delle altre linee cellulari embrionali . Come un controllo importante per verificare se la funzione del gene è necessaria in DFC / KV o anche nel tuorlo 12,16, è anche possibile limitare MO alla cella tuorlo iniettando tra le fasi epiboly dome-30% (~ 4,5 HPF) dopo tutti i ponti citoplasmatici hanno chiuso (figure 1C, F, F &#39;). Queste iniezioni sono state utilizzate in combinazione per analizzare la funzione genica in DFC / KV cellule 17-24. Per valutare il flusso di fluido in KV, microsfere fluorescenti vengono iniettati nel lume KV tra le fasi 6-10 somite hpf (12-14) e poi subito ripreso usando videomicroscopia (Figure 1G-J). Microperline sono disponibili che emettono fluorescenza rossa o verde (o entrambi), quindi è possibile utilizzare diversicanali di immagine fluorescente microsfere e MO. 1. Stadio-specifica di iniezione Morpholinos (MO) Iniezione di MO in embrioni di zebrafish è stato dimostrato in precedenza 25,26. Qui, descriviamo brevemente fase-specifici iniezioni MO. Seguendo tutte le iniezioni, gli embrioni vengono trasferiti ad una piastra di Petri e incubato a 28,5 ° C. <ol><li> Globale iniezione MO: Raccogliere gli embrioni subito dopo la fecondazione e di caricarli in una piastra di iniezione. Caricare fluorescente MO in un ago capillare e montare l&#39;ago su un microiniettore (ad esempio Harvard Apparatus PLI-90 Pico-iniettore). Rompere la punta dell&#39;ago e regolare le impostazioni (pressione di iniezione e / o tempo) per creare una caduta di iniezione che ha un volume di 1 nl come descritto 25,26. Utilizzando uno stereomicroscopio dissezione, iniettare 1 nl di MO nel tuorlo (Figura 1A) di ≥ 50 embrioni per esperimento. Lavorare velocemente per completare iniezioni dalla 4 celle cervoe. </li><li> DFC-mirata iniezione MO: Raccogliere gli embrioni subito dopo la fecondazione e incubare a 28,5 ° C. Quando gli embrioni hanno raggiunto la fase a 256 cellule (~ 2,5 HPF), rapidamente caricare in una piastra di iniezione e iniettare 1 nl fluorescente MO nel tuorlo di ≥ 100 embrioni tra le fasi a 256 celle e 1.000 celle (Figura 1B). </li><li> Tuorlo-mirata MO iniezione: Raccogliere gli embrioni subito dopo la fecondazione e incubare a 28,5 ° C. Nella fase cupola (~ 4 HPF), caricare gli embrioni in una piastra di iniezione e quindi iniettare 1 nl fluorescente MO nel tuorlo di ≥ 100 embrioni tra la cupola e le fasi epiboly 30% (Figura 1C). </li></ol> 2. Selezione di embrioni iniettati per l&#39;analisi <ol><li> Quando MO iniettato embrioni raggiungere il 75%-epiboly stadio (8 HPF), rimuovere gli embrioni non fecondate e morto e poi embrioni schermo che vivono sotto un microscopio a fluorescenza dissezione per la distribuzione del MO fluorescente. Poi allow selezionato embrioni a svilupparsi a 28.5 ° C. <ol><li> Per globale MO iniezioni, selezionare gli embrioni che hanno fluorescenza uniformemente distribuiti in tutta tutte le cellule embrionali (Figura 1D; Figura 2A). </li><li> Per DFC-MO iniezioni mirate, selezionare gli embrioni in cui fluorescente MO è diffusa in tutto il tuorlo e sembra concentrata solo al margine dorsale blastoderma (DFCS) (Figura 1E; Figura 2B). In questa fase, può essere difficile visualizzare DFCS fluorescenti causa fluorescenza nel tuorlo sottostante. Fare attenzione ad escludere embrioni in cui la fluorescenza MO ha inserito nelle cellule embrionali diverse DFCS o rimane aggregati al sito di iniezione (Figura 2D). </li><li> Per tuorlo-MO iniezioni mirate, selezionare gli embrioni in cui MO fluorescenza è uniformemente distribuiti su tutto il tuorlo e non osservato in tutte le cellule embrionali (Figura 1F; Figura 2C). Anche in questo caso, rimuovere gli embrioni in cui la fluorescente MO rimaneaggregati al sito di iniezione. </li></ol></li><li> Tra i 2-4-somite stadi (~ 11 HPF), schermo embrioni una seconda volta sotto un microscopio a fluorescenza utilizzando un ingrandimento maggiore. Poi permettono embrioni selezionati di sviluppare a 28.5 ° C. <ol><li> Per globali iniezioni MO, garantire gli embrioni selezionati hanno MO fluorescenza uniformemente distribuiti in KV e tutte le cellule embrionali (Figura 1D &#39;; Figura 2E). </li><li> Per DFC-MO iniezioni mirate, selezionare con attenzione gli embrioni che presentano fluorescenza MO solo nelle cellule KV e il tuorlo (Figura 1E &#39;). Embrioni transgenici che esprimono GFP in cellule KV, come Tg (Dusp6: d2EGFP) 27, possono aiutare nella identificazione di embrioni in cui MO è stato recapitato a cellule KV (Figura 2F). Scartare gli embrioni con MO fluorescenza nelle cellule embrionali di altri che le cellule KV. </li><li> Per tuorlo-MO iniezioni mirate, selezionare gli embrioni che hanno fluorescenza MO esclusivamente nel (tuorlo Figura 1F &#39;: Figura2G). </li></ol></li></ol> 3. Gli embrioni di montaggio per analizzare il flusso del fluido in KV <ol><li> Per analizzare il flusso asimmetrico fluido in KV globale di MO, DFC-mirata MO o tuorlo-MO mirati embrioni iniettati, con attenzione dechorionate ~ 20 embrioni di 4-6 stadi somite (~ 11-12 hpf) con pinze taglienti. </li><li> Preparare 50 ml di 1% a basso punto di fusione agarosio e aliquotare in provette da 5 ml di essere mantenuto come un liquido in un bagno a secco a 42 ° C. </li><li> Trasferire un embrione a una diapositiva depressione vetro (Supplies Italia Scientific, Inc.) e rimuovere la maggior quantità di acqua possibile. Immobilizzare l&#39;embrione aggiungendo abbastanza caldo 1% a basso punto di fusione agarosio a coprire appena l&#39;embrione (troppo agarosio può interferire con immagini successive). Come l&#39;agarosio si solidifica, utilizzare pinze per posizionare l&#39;embrione in modo che sia rivolto verso l&#39;alto KV (vista dorsale) (Figura 1 H). Montare 10 embrioni con un embrione per diapositiva. Lavorare rapidamente per garantire tutte le iniezioni nel passaggio successivo sono completed dalla stadio 10 somite (14 HPF). </li></ol> 4. Iniezione di microsfere fluorescenti in KV <ol><li> Diluire Fluoresbrite Multifuorescent 0,5 micron di diametro Microsfere (Polysciences, Inc.) a 1:50 in acqua sterile in una provetta Eppendorf da 1,5 ml. </li><li> Mescolare la diluizione 1:50 di microsfere a fondo toccando il tubo e caricare 3 ml in un ago capillare. Utilizzando lo stesso utilizzato per microiniettore MO iniezioni (ad esempio Harvard Apparatus PLI-90 Pico-iniettore), rompere la punta dell&#39;ago con una pinza e regolare le impostazioni di iniezione per generare il più piccolo possibile (&lt;0,5 nl) goccia iniezione. </li><li> Sotto un microscopio per dissezione, allineare l&#39;ago con il lume KV dell&#39;embrione prima montato. Inserire l&#39;ago nel lume e iniettare una piccola quantità (&lt;0,5 nl) di microsfere (Figura 1G). Un suggerimento piccolo ago e il volume di iniezione piccole sono fondamentali per evitare di danneggiare KV. </li><li> Iniettare tutti gli embrioni montati. Una volta che un embrione è injected, una goccia di acqua sterile può essere aggiunto in cima al agarosio per evitare che si secchi. Nel corso delle iniezioni, le perline spesso ostruire l&#39;apertura dell&#39;ago. Questo richiede nuovamente rompere la punta dell&#39;ago e la regolazione del volume di iniezione modulando la pressione di taratura o tempo dell&#39;apparato microiniezione. Sostituire l&#39;ago se diventa ottuso sufficiente a causare danni significativi durante l&#39;iniezione. </li><li> Schermo gli embrioni iniettati per la consegna di successo di perle con un microscopio in posizione verticale composto fluorescente (per esempio Zeiss AxioImager M1) con un obiettivo 20X. In primo luogo, verificare se la struttura KV è intatto con campo chiaro illuminazione (Figura 1J), e quindi utilizzare fluorescenza per osservare le perline. Selezionare embrioni in cui perle sono presenti e lo spostamento all&#39;interno del lume KV. Scartare gli embrioni con danni KV o nessuna perline galleggianti in KV. E &#39;facile perdere KV e fornire perline per tessuti circostanti o il tuorlo sottostante. In caso di insuccesso, montaggio another 10 embrioni e ripetere la procedura di iniezione. </li></ol> 5. Visualizzazione e analisi di flusso del fluido KV <ol><li> Per ogni embrione selezionato con perline all&#39;interno KV, aggiungere una goccia di acqua sterile per coprire l&#39;agarosio e poi osservare KV al microscopio in posizione verticale composto fluorescente usando un obiettivo 63X acqua immersione (Figura 1I). In alternativa, un vetrino coprioggetto può essere applicata per l&#39;uso con obiettivi immersione non acquosa e / o microscopi invertiti. </li><li> Utilizzare una telecamera ad alta velocità montata sul microscopio (es. Zeiss AxioCamHSm) per registrare un film di 10 sec. Registrare entrambe le perline utilizzando il canale fluorescente (circa 70 fotogrammi / sec) e il lume KV utilizzando un campo chiaro e contrasto interferenziale differenziale (DIC) canale. </li><li> Per visualizzare tutti i movimenti del tallone nel corso del tempo, importare il filmato di perline fluorescenti in ImageJ software (scaricabile gratuitamente dal <a href="http://rsb.info.nih.gov/ij/" target="_blank">http://rsb.info.nih.gov/ij/</a> ) unand creare una sporgenza massima di tutti i segnali fluorescenti nel film. Per eseguire la proiezione massima ImageJ, fare clic su &quot;Immagine → Pile → progetto Z&quot;. Nel &quot;Progetto Z&quot; finestra, proiettare tutte le fette con il tipo di proiezione di &quot;intensità Max&quot;. Questa immagine può essere sovrapposto un&#39;immagine DIC del KV in cui sono stati ripresi i talloni (Figure 3A-B). </li><li> I movimenti di perle singoli si possono seguire con ImageJ. Un plugin (&quot;Allineamento manuale&quot;) può essere scaricato dal sito <a href="http://rsbweb.nih.gov/ij/plugins/track/track.html" target="_blank">http://rsbweb.nih.gov/ij/plugins/track/track.html</a> . Aprire il film fluorescente perline in ImageJ ed eseguire la funzione di tracking manuale. Nella finestra di Allineamento manuale, controllare i parametri &quot;show&quot; e dei parametri di input per &quot;intervalli di tempo&quot; e &quot;x / y di calibrazione&quot;. Selezionare manualmente perle che rimangono nel piano focale del film per ≥ 50 fotogrammi e quindi, di monitorare ≥ 5 perle per embrione. Il percorso e la velocità di ciascuna sfera è generato da the software (figure 3 CD). </li></ol>

<ol>
	<li>Sutherland, M. J., Ware, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disorders+of+left-right+asymmetry:+heterotaxy+and+situsinversus.">Disorders of left-right asymmetry: heterotaxy and situsinversus.</a> Am. J. Med. Genet. C Semin. Med. Genet. 151C (4), 307-317 (2009).</li><li>Essner, J. J., Amack, J. D., Nyholm, M. K., Harris, E. B., Yost, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kupffer's+vesicle+is+a+ciliated+organ+of+asymmetry+in+the+zebrafish+embryo+that+initiates+left-right+development+of+the+brain,+heart+and+gut.">Kupffer's vesicle is a ciliated organ of asymmetry in the zebrafish embryo that initiates left-right development of the brain, heart and gut.</a> Development. 132 (6), 1247-1260 (2005).</li><li>Nonaka, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Randomization+of+left-right+asymmetry+due+to+loss+of+nodal+cilia+generating+leftward+flow+of+extraembryonic+fluid+in+mice+lacking+KIF3B+motor+protein.">Randomization of left-right asymmetry due to loss of nodal cilia generating leftward flow of extraembryonic fluid in mice lacking KIF3B motor protein.</a> Cell. 95 (6), 829-837 (1998).</li><li>Essner, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+function+for+embryonic+nodal+cilia.">Conserved function for embryonic nodal cilia.</a> Nature. 418 (6893), 37-38 (2002).</li><li>Kramer-Zucker, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cilia-driven+fluid+flow+in+the+zebrafish+pronephros,+brain+and+Kupffer's+vesicle+is+required+for+normal+organogenesis.">Cilia-driven fluid flow in the zebrafish pronephros, brain and Kupffer's vesicle is required for normal organogenesis.</a> Development. 132 (8), 1907-1921 (2005).</li><li>Schweickert, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cilia-driven+leftward+flow+determines+laterality+in+Xenopus.">Cilia-driven leftward flow determines laterality in Xenopus.</a> Curr. Biol. 17 (1), 60-66 (2007).</li><li>Tabin, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+key+to+left-right+asymmetry.">The key to left-right asymmetry.</a> Cell. 127 (1), 27-32 (2006).</li><li>Cooper, M. S., D'Amico, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cluster+of+noninvolutingendocytic+cells+at+the+margin+of+the+zebrafish+blastoderm+marks+the+site+of+embryonic+shield+formation.">A cluster of noninvolutingendocytic cells at the margin of the zebrafish blastoderm marks the site of embryonic shield formation.</a> Dev. Biol. 180 (1), 184-198 (1996).</li><li>Melby, A. E., Warga, R. M., Kimmel, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specification+of+cell+fates+at+the+dorsal+margin+of+the+zebrafish+gastrula.">Specification of cell fates at the dorsal margin of the zebrafish gastrula.</a> Development. 122 (7), 2225-2237 (1996).</li><li>Amack, J. D., Wang, X., Yost, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+T-box+genes+play+independent+and+cooperative+roles+to+regulate+morphogenesis+of+ciliated+Kupffer's+vesicle+in+zebrafish.">Two T-box genes play independent and cooperative roles to regulate morphogenesis of ciliated Kupffer's vesicle in zebrafish.</a> Dev. Biol. 310 (2), 196-210 (2007).</li><li>Oteiza, P., Koppen, M., Concha, M. L., Heisenberg, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+shaping+of+the+laterality+organ+in+zebrafish.">Origin and shaping of the laterality organ in zebrafish.</a> Development. 135 (16), 2807-2813 (2008).</li><li>Amack, J. D., Yost, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+T+box+transcription+factor+no+tail+in+ciliated+cells+controls+zebrafish+left-right+asymmetry.">The T box transcription factor no tail in ciliated cells controls zebrafish left-right asymmetry.</a> Curr. Biol. 14 (8), 685-690 (2004).</li><li>Nasevicius, A., Ekker, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+targeted+gene+'knockdown'+in+zebrafish.">Effective targeted gene 'knockdown' in zebrafish.</a> Nat. Genet. 26 (2), 216-220 (2000).</li><li>Bill, B. R., Petzold, A. M., Clark, K. J., Schimmenti, L. A., Ekker, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+primer+for+morpholino+use+in+zebrafish.">A primer for morpholino use in zebrafish.</a> Zebrafish. 6 (1), 69-77 (2009).</li><li>Kimmel, C. B., Law, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lineage+of+zebrafish+blastomeres.+I.+Cleavage+pattern+and+cytoplasmic+bridges+between+cells.">Cell lineage of zebrafish blastomeres. I. Cleavage pattern and cytoplasmic bridges between cells.</a> Dev. Biol. 108 (1), 78-85 (1985).</li><li>Arrington, C. B., Yost, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extra-embryonic+syndecan+2+regulates+organ+primordia+migration+and+fibrillogenesis+throughout+the+zebrafish+embryo.">Extra-embryonic syndecan 2 regulates organ primordia migration and fibrillogenesis throughout the zebrafish embryo.</a> Development. 136 (18), 3143-3152 (2009).</li><li>Caron, A., Xu, X., Lin, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt/beta-catenin+signaling+directly+regulates+Foxj1+expression+and+ciliogenesis+in+zebrafish+Kupffer's+vesicle.">Wnt/beta-catenin signaling directly regulates Foxj1 expression and ciliogenesis in zebrafish Kupffer's vesicle.</a> Development. 139 (3), 514-524 (2012).</li><li>Wang, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Rho+kinase+Rock2b+establishes+anteroposterior+asymmetry+of+the+ciliated+Kupffer's+vesicle+in+zebrafish.">The Rho kinase Rock2b establishes anteroposterior asymmetry of the ciliated Kupffer's vesicle in zebrafish.</a> Development. 138 (1), 45-54 (2011).</li><li>Aamar, E., Dawid, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sox17+and+chordin+are+required+for+formation+of+Kupffer's+vesicle+and+left-right+asymmetry+determination+in+zebrafish.">Sox17 and chordin are required for formation of Kupffer's vesicle and left-right asymmetry determination in zebrafish.</a> Dev. Dyn. 239 (11), 2980-2988 (2010).</li><li>Neugebauer, J. M., Amack, J. D., Peterson, A. G., Bisgrove, B. W., Yost, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF+signalling+during+embryo+development+regulates+cilia+length+in+diverse+epithelia.">FGF signalling during embryo development regulates cilia length in diverse epithelia.</a> Nature. 458 (7238), 651-654 (2009).</li><li>Schneider, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+Nkd1+promotes+Dvl+degradation+and+is+required+for+left-right+patterning.">Zebrafish Nkd1 promotes Dvl degradation and is required for left-right patterning.</a> Dev. Biol. 348 (1), 22-33 (2010).</li><li>Matsui, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Canopy1,+a+positive+feedback+regulator+of+FGF+signaling,+controls+progenitor+cell+clustering+during+Kupffer's+vesicle+organogenesis.">Canopy1, a positive feedback regulator of FGF signaling, controls progenitor cell clustering during Kupffer's vesicle organogenesis.</a> Proc. Natl. Acad. Sci. U.S.A. 108 (24), 9881-9886 (2011).</li><li>Shu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Na,K-ATPase+alpha2+and+Ncx4a+regulate+zebrafish+left-right+patterning.">Na,K-ATPase alpha2 and Ncx4a regulate zebrafish left-right patterning.</a> Development. 134 (10), 1921-1930 (2007).</li><li>Esguerra, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ttrap+is+an+essential+modulator+of+Smad3-dependent+Nodal+signaling+during+zebrafish+gastrulation+and+left-right+axis+determination.">Ttrap is an essential modulator of Smad3-dependent Nodal signaling during zebrafish gastrulation and left-right axis determination.</a> Development. 134 (24), 4381-4393 (2007).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+Zebrafish+Embryos+to+Analyze+Gene+Function.">Microinjection of Zebrafish Embryos to Analyze Gene Function.</a> J. Vis. Exp. (25), e1115 (2009).</li><li>Yuan, S., Sun, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+mRNA+and+Morpholino+Antisense+Oligonucleotides+in+Zebrafish+Embryos.">Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.</a> J. Vis. Exp. (27), e1113 (2009).</li><li>Molina, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+chemical+screening+reveals+an+inhibitor+of+Dusp6+that+expands+cardiac+cell+lineages.">Zebrafish chemical screening reveals an inhibitor of Dusp6 that expands cardiac cell lineages.</a> Nat. Chem. Biol. 5 (9), 680-687 (2009).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev. Dyn. 203 (3), 253-310 (1995).</li><li>Schneider, I., Houston, D. W., Rebagliati, M. R., Slusarski, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+fluxes+in+dorsal+forerunner+cells+antagonize+beta-catenin+and+alter+left-right+patterning.">Calcium fluxes in dorsal forerunner cells antagonize beta-catenin and alter left-right patterning.</a> Development. 135 (1), 75-84 (2008).</li><li>Clement, A., Solnica-Krezel, L., Gould, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cdc14B+phosphatase+contributes+to+ciliogenesis+in+zebrafish.">The Cdc14B phosphatase contributes to ciliogenesis in zebrafish.</a> Development. 138 (2), 291-302 (2011).</li><li>Matsui, T., Bessho, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Left-right+asymmetry+in+zebrafish.">Left-right asymmetry in zebrafish.</a> Cell Mol. Life Sci. , (2012).</li></ol>

Nessun conflitto di interessi dichiarati.

Utilizzo di fase-specifici iniezioni di indirizzare MO al DFC / KV linea cellulare è un approccio utile per lo studio delle cellule-autonomia della funzione del gene e di evitare fenotipi pleiotropici causati da knockdown gene globale. Tuttavia, queste iniezioni possono essere tecnicamente impegnativo. Iniezione di MO tra le fasi 256-cellula e cellula-1000 può portare a tre possibili esiti: 1) la MO rimane aggregati nel sito di iniezione, 2) si diffonde MO tutto il tuorlo ed entra DFC / KV cellule o 3) il MO si diffon...

<table border="1"><tbody><tr><td> Nome del reagente / materiale </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> Standard di controllo oligo-lissamina tag </td><td> Gene Tools, LLC </td><td></td></tr><tr><td> Personalizzato Rock2b morfolino oligo </td><td> Gene Tools, LLC </td><td></td></tr><tr><td> Fluoresbrite Multifluorescent 0,5 micron Microsfere </td><td> Polysciences, Inc. </td><td> 24054 </td></tr></tbody></table>

analysis of gene function and visualization of cilia-generated fluid flow in kupffer's vesicle

Gli organi interni come il cuore, il cervello, e nell&#39;intestino sviluppare sinistra-destra (LR) asimmetrie che sono fondamentali per le loro normali funzioni 1. Ciglia mobili sono coinvolti nella creazione LR asimmetria negli embrioni dei vertebrati, tra cui mouse, rana, e zebrafish 2-6. Questi &#39;cilia LR &quot;generano flusso asimmetrico fluido che è necessario per attivare una conservata asimmetrica Nodal (TGF-β superfamiglia) di segnalazione cascata nel mesoderma piastra laterale sinistra, che è pensato per fornire informazioni patterning LR per lo sviluppo di organi 7. Così, per comprendere i meccanismi sottostanti patterning LR, è essenziale per identificare i geni che regolano l&#39;organizzazione delle cellule ciliate LR, la motilità e la lunghezza delle ciglia LR e la loro capacità di generare robusto flusso asimmetrico. In zebrafish, ciglia LR si trovano in vescicole di Kupffer (KV) 2,4,5. KV è costituito da un singolo strato di cellule epiteliali monociliatedche racchiudono un lume piene di liquido. Mappatura destino KV ha dimostrato che è derivato da un gruppo di ~ 20-30 cellule conosciute come cellule precursore dorsali (DFCS) che migrano al margine blastoderm dorsale durante fasi epiboly 8,9. Durante le fasi iniziali somite, cluster DFCS e differenziarsi in cellule epiteliali ciliate per formare KV nel tailbud dell&#39;embrione 10,11. La capacità di identificare e tenere traccia di DFCS-in combinazione con trasparenza ottica e il rapido sviluppo del pesce zebra dell&#39;embrione-make zebrafish KV un sistema ottimo modello per studiare le cellule ciliate LR. È interessante notare che, progenitori della DFC / KV linea cellulare mantenere ponti citoplasmatici tra la cellula uovo fino a 4 ore post-fecondazione (HPF), mentre ponti citoplasmatici tra la cellula uovo e altre cellule embrionali vicino dopo 2 HPF 8. Approfittando di questi ponti citoplasmatici, abbiamo sviluppato una fase specifica strategia di iniezione per fornire morfolino oligonucleotides (MO) esclusivamente a DFCS smontabili e la funzione di un gene mirato in queste cellule 12. Questa tecnica crea embrioni chimerici in cui viene buttato funzione genica giù nella DFC / KV lignaggio sviluppare nel contesto di un embrione wild-type. Per analizzare il flusso asimmetrico fluido in KV, iniettiamo microsfere fluorescenti nel lume KV e movimento del disco perlina con videomicroscopia 2. Flusso del fluido è facilmente visualizzato e può essere quantificata inseguimento spostamento tallone nel tempo. Qui, con lo stadio-specifico DFC-targeting tecnica knockdown gene e iniezione di microsfere fluorescenti in KV per visualizzare il flusso, presentiamo un protocollo che fornisce un approccio efficace per caratterizzare il ruolo di un particolare gene durante lo sviluppo e la funzione KV.

Fase-specifici iniezioni MO fornire un approccio utile per analizzare la funzione genica in specifici compartimenti dell&#39;embrione. Figura 1 presenta un diagramma di flusso delle strategie di iniezione utilizzato per testare la funzione del gene in DFC / KV cellule e come introdurre perline fluorescenti per visualizzare il flusso del fluido in KV. La distribuzione di MO fluorescente successo in embrioni iniettati fase-specifici è mostrato schematicamente nelle figure 1D-F e in embri...

Watch this Scientific Journal Video about Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer's Vesicle at JoVE.com

Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer&#039;s Vesicle

Cilia generato il flusso del fluido in vescicole di Kupffer (KV) controlla sinistra-destra patterning dell&#39;embrione zebrafish. Qui, si descrive una tecnica per modulare la funzione del gene specificamente in cellule KV. Inoltre, mostriamo come fornire perline fluorescenti in KV per visualizzare il flusso del fluido.

Analisi della funzione del gene e visualizzazione di Cilia-Generated flusso di fluido in vescicole di Kupffer

Internal organs such as the  heart, brain, and gut develop left-right (LR) asymmetries that are critical for  their normal functions1. Motile cilia are ...

analysis-gene-function-visualization-cilia-generated-fluid-flow

Research

JoVE Journal

Biology

Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer's Vesicle

14.7K Views.  Upstate Medical University. Cilia generato il flusso del fluido in vescicole di Kupffer (KV) controlla sinistra-destra patterning dell'embrione zebrafish. Qui, si descrive una tecnica per modulare la funzione del gene specificamente in cellule KV. Inoltre, mostriamo come fornire perline fluorescenti in KV per visualizzare il flusso del fluido.

Video: Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer's Vesicle

Microinjection of Zebrafish Embryos to Analyze Gene Function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

Microiniezione di embrioni di zebrafish per analizzare la funzione del gene

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

Ricerca

Biologia

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

Induzione e test di ipossia in coltura cellulare

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

Annotation of Plant Gene Function via Combined Genomics, Metabolomics and Informatics

Given the ever expanding number of model plant species for which complete genome sequences are available and the abundance of bio-resources such as knockout mutants, wild accessions and advanced breeding populations, there is a rising burden for gene functional annotation. In this protocol, annotation of plant gene function using combined co-expression gene analysis, metabolomics and informatics is provided (Figure 1). This approach is based on the theory of using target genes of known function to allow the identification of non-annotated genes likely to be involved in a certain metabolic process, with the identification of target compounds via metabolomics. Strategies are put forward for applying this information on populations generated by both forward and reverse genetics approaches in spite of none of these are effortless. By corollary this approach can also be used as an approach to characterise unknown peaks representing new or specific secondary metabolites in the limited tissues, plant species or stress treatment, which is currently the important trial to understanding plant metabolism.

Combination of genomics, co-expression gene analysis and the identification of target compounds via metabolism give gene functional annotation.

Annotazione della funzione impianto Gene via Genomics combinati, la metabolomica e Informatica

Given the ever expanding number of model plant species for which complete genome sequences are available and the abundance of bio-resources such as ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Isolamento dei ribosomi Polipeptidi Bound nascenti<em&gt; In vitro</em&gt; Per identificare i siti di pausa traslazione lungo mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualizzazione e l&#39;analisi di molecole di mRNA mediante fluorescenza<em&gt; In Situ</em&gt; Ibridazione in<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

Ricostituzione funzionale e attività Canale Misure di purificata di tipo selvatico e mutante CFTR proteine

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

L&#39;Assemblea e l&#39;applicazione di &quot;Shear Anelli &#39;: A Novel endoteliale modello per Orbital, unidirezionale e periodica del fluido di flusso e lo stress di taglio

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

Analisi microbiota utilizzando PCR in due fasi e sequenziamento genico rRNA 16S di nuova generazione

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

In situ Hybridization for Sipunculus nudus Coelomic Fluid

In situ hybridization (ISH) is a very informative technique to present cellular distribution patterns of specific genes (e.g., mRNA and ncRNA) in tissues. The sipunculid worm Sipunculus nudus is a crucial fishery resource as it has high nutritional and medicinal values. Currently, the research on the molecular biology of Sipunculus nudus is still in its infancy. The purpose of this article is to develop a sensitive method for localizing specific mRNA in Sipunculus nudus coelomic fluid. The protocol includes detailed steps of ISH, including digoxigenin-labeled antisense and sense riboprobe preparation, coelomic fluid collection and section preparation, specific riboprobe hybridization, antibody incubation, coloration and post-coloration treatments. The representative results obtained from a successful experiment using this method are demonstrated. The protocol should be applicable to other Sipuncula species as well.

In situ Hybridization for Sipunculus nudus Coelomic Fluid

This protocol describes an effective in situ hybridization approach to detect the mRNA expression levels and spatial patterns of target genes in Sipunculus nudus coelomic fluid.

Ibridazione in situ per Sipunculus nudus Coelomic Fluid

In situ hybridization (ISH) is a very informative technique to present cellular distribution patterns of specific genes (e.g., mRNA and ncRNA) in tissues. ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

In vitro Analisi della funzione E3 Ubiquitina ligasi

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...