Questo lavoro è stato supportato dal NIH (DK093625 di LH e DK069909 e DK047757 a JHL) e la VA (Merit Award I01BX000820 a JHL). Vorremmo ringraziare il Dr. Michael Pack and Dr. Jie Egli presso l&#39;Università della Pennsylvania Zebrafish core per fornire supporto essenziale.

NOTA: Etica Dichiarazione: Tutti gli esperimenti di zebrafish sono stati approvati dalla cura e l&#39;uso degli animali Comitato Istituzionale presso la Virginia Medical School orientale. 1. Morpholino Preparazione <ol><li> Progettazione e sintesi di traduzione-bloccante (AUG-) e-giunzione inibizione (Splice-) antisenso morfolino (MO) oligonucleotidi per il gene di interesse secondo le istruzioni del produttore (Figura 1A). Si prega di consultare le informazioni del produttore nella tabella 1. NOTA: MO vengono spediti come scorte liofilizzati in bottiglie di vetro. </li><li> Aggiungere acqua sterile alta qualità per le bottiglie di vetro per risospendere MOs ad una concentrazione finale di 25 mg / mL. Assicurarsi che l&#39;oligonucleotide è completamente sciolto. Se alcuni resti solidi, riscaldare il flaconcino contenente la soluzione di riserva oligonucleotide a 65 ° C per 5 a 10 minuti e vortex brevemente. </li><li> Conservare la soluzione di riserva MO a RT. Non riporli a -20 ° 4 ° C o; C perché le temperature più basse possono provocare l&#39;oligonucleotide MO di legarsi alla parete del contenitore. Misurare la concentrazione della soluzione madre utilizzando uno spettrofotometro (fare riferimento alla Tabella 1) ogni volta che un nuovo MO soluzione madre è fatto. </li><li> Preparare la soluzione di lavoro MO il giorno dell&#39;iniezione diluendo la soluzione madre con acqua sterile alta qualità alla dose desiderata. Aggiungere 0,5% di fenolo-rosso per raggiungere una concentrazione di 0,05%. Ad esempio, per rendere soluzione di lavoro 5 microlitri con una concentrazione di 15 ng / nL, aggiungere 3 microlitri soluzione stock MO e 0,5 microlitri 0,5% di fenolo-rosso a 1,5 ml di acqua. NOTA: Con questa concentrazione di lavoro, ogni goccia (500 pl) di iniezione contiene 7,5 ng di MO e due gocce contiene 15 ng di MO. </li></ol> 2. Preparazione del Apparato Injection <ol><li> Vetro Acquisto aghi pre-tirato (vedi Tabella 1 per informazioni dettagliate). In alternativa, tirare l&#39;iniezione di vetro ago with un estrattore ago. </li><li> Accendere il compressore d&#39;aria e regolare l&#39;impostazione di 50 psi pressione. Accendere la sorgente di luce microscopio da dissezione e Pico pompa micro-iniezione e regolare le impostazioni come segue: mantenere la pressione 20 psi, espellere pressione 10 psi, valore periodo di 2,5 e 100 msec gamma di gating. </li><li> Caricare il bicchiere ago pre-stirato con 5 ml di soluzione di iniezione e inserire l&#39;ago in posizione verticale con la punta rivolta verso il basso. Attendere che tutta la soluzione raggiunge la punta dell&#39;ago con bolle d&#39;aria visibili. Posizionare il porta-aghi in una posizione appropriata vicino al microscopio e inserire l&#39;ago di vetro nel supporto. Regolare l&#39;angolo di iniezione a 45 gradi. </li><li> Portare la punta dell&#39;ago in vista al microscopio, alta dal palco, e concentrarsi sulla regione più sottile della punta. Spezzare la punta dell&#39;ago con una pinzetta punta fine. </li><li> Posizionare un righello e un tubo capillare con diametro interno di 0,15 mm di lato a fianco al microscopio;iniettare la soluzione in una estremità del tubo capillare per effettuare una colonna di liquido. Regolare il tempo di espulsione per raggiungere ogni 17 gocce per 1 mm di colonna lunghezza di liquido, poi il volume di ogni goccia è 1 NL (volume goccia = π x raggio 2 x lunghezza (di colonna di liquido) / count (di gocce). NOTA: Una goccia di diametro 0,1 mm restituisce un volume di iniezione di circa 500 pl (volume goccia = 4/3 x π x raggio 3). </li></ol> 3. Preparazione di fertilizzato Zebrafish embrioni e iniezione con Morpholinos <ol><li> Impostare Tg (wt1b: GFP) transgenici coppie nidificanti zebrafish secondo protocolli pubblicati per la zootecnia zebrafish di serie e la manutenzione. Raccogliere gli embrioni dopo progenie naturale, e tenerli in una piastra di Petri 10 centimetri riempito con acqua E3 (5mm NaCl, KCl 0,17 mm, 0,33 mM CaCl 2, 0,33 MgSO mm 4). Inizia iniezione MO subito 13. Monitorare dell&#39;embrione morfologia al microscopio e assicurarsi iniezione MO è al onefase delle cellule, o al più tardi nella fase quattro celle. <ol><li> Trasferire gli embrioni di una capsula di Petri 10 centimetri con il cuneo a forma di agar depressioni. Aspirare l&#39;acqua E3 supplementare e premere delicatamente gli embrioni nelle depressioni. </li><li> Manipolare gli embrioni con la micropipetta di visualizzare 1-cellule di embrioni stadio sotto il microscopio da dissezione. Penetrare il corion e poi il tuorlo di iniettare una o due gocce di MO nel tuorlo (1 goccia = 500 pl). Trasferire gli embrioni iniettati di 10 cm capsula di Petri con acqua E3 e incubare a 28,5 ° C. </li></ol></li><li> Dopo le iniezioni, rimuovere gli embrioni morti e registrare il numero di embrioni iniettati. Sostituire l&#39;acqua E3 nel piatto ogni 24-48 ore. </li></ol> 4. Esperimenti fenotipo di soccorso <ol><li> Selezionare un ortologo da un&#39;altra specie che ha una diversa struttura di coppia di base principale (di solito 3-7 coppie di basi disallineamenti) ed è, quindi, resistenti al MO, per il soccorso. Ad esempio, in questo esperimento, abbiamo sceltoMouse perché la sequenza del mouse Wnt5a mRNA è differente dalla sequenza zebrafish. </li><li> Progettare un set di primer con il primer forward partenza dal sito traslazionale e il primer reverse da vicino alla fine della regione codificante mRNA. Aggiungere una sequenza promotore T7 alla 5&#39;fine della sequenza primer forward. In questo esperimento, abbiamo utilizzato le seguenti sequenze di primer; avanti: 5&#39;-TAA tac gac tca tag cta cta gga tga TGC TGC tga agc tga a- 3 &#39;e retromarcia: 5&#39;-TCA ctt gca gac gta ctg gtc- 3&#39;. </li><li> Eseguire Polymerase Chain Reaction 50 microlitri (PCR) con il set di primer (0,5 mM di ciascun primer) progettati sopra e 0.1 mg DNA stampo contenente il mouse Wnt5a sequenza cDNA con il seguente programma PCR: 1 ciclo a 95 ° C per 5 min; 35 cicli di 95 ° C (30 sec), 58,5 ° C (30 sec), 72 ° C (1 minuto); e passo estensione finale a 72 ° C per 7 minuti. </li><li> Dopo aver terminato tegli ciclo, eseguito 2 ml di prodotto di PCR su 1% gel per assicurarsi che la banda è la dimensione giusta. Purificare il prodotto di PCR con un kit di purificazione PCR secondo le istruzioni del produttore. Controllare concentrazione di DNA con uno spettrofotometro. Conservare il prodotto di PCR purificato a -20 ° C o utilizzare direttamente capped trascrizione dell&#39;RNA nel passaggio successivo. </li><li> Sintetizzare in vitro capped mRNA con un kit di sintesi di RNA capped secondo le istruzioni del produttore. Diluire il mRNA in 20 microlitri RNasi acqua gratuita e determinare la concentrazione. Aliquota della RNA e conservare a -80 ° C. </li><li> Il giorno dell&#39;iniezione, preparare una soluzione di lavoro del mRNA con RNasi acqua sterile libera. Si noti che il 40 pg è generalmente la dose massima RNA in cui effetti non specifici o tossici del RNA non si verificano. Mantenere la soluzione di lavoro su ghiaccio. Iniettare l&#39;embrione con MO e quindi l&#39;mRNA di salvataggio, o co-iniezione di entrambi insieme. Ad esempio, se la concentrazione del prepar mRNAed al passo 4.5 è 0,6 mg / mL, aggiungere 0,67 ml di mRNA (0,4 mg) di 4,33 ml di acqua RNasi libero di fare una concentrazione finale di 0,08 mg / mL, allora una goccia (500 pl) di ciascuna iniezione contiene 40 pg di mRNA. Preparare MO come descritto al punto 3. </li></ol> 5. Preparazione di embrioni iniettati per Imaging fluorescente <ol><li> Dopo incubazione a 28,5 ° CO / N, aggiungere 0,003% N-feniltiourea (PTU) all&#39;acqua E3 per prevenire melanizzazione, che colpisce l&#39;osservazione della struttura pronephros mediante microscopia a fluorescenza. Tuttavia, non aggiungere PTU prima gastrulation, perché colpisce sviluppo embrionale precoce. </li><li> Al 48 HPF, dechorionate manualmente gli embrioni con pinzette sottili. Prendere le immagini di 48 e di 72 embrioni HPF al microscopio dissezione luce. NOTA: Queste immagini possono essere prese senza anestesia. </li><li> Circa 10 minuti prima di imaging al microscopio a fluorescenza, anestetizzare gli embrioni mettendoli inuna capsula di Petri 10 centimetri contenente 160 mg / ml tamponata Tricaine. Attendere 5-10 minuti, quindi toccare leggermente il zebrafish per verificare che non si muovono e quindi l&#39;anestesia sta lavorando. </li><li> Dopo gli embrioni vengono anestetizzati, montarli in 3% metilcellulosa e li orientano in posizione prona sotto un microscopio da dissezione (fare riferimento alla Tabella 1). </li><li> Osservare la struttura pronephros sotto un microscopio a fluorescenza (fare riferimento alla Tabella 1) e scattare foto a diversi ingrandimenti (10X e 20X). </li></ol>

<ol>
	<li>Choi, S. Y., 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=Cdc42+deficiency+causes+ciliary+abnormalities+and+cystic+kidneys.">Cdc42 deficiency causes ciliary abnormalities and cystic kidneys.</a> J Am Soc Nephrol. 24 (9), 1435-1450 (2013).</li><li>Hostetter, C. L., Sullivan-Brown, J. L., Burdine, 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=Zebrafish+pronephros:+a+model+for+understanding+cystic+kidney+disease.">Zebrafish pronephros: a model for understanding cystic kidney disease.</a> Dev Dyn. 228 (3), 514-522 (2003).</li><li>Nyengaard, J. R., Bendtsen, 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=Glomerular+number+and+size+in+relation+to+age,+kidney+weight,+and+body+surface+in+normal+man.">Glomerular number and size in relation to age, kidney weight, and body surface in normal man.</a> Anat Rec. 232 (2), 514-201 (1992).</li><li>Keller, G., Zimmer, G., Mall, G., Ritz, E., Amann, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nephron+number+in+patients+with+primary+hypertension.">Nephron number in patients with primary hypertension.</a> N Engl J Med. 348 (2), 101-108 (2003).</li><li>Cullen-McEwen, L. A., Kett, M. M., Dowling, J., Anderson, W. P., Bertram, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nephron+number,+renal+function,+and+arterial+pressure+in+aged+GDNF+heterozygous+mice.">Nephron number, renal function, and arterial pressure in aged GDNF heterozygous mice.</a> Hypertension. 41 (2), 335-340 (2003).</li><li>Gerlach, G. F., Wingert, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kidney+organogenesis+in+the+zebrafish:+insights+into+vertebrate+nephrogenesis+and+regeneration.">Kidney organogenesis in the zebrafish: insights into vertebrate nephrogenesis and regeneration.</a> Wiley Interdiscip Rev Dev Biol. 2 (5), 559-585 (2013).</li><li>Igarashi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview:+nonmammalian+organisms+for+studies+of+kidney+development+and+disease.">Overview: nonmammalian organisms for studies of kidney development and disease.</a> J Am Soc Nephrol. 16 (2), 296-298 (2005).</li><li>Zhou, W., Boucher, R. C., Bollig, F., Englert, C., Hildebrandt, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+mesonephric+development+and+regeneration+using+transgenic+zebrafish.">Characterization of mesonephric development and regeneration using transgenic zebrafish.</a> Am J Physiol Renal Physiol. 299 (5), 1040-1047 (2010).</li><li>Liu, 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=A+defect+in+a+novel+Nek-family+kinase+causes+cystic+kidney+disease+in+the+mouse+and+in+zebrafish.">A defect in a novel Nek-family kinase causes cystic kidney disease in the mouse and in zebrafish.</a> Development. 129 (24), 5839-5846 (2002).</li><li>Slanchev, K., Putz, M., Schmitt, A., Kramer-Zucker, A., Walz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nephrocystin-4+is+required+for+pronephric+duct-dependent+cloaca+formation+in+zebrafish.">Nephrocystin-4 is required for pronephric duct-dependent cloaca formation in zebrafish.</a> Hum Mol Genet. 20 (16), 3119-3128 (2011).</li><li>Huang, L., 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+role+of+Wnt5a+in+prostate+gland+development.">The role of Wnt5a in prostate gland development.</a> Dev Biol. 328 (2), 188-199 (2009).</li><li>Andre, P., 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+Wnt+coreceptor+Ryk+regulates+Wnt/planar+cell+polarity+by+modulating+the+degradation+of+the+core+planar+cell+polarity+component+Vangl2.">The Wnt coreceptor Ryk regulates Wnt/planar cell polarity by modulating the degradation of the core planar cell polarity component Vangl2.</a> J Biol Chem. 287 (53), 44518-44525 (2012).</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>Torres, V. E., Harris, P. C., Pirson, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autosomal+dominant+polycystic+kidney+disease.">Autosomal dominant polycystic kidney disease.</a> Lancet. 369 (9569), 1287-1301 (2007).</li></ol>

The authors have nothing to disclose.

Rene policistico (PKD) è una delle principali cause di malattia renale allo stadio terminale nell&#39;uomo ed è caratterizzata da progressiva formazione di cisti, l&#39;allargamento renale, e lo sviluppo tubulo anormale 14. Autosomica dominante PKD (ADPKD) è una malattia genetica in cui la mutazione di una PKD1, codifica policistina-1 (PC1), o PKD2, codifica policistina-2 (PC2), i risultati di reni policistiche. Molti altri geni, in particolare quelli che codificano proteine ​​che si trovano nel cilium...

Zebrafish (Danio rerio) gli embrioni sono stati ampiamente utilizzati come modello per lo studio dello sviluppo del rene e rene policistico. Ci sono molti vantaggi di utilizzare zebrafish come un modello animale: la fattibilità di studiare le interazioni genetiche, la capacità di utilizzare Morpholinos antisenso (MO) per le proteine ​​atterramento, l&#39;opportunità di saggiare rapidamente un gran numero di embrioni, e la facilità di visualizzazione fenotipi organo in larve 1 vivono. I pronephros è il primo rene a sviluppare nei vertebrati ed è funzionale in zebrafish larvale 2. La struttura dei pronephros zebrafish è relativamente semplice rispetto alle metanefro mammiferi, la terza e ultima rene sviluppare nei mammiferi. Il nefrone è l&#39;unità di lavoro del rene, con ogni rene umano contenente tra 500,000-1,000,000 nefroni 3,4 e ciascun rene del mouse con circa 13.000 nefroni 5, il che rende difficile osservare la struttura singola nefrone in reni umani o il mouse. Il pesce zebra ha solo due nefroni, e ogni nefrone zebrafish contiene tutti i principali componenti presenti nel glomerulo e tubuli di topi e gli esseri umani 6 e tipi di cellule renali specializzati simili. Rispetto ad altri modelli di vertebrati, come Xenopus, nefrone zebrafish assomiglia più da vicino il nefrone mammiferi perché ha un sistema chiuso 7. Negli ultimi anni, il genoma zebrafish è stato sequenziato, permettendo l&#39;ampia introduzione di strumenti genetici, ampie risorse mutanti, e raccolte di linee giornalista transgeniche nei modelli di zebrafish. Pronefro zebrafish forme tra 12-72 ore dopo la fecondazione (HPF) e possono essere visualizzati facilmente negli embrioni trasparenti. Proteina tumore del Wilm WT1 è un fattore essenziale per lo sviluppo del rene. Linee di zebrafish transgenici che esprimono la proteina fluorescente verde (GFP) sotto il controllo del promotore wt1b Tg (wt1b: GFP) mostrano GFP espresfissione specificamente trova nelle regioni pronephric in embrioni di zebrafish, a partire dal 17 hpf 8. Nefronoftisi (NPHP), una malattia renale cistica autosomica recessiva, è causata da mutazioni di geni NPHP 9. NPHP4 atterramento da morpholino causato la formazione di cisti nel Tg (wt1b: GFP). Pesce 10 Pertanto, questo pesce transgenico è un modello adatto per osservare le strutture renali e formazione di cisti durante lo sviluppo del rene. È importante sottolineare che l&#39;influenza di modulatori di sviluppo rene può essere studiata utilizzando questo ceppo in un tempo e manodopera modo efficiente. Il nostro documento descrive l&#39;uso di Tg (wt1b: GFP) pesce come modello per visualizzare rene formazione di cisti dopo la modulazione del gene. Abbiamo usato inizio e splice-site anti-senso MOs per abbattere il gene Wnt5a in zebrafish. Wnt5a è una glicoproteina secreta non canonica della famiglia Wnt che svolge un ruolo importante nello sviluppo dei vari organi e postnatale cellularefunzione 11. Wnt5a opera attraverso percorsi Wnt non canonici, tra cui il percorso di polarità planare delle cellule (PCP), che è stato trovato a svolgere un ruolo nella divisione cellulare orientato durante renale allungamento tubolare. Wnt5a regola l&#39;/ PCP pathway Wnt formando un complesso con il recettore come tirosina chinasi (Ryk), che trasduce ulteriormente segnalazione Wnt5a formando un complesso con la proteina VANGL polarità cellulare planare 2 (Vangl2), promuovendo in tal modo la stabilità Vangl2 12. Difetti nella via PCP possono provocare divisione cellulare casuale e causare la formazione di cisti renale. Abbiamo usato il Tg (wt1b: GFP) Linea zebrafish per osservare la formazione di cisti renali dopo Wnt5a atterramento. Il Tg (wt1b: GFP) modello di zebrafish permette l&#39;imaging dal vivo e l&#39;osservazione puntuale della struttura del rene. Dopo Wnt5a atterramento, formazione di cisti renali è stato trovato con inizio alle 24 HPF; a 72 HPF, cisti potrebbero essere trovati nei glomeruli e tubuli prossimali. Questo metodo può essere utilizzato anche per sCreen altri geni che possono causare la formazione di cisti renali.

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			<td height="21" style="height:21px;width:221px;">0.5% Phenol-Red</td>
			<td style="width:229px;">Sigma</td>
			<td style="width:195px;">P0290</td>
			<td style="width:491px;">Color indicator for injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Morpholino</td>
			<td style="width:229px;">Genn Tools, LLC</td>
			<td style="width:195px;">(customized)</td>
			<td>Customized designed to the gene of interest</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:221px;">Pre-pulled needle</td>
			<td style="width:229px;">Tritech Research</td>
			<td style="width:195px;">MINJ-PP</td>
			<td style="width:491px;">If large amount of needle is required, you can also purchase a needle puller and prepare the needle in the lab</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">T7 mMessage Kit</td>
			<td style="width:229px;">Ambion</td>
			<td style="width:195px;">1344</td>
			<td>For in vitro transcription to make capped mRNA for rescue experiment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">N-Phenylthiourea</td>
			<td style="width:229px;">Sigma</td>
			<td style="width:195px;">P7629</td>
			<td>For prevention of melanization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Tricaine</td>
			<td style="width:229px;">Sigma</td>
			<td style="width:195px;">A5040</td>
			<td>Also called ethyl 3-aminobenzoate, for zebrafish anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Methyl Cellulose</td>
			<td style="width:229px;">Sigma</td>
			<td style="width:195px;">M-0387</td>
			<td>For position of zebrafish&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">QIAquick PCR Purification Kit&#160;</td>
			<td style="width:229px;">Qiagen</td>
			<td style="width:195px;">28104</td>
			<td>For purification of PCR product for cap RNA synthesis.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">dNTP mix</td>
			<td style="width:229px;">Promega</td>
			<td style="width:195px;">U1511</td>
			<td>For PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Tag DNA Polymerase</td>
			<td style="width:229px;">Invitrogen</td>
			<td style="width:195px;">10342-053</td>
			<td>For PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;"></td>
			<td style="width:229px;"></td>
			<td style="width:195px;"></td>
			<td></td>
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			<td style="width:195px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:221px;">Equipment</td>
			<td style="width:229px;"></td>
			<td style="width:195px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">NanoDrop sepctrophotometer</td>
			<td style="width:229px;">Thermo Scientific</td>
			<td style="width:195px;">ND-1000</td>
			<td>For measure morpholino and RNA concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Air Compressor</td>
			<td style="width:229px;">Werther International, Inc.</td>
			<td style="width:195px;">Panther Compact 106</td>
			<td>Air source for injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Pico micro-injection pump</td>
			<td style="width:229px;">World Precision Instruments Inc</td>
			<td style="width:195px;">PV830 Pnematic PicoPump</td>
			<td>Other types of microinjection system can be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Micro-manuplator</td>
			<td style="width:229px;">World Precision Instruments Inc</td>
			<td style="width:195px;">MMJR</td>
			<td>Right-handed (MMJL for left handed)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Needle holder</td>
			<td style="width:229px;">World Precision Instruments Inc</td>
			<td style="width:195px;">5430-ALL</td>
			<td>To hold needle for micromanipulation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Dumont Tweezers</td>
			<td style="width:229px;">Fine Surgical Tools</td>
			<td style="width:195px;">11253-20</td>
			<td>For breaking off the needle tip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Dissecting microscope</td>
			<td>Leica M205C&#160;</td>
			<td style="width:195px;"></td>
			<td>For observing and imaging zebrafish embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Fluorscence microscope</td>
			<td>Zeiss Axio Obserer D1m &#160;</td>
			<td style="width:195px;"></td>
			<td>For imaging zebrafish pronephros</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Capillary tube (I.D 0.15 mm)</td>
			<td>VitroCom</td>
			<td style="width:195px;">CV1525Q-100</td>
			<td>For measure the volume of each injected drop</td>
		</tr>
	</tbody>
</table>

a possible zebrafish model of polycystic kidney disease: knockdown of wnt5a causes cysts in zebrafish kidneys

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to &#8220;off-target&#8221; effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.

Wnt5a abbattere è stato ottenuto con l&#39;introduzione di traduzione bloccando MO (AUG-MO) o esone / introni splice confine MO (giuntura-MO) per zebrafish embrioni nella fase di una cella. Il AUG-MO rivolge il codone di inizio e, quindi, inibisce entrambi messaggio Wnt5a materna e zigotico. La giunzione-MO rivolge il terzo sito donatore di splicing e inibisce solo la trascrizione zygotic di Wnt5a (Figura 1A). Il AUG- e morphants splice- phenocopied vicenda con più difetti, tra cui l...

Watch this Scientific Journal Video about A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys at JoVE.com

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish.

Un possibile modello Zebrafish della malattia del rene policistico: Knockdown di<em&gt; Wnt5a</em&gt; Provoca cisti in Zebrafish Reni

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The ...

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Research

JoVE Journal

Medicine

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

11.0K Views.  Eastern Virginia Medical School. We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish. 

Video: A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

Induction of Myocardial Infarction in Adult Zebrafish Using Cryoinjury

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction1. By contrast, urodele amphibians and teleost fish retain a remarkable capacity for cardiac regeneration with little or no scarring throughout life2,3. It is not known why only some non-mammalian vertebrates can recreate a complete organ from remnant tissues4,5. To understand the molecular and cellular differences between regenerative responses in different species, we need to use similar approaches for inducing acute injuries.
In mammals, the most frequently used model to study cardiac repair has been acute ischemia after a ligation of the coronary artery or tissue destruction after cryoinjury6,7. The cardiac regeneration in newts and zebrafish has been predominantly studied after a partial resection of the ventricular apex2,3. Recently, several groups have established the cryoinjury technique in adult zebrafish8-10. This method has a great potential because it allows a comparative discussion of the results obtained from the mammalian and non-mammalian species.
Here, we present a method to induce a reproducible disc-shaped infarct of the zebrafish ventricle by cryoinjury. This injury model is based on rapid freezing-thawing tissue, which results in massive cell death of about 20% of cardiomyocytes of the ventricular wall. First, a small incision was made through the chest with iridectomy scissors to access the heart. The ventricular wall was directly frozen by applying for 23-25 seconds a stainless steel cryoprobe precooled in liquid nitrogen. To stop the freezing of the heart, fish water at room temperature was dropped on the tip of the cryoprobe. The procedure is well tolerated by animals, with a survival rate of 95%.
To characterize the regenerative process, the hearts were collected and fixed at different days after cryoinjury. Subsequently, the specimen were embedded for cryosectioning. The slides with sections were processed for histological analysis, in situ hybridization and immunofluorescence. This undertaking enhances our understanding of the factors that are required for the regenerative plasticity in the zebrafish, and provide new insights into the machinery of cardiac regeneration. A conceptual and molecular understanding of heart regeneration in zebrafish will impact both developmental biology and regenerative medicine.

Zebrafish represents a valuable model to study the mechanisms of heart regeneration in vertebrates. Here, we present a protocol for induction of a heart infarct in adult zebrafish using cryoinjury. This method results in massive cell death within 20% of the ventricular wall, similar to that observed in mammalian infarcts.

L&#39;induzione di infarto miocardico in Zebrafish adulti usando Cryoinjury

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction1. By contrast, urodele amphibians and ...

Ricerca

Medicina

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Valutare i cambiamenti teratogeni in un modello di Zebrafish di esposizione alcolica fetale

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

Cariotipo spettrale allo studio anomalie cromosomiche negli esseri umani e nei topi con malattia del rene policistico

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

Screening per Modificatori melanoma utilizzando un modello Zebrafish Tumore autoctone

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

A Zebrafish Model of Diabetes Mellitus and Metabolic Memory

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory and large scale clinical trials has revealed that diabetic complications progress unimpeded via the phenomenon of metabolic memory even when glycemic control is pharmaceutically achieved. Gene expression can be stably altered through epigenetic changes which not only allow cells and organisms to quickly respond to changing environmental stimuli but also confer the ability of the cell to "memorize" these encounters once the stimulus is removed. As such, the roles that these mechanisms play in the metabolic memory phenomenon are currently being examined.
We have recently reported the development of a zebrafish model of type I diabetes mellitus and characterized this model to show that diabetic zebrafish not only display the known secondary complications including the changes associated with diabetic retinopathy, diabetic nephropathy and impaired wound healing but also exhibit impaired caudal fin regeneration. This model is unique in that the zebrafish is capable to regenerate its damaged pancreas and restore a euglycemic state similar to what would be expected in post-transplant human patients. Moreover, multiple rounds of caudal fin amputation allow for the separation and study of pure epigenetic effects in an in vivo system without potential complicating factors from the previous diabetic state. Although euglycemia is achieved following pancreatic regeneration, the diabetic secondary complication of fin regeneration and skin wound healing persists indefinitely. In the case of impaired fin regeneration, this pathology is retained even after multiple rounds of fin regeneration in the daughter fin tissues. These observations point to an underlying epigenetic process existing in the metabolic memory state. Here we present the methods needed to successfully generate the diabetic and metabolic memory groups of fish and discuss the advantages of this model.

Metabolic memory is the phenomenon by which diabetic complications persist and progress unimpeded even after euglycemia is achieved pharmaceutically. Here we describe a diabetes mellitus zebrafish model which is unique in that it allows for the examination of the mitotically transmissible epigenetic components of metabolic memory in vivo.

Un modello Zebrafish di diabete mellito e di memoria metabolica

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory ...

Assessment of Vascular Function in Patients With Chronic Kidney Disease

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA, aPWV, and vascular endothelial cell protein expression.

The degree of vascular dysfunction and contributing physiological mechanisms can be assessed in patients with chronic kidney disease by measuring brachial artery flow-mediated dilation, aortic pulse-wave velocity, and vascular endothelial cell protein expression.

Valutazione della funzione vascolare nei pazienti con malattia renale cronica

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this ...

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

L&#39;uso di ultra-alto campo MRI in piccolo roditore modelli di malattia del rene policistico per<em&gt; In Vivo</em&gt; Fenotipizzazione and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish

The genetically accessible adult zebrafish (Danio rerio) has been increasingly used as a vertebrate model for understanding human diseases such as cardiomyopathy. Because of its convenience and amenability to high throughput genetic manipulations, the generation of acquired cardiomyopathy models, such as the doxorubicin-induced cardiomyopathy (DIC) model in adult zebrafish, is opening the doors to new research avenues, including discovering cardiomyopathy modifiers via forward genetic screening. Different from the embryonic zebrafish DIC model, both initial acute and later chronic phases of cardiomyopathy can be determined in the adult zebrafish DIC model, enabling the study of stage-dependent signaling mechanisms and therapeutic strategies. However, variable results can be obtained with the current model, even in the hands of experienced investigators. To facilitate future implementation of the DIC model, we present a detailed protocol on how to generate this DIC model in adult zebrafish and describe two alternative ways of intraperitoneal (IP) injection. We further discuss options on how to reduce variations to obtain reliable results and provide suggestions on how to appropriately interpret the results.

A method to generate a doxorubicin-induced cardiomyopathy model in adult zebrafish (Danio rerio) is described here. Two alternative ways of intraperitoneal injection are presented and conditions to reduce variations among different experimental groups are discussed.

Un modello di cardiomiopatia indotta da doxorubicina in Zebrafish adulto

The genetically accessible adult zebrafish (Danio rerio) has been increasingly used as a vertebrate model for understanding human diseases such as ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

Valutazione della funzione del rene in modelli murini di malattia glomerulare

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Misurazione di contrattilità isometrica dell'arteria mesenterica Mouse con filo Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...