Diese Arbeit wurde durch die NIH (DK093625 an LH und DK069909 und DK047757 zu JHL) und die VA (Merit Award I01BX000820 zu JHL). Wir möchten Dr. Michael Pack und Dr. Jie er für die Bereitstellung wesentlicher Unterstützung danken an der Universität von Pennsylvania Zebrafisch-Core.

HINWEIS: Ethik Hinweise: Alle Zebrafisch-Experimente wurden von der Institutional Animal Care und Verwenden Committee an der Eastern Virginia Medical School zugelassen. 1. Morpholino Vorbereitung <ol><li> Design und Synthese Übersetzung blockier (Aug-) und Spleiß-Hemmung (Spleiß-) Antisense-Morpholino (MO) Oligonukleotide für das Gen von Interesse nach Herstellerangaben (1A). Bitte lesen Sie Informationen des Herstellers, die in Tabelle 1. HINWEIS: MOs werden als lyophilisierte Aktien in Glasflaschen geliefert. </li><li> Hinzufügen hochwertigen sterilem Wasser auf die Glasflasche zu resuspendieren MOs zu einer Endkonzentration von 25 ug / ul. Stellen Sie sicher, dass das Oligonukleotid vollständig gelöst ist. Wenn einige solide bleibt, heizen das Fläschchen mit dem Lager Oligonukleotid-Lösung bei 65 ° C für 5 bis 10 Minuten und kurz vortexen. </li><li> Bewahren Sie die MO-Stammlösung bei RT. Lagern Sie diese nicht bei 4 ° C oder -20 °; C, weil geringere Temperaturen können dazu führen, die MO-Oligonukleotids an der Behälterwand zu binden. Messen Sie die Konzentration der Stammlösung mit einem Spektralphotometer jedes Mal eine neue MO-Stammlösung hergestellt wird (siehe Tabelle 1 beziehen). </li><li> Bereiten Sie die MO-Arbeitslösung am Tag der Injektion durch Verdünnung der Stammlösung mit hochgradigen sterilem Wasser auf die gewünschte Dosis. Mit 0,5% Phenol-Rot zu einer Konzentration von 0,05% zu erreichen. Wenn Sie beispielsweise 5 ul Arbeitslösung mit einer Konzentration von 15 ng / nL, fügen 3 ul MO Stammlösung und 0,5 ul 0,5% Phenol-rot bis 1,5 ul Wasser. HINWEIS: Mit dieser Arbeitskonzentration, jeder Tropfen (500 pl) der Injektion enthält 7,5 ng MO und zwei Tropfen enthalten 15 ng von MO. </li></ol> 2. Herstellung der Einspritzvorrichtung <ol><li> Kauf Glas vorgezogen Nadeln (siehe Tabelle 1 für weitere Informationen). Alternativ ziehen Sie das Glas Injektionsnadel with eine Nadel Magnet. </li><li> Schalten Sie den Kompressor und stellen Sie die Druckeinstellung auf 50 psi. Schalten Sie den Binokular-Lichtquelle und Pico Mikroeinspritzpumpe und passen Sie die Einstellungen wie folgt: Halten Druck 20 psi, werfen Druck 10 psi, Zeit-Wert von 2,5 und 100 &amp; mgr; s Palette von Gating. </li><li> Legen Sie das Glas vor, zog Nadel mit 5 ul der Injektionslösung und setzen Sie die Nadel in vertikaler Position mit der Spitze nach unten. Warten Sie, bis die gesamte Lösung der Nadelspitze ohne sichtbare Luftblasen erreicht. Setzen Sie den Nadelhalter in einer passenden Position neben dem Mikroskop und legen Sie die Glasnadel in die Halterung. Ist der Einspritzwinkel um 45 Grad. </li><li> Bringen Sie die Nadelspitze in Sicht unter dem Mikroskop, hoch über der Bühne, und konzentrieren sich auf die dünnsten Bereich der Spitze. Brechen Sie die Nadelspitze mit feinen Spitze Pinzette. </li><li> Legen Sie ein Lineal und ein Rohr oder Schlauch mit Innendurchmesser von 0,15 mm nebeneinander unter dem Mikroskop;Injizieren der Lösung in ein Ende des Kapillarröhrchens einen Flüssigkeitssäule bilden. Stellen Sie die Auswurfzeit alle 17 Tropfen je 1 mm Länge Flüssigkeitssäule zu erreichen, wird das Volumen jeder Tropfen ist 1 nl (Tropfenvolumen = π x Radius 2 x Länge (der Flüssigkeitssäule) / count (Tropfen). HINWEIS: Ein Tropfen mit einem Durchmesser von 0,1 mm ergibt einen Injektionsvolumen von etwa 500 pl (Tropfenvolumen = 4/3 x π x Radius 3). </li></ol> 3. Herstellung der befruchteten Zebrafischembryonen und Injektion mit Morpholinos <ol><li> Transgenen Zebrafisch Zuchtpaare nach den veröffentlichten Protokollen für Standard Zebrafischhaltung und Wartung: bis Tg (GFP wt1b) ein. Sammeln Embryonen nach Naturbrut, und halten sie in einer 10 cm Petrischale mit E3 Wasser gefüllt (5 mM NaCl, 0,17 mm KCl, 0,33 mM CaCl 2, 0,33 mM MgSO 4). Starten MO Injektion sofort 13. Überwachen Embryo Morphologie unter dem Mikroskop und stellen Sie sicher, MO Injektion ist im Ein-Zell-Stadium, oder spätestens am Vierzellstadium. <ol><li> Transfer der Embryonen in einen 10-cm-Petrischale mit Agar Tröge keilförmig. Saugen Sie das zusätzliche E3 Wasser und drücken Sie vorsichtig die Embryonen in die Tröge. </li><li> Bearbeiten Sie die Embryonen mit der Mikropipette 1-Zell-Stadium Embryos unter dem Binokular visualisieren. Durchdringen die Chorion und dann das Eigelb, ein oder zwei Tropfen MO in den Dotter (1 Tropfen = 500 pl) zu injizieren. Übertragen Sie die injizierten Embryonen zu einer 10 cm Petrischale mit E3 Wasser und Inkubation bei 28,5 ° C. </li></ol></li><li> Nach den Injektionen, entfernen Sie die toten Embryonen und notieren Sie die Anzahl der injizierten Embryonen. Ersetzen Sie die E3 Wasser in der Schale alle 24 bis 48 Stunden. </li></ol> 4. Phänotyp Rettungsversuche <ol><li> Auszuwählen, die ein Ortholog von einer anderen Spezies, die eine andere primäre Basispaarstruktur (üblicherweise 3-7 Basenpaar Mismatches) aufweist und daher beständig gegen das MO, zur Rettung. Beispielsweise wird in diesem Experiment haben wir unsMaus weil die Maus Wnt5a mRNA-Sequenz unterscheidet sich von der Zebrafisch-Sequenz. </li><li> Entwerfen eines Primers mit der Vorwärtsprimer beginnend beim Translationsstelle und der Reverse-Primer in der Nähe des Endes der mRNA codierende Region eingestellt. Hinzufügen eines T7-Promotorsequenz am 5&#39;-Ende des Vorwärtsprimer-Sequenz. In diesem Experiment verwendeten wir die folgenden Primersequenzen; vorwärts: 5&#39;-taa tac gac tca cta Tag GGA cta tga tgc tgc tga agc tga a- 3 &#39;und umgekehrt: 5&#39;-TCA ctt gca gac gta ctg gtc- 3&#39;. </li><li> Durchführen einer 50 ul Polymerase-Kettenreaktion (PCR) mit den Primersatz (0,5 &amp; mgr; M von jedem Primer) vorstehend ausgebildet und 0,1 &amp; mgr; g Matrizen-DNA, die das Maus Wnt5a cDNA-Sequenz mit der folgenden PCR-Programm: 1 Zyklus bei 95 ° C für 5 min; 35 Zyklen von 95 ° C (30 sec), 58,5 ° C (30 sec), 72 ° C (1 min); und abschließende Extension bei 72 ° C für 7 min. </li><li> Nach Abschluss ter Zyklus ausführen 2 ul des PCR-Produkts auf 1% Agarosegel, um sicherzustellen, dass die Band die richtige Größe. Man reinige das PCR-Produkt mit einem PCR-Purification-Kit nach den Anweisungen des Herstellers. Überprüfen DNA-Konzentration mit einem Spektralphotometer. Speicherung des gereinigten PCR-Produkts bei -20 ° C oder direkt in verkappter RNA Transkription Verwendung im nächsten Schritt. </li><li> Synthetisieren in vitro mRNA mit Cap mit einem verkappten RNA-Synthese-Kits nach den Anweisungen des Herstellers. Verdünnen Sie die mRNA in 20 ul RNase freies Wasser und Bestimmung der Konzentration. Aliquot der RNA und bei -80 ° C. </li><li> Am Tag der Injektion, bereiten eine Arbeitslösung der mRNA mit RNase frei sterilem Wasser. Man beachte, daß 40 pg ist im allgemeinen die höchste RNA Dosis, bei der unspezifische oder toxische Wirkung des RNA treten nicht auf. Halten Sie die Arbeitslösung auf Eis. Spritzen Sie die Embryos mit MO und dann die Rettung mRNA, oder Co-spritzen beide zusammen. Wenn beispielsweise die Konzentration der mRNA prepared in Schritt 4.5 ist 0,6 ug / ul, fügen 0,67 ul mRNA (0,4 ug) bis 4,33 &amp; mgr; l RNase-freies Wasser bis zu einer Endkonzentration von 0,08 ug / ul zu ergeben, und ein Tropfen (500 pl) jeder Injektion enthält 40 pg mRNA. Bereiten MO, wie in Schritt 3 beschrieben. </li></ol> 5. Vorbereitung der injizierten Embryonen für Fluorescent Imaging <ol><li> Nach der Inkubation bei 28,5 ° CO / N, fügen Sie 0,003% N-Phenylthioharnstoff (PTU) mit dem E3 Wasser Melanisierung, die Beobachtung der Vorniere Struktur mittels Fluoreszenzmikroskopie wirkt verhindern. Allerdings PTU nicht vor Gastrulation abgeben, weil es die frühe embryonale Entwicklung betrifft. </li><li> Bei 48 HPF, manuell dechorionate die Embryonen mit feinen Pinzette. Nehmen Sie Bilder von 48 und 72 HPF Embryonen unter einem Licht Dissektionsmikroskop. Hinweis: Diese Bilder sind ohne Narkose entnommen werden. </li><li> Etwa 10 Minuten vor der unter dem Fluoreszenzmikroskop Bildgebung, betäuben die Embryonen, indem Sie sie in10 cm Petrischale mit 160 ug / ml gepufferte Tricaine. Warten Sie 5-10 Minuten, und dann locker am Zebrafisch zu bestätigen, dass sie sich nicht bewegen und damit die Narkose wirkt. </li><li> Nachdem die Embryos betäubt werden, montieren sie in 3% Methylcellulose und orientieren sie in Bauchlage unter einem Binokular (siehe Tabelle 1). </li><li> Beachten Sie die Vorniere Struktur unter einem Fluoreszenzmikroskop (siehe auch Tabelle 1) und nehmen Sie Bilder mit unterschiedlicher Vergrößerung (10X und 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.

Polyzystische Nierenerkrankung (PKD) ist eine der führenden Ursachen für end-stage renal Krankheit beim Menschen und wird durch fortschreitende Zysten, Nieren Erweiterung und abnorme Entwicklung Röhrchen 14 aus. Autosomal dominante PKD (ADPKD) ist eine Erbkrankheit, bei der die Mutation von entweder PKD1 kodiert Polycystin-1 (PC1) oder PKD2, Polycystin-2 (PC2) kodiert, führt zu Zystennieren. Viele andere Gene, vor allem Proteine, die in der primären Zilien gefunden codiert, wird angenommen, dass bei der ...

Zebrafisch (Danio rerio) Embryonen wurden allgemein als Modell für die Untersuchung der Nierenentwicklung und polyzystische Nierenerkrankung verwendet. Es gibt viele Vorteile der Verwendung von Zebrafisch als Tiermodell: Die Durchführbarkeit Studium genetischen Wechselwirkungen, die Fähigkeit, Antisense-Morpholino (MO) für die Proteinzuschlags verwenden, die Möglichkeit, schnell zu testen große Anzahl von Embryos und die Betrachtung Organ Phänotypen lebenden Larven 1. Die Vorniere ist die erste Niere bei Wirbeltieren entwickeln und ist funktional in Zebrafisch-Larven 2. Die Struktur der Zebrafisch pronephros ist relativ einfach im Vergleich zu den Säuger Metanephros, um die dritte und letzte Niere in Säugetieren zu entwickeln. Nephronanalog ist die Arbeitseinheit der Niere, wobei jede humane Nieren enthaltend zwischen 500.000-1.000.000 Nephronen 3,4 und jeder Maus Niere mit ungefähr 13.000 Nephronen 5, was es schwierig macht, einzelne Nephron Struktur beobachten in Mensch oder Maus Nieren. Der Zebrafisch besitzt nur zwei Nephronen und jedes Zebrabärbling Nephron enthält alle Hauptkomponenten im Glomerulus und Tubuli von Mäusen und Menschen und 6 ähnlich speziellen Nierenzelltypen gefunden. Im Vergleich zu anderen Wirbeltiermodellen wie Xenopus, Zebrafisch Nephron mehr ähnelt die Säuger Nephron weil es ein geschlossenes System 7 hat. In den letzten Jahren hat sich die Zebrafisch wurde sequenziert, was die breite Einführung von genetischen Werkzeugen umfangreichen Mutanten Ressourcen und Sammlungen von transgenen Reporterlinien in Zebrafisch-Modelle. Die Zebrafisch Vorniere bildet sich zwischen 12 bis 72 Stunden nach der Befruchtung (HPF) und kann leicht in den durchsichtigen Embryos visualisiert werden. Die Wilms-Tumor Protein WT1 ist ein wesentlicher Faktor für die Nierenentwicklung. Transgenic Zebrafischlinien grün fluoreszierende Protein (GFP) unter der Kontrolle des Promotors wt1b Tg Ausdruck (wt1b: GFP) zeigen GFP expression speziell in pronephric Regionen in Zebrafischembryonen, ab 17 HPF 8. Nephronophthisis (NPHP), eine autosomal-rezessive zystischen Nierenerkrankungen, wird durch Mutationen der Gene NPHP 9 verursacht. NPHP4 durch Morpholino Knockdown verursacht Zystenbildung in der Tg (wt1b: GFP). Fisch 10 Daher ist dieser transgene Fische ein geeignetes Modell zur Beobachtung Nieren Strukturen und Zystenbildung während der Nierenentwicklung. Wichtig ist, dass der Einfluss von Modulatoren der Nierenentwicklung Verwendung dieses Stammes in einer Zeit und Arbeit effizient untersucht werden. Unser Papier beschreibt die Verwendung von Tg (wt1b: GFP) Fisch als Vorbild zu Nierenzystenbildung nach Genmodulation visualisieren. Wir benutzten Start- und Spleißstellen Antisense-MOs um knock down der Wnt5a Gen in Zebrafisch. Wnt5a ist eine nicht-kanonische sekretierte Glykoprotein des Wnt-Familie, die in der Entwicklung von verschiedenen Organen und postnatalen zellulären eine wichtige Rolle spielt,Funktion 11. Wnt5a wirkt durch nicht-kanonischen Wnt-Signalwege, einschließlich der planaren Zellpolarität (PCP) Weg, die gefunden wurde, um eine Rolle in orientierte Zellteilung während der Nierentubuli Dehnung zu spielen. Wnt5a regelt die Wnt / PCP-Weg, indem ein Komplex mit dem Rezeptor ähnliche Tyrosinkinase (Ryk), die weiter transduziert Wnt5a Signalisierung durch Bildung eines Komplexes mit dem VANGL planaren Zellpolarität protein 2 (Vangl2), wodurch Vangl2 Stabilität 12 fördern. Defekte in der PCP-Weg kann in zufälliger Zellteilung verursachen und eine Nierenzystenbildung. Wir nutzten die Tg (wt1b: GFP) Zebrafisch Linie zu Nierenzystenbildung folgenden Wnt5a Zuschlags beobachten. Die Tg (wt1b: GFP) Zebrafisch-Modell ermöglicht die Live-Darstellung und zeitnahe Beobachtung der Nierenstruktur. Nach Wnt5a Zuschlags wurde Nierenzystenbildung gefunden, beginnend bei 24 HPF; bei 72 HPF, könnte Zysten in den Glomeruli und den proximalen Tubuli gefunden werden. Diese Methode könnte auch zu s verwendet werdencreen andere Gene, die Nierenzystenbildung verursachen könnten.

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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>
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		<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>
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		<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>
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		<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 niederschlagen wurde durch die Einführung Übersetzung blockieren MO erreicht (August-MO) oder Exon / Intron-Grenze Splice-MO (Splice-MO) auf Zebrafischembryonen an dem einen Zellstadium. Die August-MO zielt auf das Start-Codon und daher hemmt sowohl von Müttern und zygotischen Wnt5a Nachricht. Der Spleiß-MO zielt die dritte Spleißdonorstelle und hemmt nur die zygotischen Transkript Wnt5a (1A). Die Aug- und Spleiß- morphants phenocopied seitig mit mehreren Mängeln, darunter...

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.

Eine mögliche Zebrafisch-Modell der Kidney Disease: Knockdown von<em&gt; Wnt5a</em&gt; Ursachen Zysten in Zebrafisch-Nieren

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 Aufrufe.  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. 

Serie: 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.

Induktion eines Myokardinfarktes in Adult Zebrafisch Mit Cryoinjury

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

Forschung

Medizin

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.

Die Beurteilung Teratogene Änderungen in einem Zebrafisch-Modell der 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, ...

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.

Spektrale Karyotypisierung zu Chromosomenanomalien in Menschen und Mäusen mit Kidney Disease Study

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 für Melanom Modifiers mit einem Zebrafisch Autochthone Tumormodell

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.

Ein Zebrafisch Modell des Diabetes mellitus und Stoffwechselkrankheiten Speicher

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.

Beurteilung der Gefäßfunktion bei Patienten mit chronischer Nierenerkrankung

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.

Verwendung von Ultrahochfeld-MRT in Kleintier Modelle aus Polyzystische Nierenerkrankung für<em&gt; In Vivo</em&gt; Phänotypisierung und 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.

Doxorubicin-induzierten Kardiomyopathie Model in Erwachsenen Zebrafisch

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.

Beurteilung der Nierenfunktion in Mausmodellen der glomerulären Erkrankung

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.

Messung der isometrischen Kontraktilität der Maus mesenterialen Arterie mit Draht Myographie

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