Ce travail a été soutenu par le NIH (DK093625 à la LH et DK069909 et DK047757 à JHL) et VA (Merit Award I01BX000820 à JHL). Nous tenons à remercier le Dr Michael Pack et le Dr Jie Il à l&#39;Université de Pennsylvanie poisson zèbre de base pour fournir un soutien essentiel.

Déclaration de l&#39;éthique: NOTE: Toutes les expériences de poisson zèbre ont été approuvés par le soin et l&#39;utilisation des animaux Commission institutionnelle à l&#39;Eastern Virginia Medical School. 1. Préparation Morpholino <ol><li> Conception et traduction synthétiser-blocage (août-) et épissage inhiber (jonction ou) anti-sens morpholino (MO) oligonucléotides pour le gène d&#39;intérêt selon les instructions du fabricant (figure 1A). Se il vous plaît voir les informations du fabricant dans le tableau 1. NOTE: OM sont expédiées comme les stocks lyophilisés dans des bouteilles en verre. </li><li> Ajouter de l&#39;eau stérile de haute qualité pour les bouteilles en verre pour remettre en suspension les OM à une concentration finale de 25 ug / ul. Assurez-vous que l&#39;oligonucléotide est complètement dissous. Si quelques restes solides, chauffer le flacon contenant la solution stock oligonucléotide à 65 ° C pendant 5 à 10 min et le vortex brièvement. </li><li> Rangez la solution stock de MO à température ambiante. Ne pas les stocker à 4 ° C ou -20 °; C car des températures plus basses peuvent provoquer l&#39;oligonucléotide MO à se lier à la paroi du récipient. Mesurer la concentration de la solution d&#39;achat d&#39;actions à l&#39;aide d&#39;un spectrophotomètre (se il vous plaît se référer à Tableau 1) à chaque fois une nouvelle solution MO boursier est faite. </li><li> Préparer la solution de travail MO sur le jour de l&#39;injection par dilution de la solution mère avec de l&#39;eau stérile de haute qualité pour la dose souhaitée. Ajouter 0,5% de phénol-rouge pour atteindre une concentration de 0,05%. Par exemple, pour rendre la solution de travail de 5 pi avec une concentration de 15 ng / nL, ajouter 3 pi MO solution stock et 0,5 ul de 0,5% de phénol-rouge pour 1,5 pi d&#39;eau. NOTE: Avec cette concentration de travail, chaque goutte (500 pl) d&#39;injection contient 7,5 ng de MO et de deux gouttes contient 15 ng de MO. </li></ol> 2. Préparation de l&#39;appareil d&#39;injection <ol><li> Achat verre aiguilles pré-tiré (se il vous plaît voir le tableau 1 pour des informations détaillées). Sinon, tirez l&#39;injection de verre aiguille with un extracteur de l&#39;aiguille. </li><li> Allumez le compresseur d&#39;air et d&#39;ajuster le réglage à 50 psi pression. Allumez la source de lumière du microscope de dissection et Pico pompe micro-injection et d&#39;ajuster les paramètres comme suit: maintenir la pression de 20 psi, éjectez la pression de 10 psi, la valeur de période de 2,5 et 100 microsecondes gamme de déclenchement. </li><li> Chargez le verre aiguille pré-tiré avec 5 pi de la solution d&#39;injection et placer l&#39;aiguille dans une position verticale avec la pointe vers le bas. Attendez jusqu&#39;à ce que toute la solution atteint la pointe de l&#39;aiguille sans bulles d&#39;air visibles. Placer le support d&#39;aiguille dans une position appropriée à côté du microscope et insérer l&#39;aiguille de verre dans le support. Ajustez l&#39;angle d&#39;injection à 45 degrés. </li><li> Apportez la pointe de l&#39;aiguille dans la vue sous le microscope, haute hors de la scène, et se concentrer sur la région la plus mince de la pointe. Casser la pointe de l&#39;aiguille avec de fines pinces ponctuelles. </li><li> Placez une règle et un tube capillaire avec diamètre intérieur de 0,15 mm côte à côte sous le microscope;injecter la solution dans une extrémité du tube capillaire à faire une colonne de liquide. Réglez le temps d&#39;éjection pour atteindre toutes les 17 gouttes par colonne de liquide de longueur de 1 mm, le volume de chaque goutte est une nl (volume de la goutte = π x rayon de 2 x de longueur (de la colonne de liquide) / nombre (de gouttes). REMARQUE: Une goutte d&#39;un diamètre de 0,1 mm donne un volume d&#39;injection de l&#39;ordre de 500 pl (volume de goutte = 4/3 π x rayon x 3). </li></ol> 3. Préparation de fécondés embryons de poissons zèbres et injection avec Morpholinos <ol><li> Mettre en place Tg (wt1b: GFP) couples reproducteurs de poisson zèbre transgénique selon des protocoles publiés pour l&#39;élevage du poisson zèbre standard et l&#39;entretien. Recueillir des embryons après spawn naturel, et de les garder dans une boîte de Pétri 10 cm rempli d&#39;eau E3 (5 mM NaCl, KCl 0,17 mm, 0,33 mM de CaCl2, 0,33 mM MgSO 4). Lancer injection MO immédiatement 13. Surveiller embryon morphologie sous le microscope et faire l&#39;injection MO sûr, ce est à la de unstade de la cellule, ou au plus tard au stade de quatre cellules. <ol><li> Transfert des embryons dans une boîte de Pétri de 10 cm avec de la gélose en forme de coin creux. Aspirer l&#39;eau E3 supplémentaire et appuyez doucement sur les embryons dans les creux. </li><li> Manipuler les embryons avec la micropipette pour visualiser embryons au stade 1 cellule sous le microscope de dissection. Pénétrer le chorion puis le jaune d&#39;injecter une ou deux gouttes de MO dans le jaune (1 goutte = 500 pl). Transférer les embryons injectés à une boîte de Pétri de 10 cm avec de l&#39;eau E3 et incuber à 28,5 ° C. </li></ol></li><li> Après les injections, retirer les embryons morts et d&#39;enregistrer le nombre d&#39;embryons injectés. Remplacer l&#39;eau E3 dans le plat tous les 24 à 48 heures. </li></ol> 4. Les expériences phénotype sauvetage <ol><li> Sélectionnez un orthologue d&#39;une autre espèce qui a une structure différente (habituellement 3-7 paires de bases de l&#39;inadéquation) de paires de base primaire et est donc résistant à la MO, pour le sauvetage. Par exemple, dans cette expérience, nous avons choisisouris parce que la séquence d&#39;ARNm de souris Wnt5a est différente de la séquence de poisson zèbre. </li><li> Conception d&#39;un jeu d&#39;amorces avec l&#39;amorce sens à partir de l&#39;emplacement de translation et l&#39;amorce inverse à proximité de l&#39;extrémité de la région codante de l&#39;ARNm. Ajouter une séquence de promoteur de T7 à l&#39;extrémité 5 &#39;de la séquence de l&#39;amorce sens. Dans cette expérience, nous avons utilisé les séquences d&#39;amorces suivantes; avant: 5&#39;-taa tac GAC TCA GGA tag CTA CTA tga tgc tgc TGA TGA AGC a- 3 &#39;et inverser: 5&#39;TCA ctt gca GAC gta CTG gtc- 3&#39;. </li><li> Effectuer une réaction en chaîne par polymerase de 50 pi (PCR) avec le jeu d&#39;amorces (0,5 uM de chaque amorce) conçu ci-dessus et 0,1 ug d&#39;ADN matrice contenant la souris séquence Wnt5a ADNc avec le programme de PCR suivant: 1 cycle à 95 ° C pendant 5 min; 35 cycles de 95 ° C (30 sec), 58,5 ° C (30 sec), 72 ° C (1 min); et l&#39;étape d&#39;extension finale à 72 ° C pendant 7 min. </li><li> A la fin de til vélo, de courir 2 pl du produit de PCR sur un gel d&#39;agarose à 1% pour se assurer que la bande est la bonne taille. Purifier le produit de la PCR avec un kit de purification PCR selon les instructions du fabricant. Vérifier la concentration de l&#39;ADN avec un spectrophotomètre. Stocker le produit PCR purifié à -20 ° C ou utiliser directement dans plafonné transcription de l&#39;ARN dans l&#39;étape suivante. </li><li> La synthèse in vitro d&#39;ARNm coiffés avec un kit de synthèse d&#39;ARN coiffée selon les instructions du fabricant. Diluer l&#39;ARNm dans 20 ul d&#39;eau libre de RNase et déterminer la concentration. Aliquoter l&#39;ARN et conserver à -80 ° C. </li><li> Le jour de l&#39;injection, préparer une solution de travail de l&#39;ARNm par la RNase de l&#39;eau stérile libre. Notez que 40 pg dose est généralement de l&#39;ARN de la plus élevée à laquelle des effets non spécifiques ou toxiques de l&#39;ARN ne se produisent pas. Gardez la solution de travail sur la glace. Injecter l&#39;embryon avec MO et l&#39;ARNm de sauvetage, ou co-injecter les deux ensemble. Par exemple, si la concentration de l&#39;ARNm de prépared à l&#39;étape 4.5 est de 0,6 ug / ul, ajouter 0,67 ul d&#39;ARNm (0,4 ug) à 4,33 pi de RNase eau libre pour obtenir une concentration finale de 0,08 ug / ul, puis une goutte (500 pl) de chaque injection contient 40 pg de l&#39;ARNm. Préparer MO comme décrit à l&#39;étape 3. </li></ol> 5. Préparation des embryons injectés pour l&#39;imagerie de fluorescence <ol><li> Après incubation à 28,5 ° CO / N, ajouter 0,003% de N-phénylthiourée (PTU) à l&#39;eau pour empêcher la mélanisation E3, ce qui affecte l&#39;observation de la structure de pronéphros en utilisant la microscopie de fluorescence. Cependant, ne ajoutez pas PTU avant gastrulation, car elle affecte le développement embryonnaire précoce. </li><li> Au 48 HPF, dechorionate manuellement les embryons avec des pincettes fines. Prenez des photos de 48 et 72 embryons hpf sous un dissection microscope optique. NOTE: Ces photos peuvent être prises sans anesthésie. </li><li> Environ 10 minutes avant l&#39;imagerie au microscope à fluorescence, anesthésier les embryons en les plaçant dansune boîte de Pétri de 10 cm contenant 160 ug / ml tamponnée tricaïne. Attendre 5 à 10 min, puis appuyez légèrement sur le poisson zèbre pour confirmer qu&#39;ils ne se déplacent pas et donc l&#39;anesthésie travaille. </li><li> Après les embryons sont anesthésiés, montez-les dans 3% de méthylcellulose et de les orienter dans une position couchée sous un microscope de dissection (se il vous plaît voir le tableau 1). </li><li> Observer la structure de pronéphros sous un microscope à fluorescence (se il vous plaît se référer au tableau 1) et prendre des photos à différents grossissements (10x et 20x). </li></ol>

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The authors have nothing to disclose.

Polykystose rénale (PKD) est l&#39;une des principales causes de maladie rénale terminale chez l&#39;homme et est caractérisée par la formation progressive d&#39;un kyste, l&#39;élargissement rénale, et le développement des tubules anormale 14. PKD autosomique dominante (ADPKD) est une maladie génétique dans laquelle une ou l&#39;autre mutation de PKD1, codant pour la polycystine-1 (PC1) ou PKD2, codant pour la polycystine-2 (PC2), les résultats dans les reins polykystiques. Beaucoup d&#39;autres g?...

Poisson zèbre (Danio rerio) embryons ont été largement utilisé comme un modèle pour étudier le développement du rein et la polykystose rénale. Il existe de nombreux avantages à l&#39;utilisation du poisson zèbre comme modèle animal: la faisabilité de l&#39;étude des interactions génétiques, la capacité d&#39;utiliser morpholinos antisens (MO) pour la protéine knockdown, la possibilité de doser rapidement un grand nombre d&#39;embryons, et la facilité de visualisation des phénotypes d&#39;organes en une larves vivantes. Les pronéphros est le premier rein à développer chez les vertébrés et est fonctionnel chez le poisson zèbre larves 2. La structure des pronéphros poisson zèbre est relativement simple par rapport à métanéphros mammifères, la troisième et dernière rein de développer chez les mammifères. Le néphron est l&#39;unité de travail du rein, rein humain avec chacun contenant entre 3,4 et 500,000-1,000,000 néphrons chaque rein de souris ayant environ 13000 néphrons 5, il est difficile d&#39;observer la structure de néphron unique in reins humains ou de souris. Le poisson zèbre n&#39;a que deux néphrons, et chaque néphron poisson zèbre contient tous les composants majeurs trouvés dans le glomérule et les tubules de souris et les humains 6 et types similaires de cellules rénales spécialisés. Par rapport à d&#39;autres modèles de vertébrés tels que Xenopus, le poisson zèbre néphron ressemble plus étroitement néphron de mammifère parce qu&#39;il a un système fermé 7. Au cours des dernières années, le génome du poisson zèbre a été séquencé, ce qui permet la mise en place de l&#39;ensemble des outils génétiques, de vastes ressources mutantes et des collections de lignées rapporteuses dans des modèles transgéniques de poisson zèbre. Les pronéphros de poisson zèbre formes entre 12-72 heures après la fécondation (HPF) et peuvent être visualisées facilement dans les embryons transparents. La protéine de tumeur du Wilm WT1 est un facteur essentiel pour le développement du rein. Lignes de poisson zèbre transgéniques exprimant la protéine fluorescente verte (GFP) sous le contrôle du promoteur de wt1b Tg (wt1b: GFP) montrer GFP EXPREssion spécifiquement situé dans les régions pronephrique dans embryons de poisson zèbre, à partir de 17 hpf 8. Néphronophtise (NPHP), une maladie du rein kystique autosomique récessive, est causée par des mutations de gènes NPHP neuf. NPHP4 knockdown morpholino causé par la formation de kystes dans la Tg (wt1b: GFP). Poisson 10 Par conséquent, ce poisson transgénique est un modèle approprié pour l&#39;observation des structures de rein et la formation de kystes au cours du développement rénal. Fait important, l&#39;influence des modulateurs de développement du rein peut être étudiée en utilisant cette souche dans un temps et du travail de manière efficace. Notre article décrit l&#39;utilisation de Tg (wt1b: GFP) poisson comme un modèle de visualiser la formation de kystes rénaux après modulation génique. Nous avons utilisé le site d&#39;épissage de début et anti-sens OM pour abattre le gène Wnt5a chez le poisson zèbre. Wnt5a est une glycoprotéine sécrétée non-canonique de la famille Wnt qui joue un rôle important dans le développement de divers organes et postnatal cellulairefonction 11. Wnt5a fonctionne par des voies Wnt non canoniques, y compris la voie de la polarité cellulaire planaire (PCP), qui se est révélé jouer un rôle dans la division cellulaire lors de l&#39;allongement orienté tubulaire rénale. Wnt5a régule la voie / PCP Wnt en formant un complexe avec le récepteur, comme la tyrosine kinase (Ryk), qui transduit en outre signalisation Wnt5a en formant un complexe avec le VANGL protéine de la polarité cellulaire plane 2 (Vangl2), favorisant ainsi Vangl2 stabilité 12. Défauts dans la voie de PCP peuvent entraîner dans la division cellulaire aléatoire et provoquer la formation de kyste rénal. Nous avons utilisé le Tg (wt1b: GFP) ligne poisson zèbre pour observer la formation de kystes rénaux suivante Wnt5a knockdown. Le Tg (wt1b: GFP) modèle de poisson zèbre permet l&#39;imagerie en direct et l&#39;observation en temps opportun de la structure du rein. Après Wnt5a knockdown, la formation de kystes rénaux a été trouvée à partir de 24 hpf; à 72 HPF, les kystes peuvent être trouvés dans les glomérules et les tubules proximaux. Cette méthode peut également être utilisée pour sCreen autres gènes qui pourraient provoquer la formation de kystes rénaux.

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	<tbody>
		<tr height="21">
			<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>
		</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>
		</tr>
		<tr height="21">
			<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 abattre a été réalisé par l&#39;introduction traduction bloquant MO (AUG-MO) ou de l&#39;exon / intron jonction des frontières MO (épissage MO) pour embryons de poisson zèbre au stade d&#39;une cellule. L&#39;AUG-MO cible le codon d&#39;initiation et, par conséquent, inhibe à la fois un message de Wnt5a maternelle et zygotique. L&#39;épissure-MO vise le troisième site donneur d&#39;épissage et inhibe seulement la transcription zygotique de Wnt5a (figure 1A). Le août-...

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 modèle de poisson zèbre possible de la maladie polykystique des reins: Knockdown de<em&gt; Wnt5a</em&gt; Causes kystes chez le poisson zèbre reins

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 ...

a-possible-zebrafish-model-polycystic-kidney-disease-knockdown-wnt5a

Research

JoVE Journal

Medicine

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

11.0K vues.  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. 

Vidéo: 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;induction de l&#39;infarctus du myocarde chez le poisson zèbre Adulte utilisant Cryoinjury

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

Recherche

Médecine

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.

Évaluer les changements tératogènes dans un modèle de poisson zèbre de l&#39;exposition d&#39;alcoolisation fœtale

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.

Caryotype spectral pour l&#39;étude d&#39;anomalies chromosomiques dans les humains et les souris de la maladie polykystique des reins

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.

Dépistage du mélanome Modificateurs l&#39;aide d&#39;un modèle de tumeur Zebrafish Autochtone

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 modèle de poisson zèbre du diabète sucré et de la mémoire métabolique

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.

L&#39;évaluation de la fonction vasculaire chez les patients atteints de maladie rénale chronique

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;utilisation de l&#39;IRM de terrain ultra-haute dans les modèles petit rongeur de la maladie polykystique des reins pour<em&gt; In Vivo</em&gt; Phénotypage 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 modèle de cardiomyopathie induite par la doxorubicine chez le poisson zèbre adulte

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.

Évaluation de la fonction rénale chez les modèles murins de la maladie glomérulaire

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.

Mesure de la contractilité isométrique de l’artère mésentérique de souris à l’aide de fil myographie

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