Les auteurs tiennent à remercier le Bureau de la recherche du Presbyterian College School of Pharmacy de soutien financier de ce projet à travers les bourses petite pharmacie pour la recherche &amp; Collaboration (SPARC) Award. Les auteurs remercient M. John Smink, directeur du Laboratoire de recherche des animaux aquatiques, Clemson University, Clemson, SC pour sa révision technique assistance manuscrit.

NOTE: Ces protocoles suivent les directives de IACUC du Presbyterian College. 1. Matériaux Acquérir énumérées dans le tableau 1. 2. Assembler le plateau 5 Chrome Selon les instructions du fabricant. Cela être utilisés pour héberger le Fonds. 3. Construction du réservoir d&#39;eau et un système de filtration (figures 1A et 2). <ol><li> Aide de la figure 1A comme un guide, mise en page le seau de 18 gallons, pompe, filtres et lampe UV pour déterminer l&#39;emplacement d&#39;installation et la taille finale. </li><li> Une fois que les pièces sont disposées, mesurer et couper le tuyau de PVC ¾ &quot;à la longueur nécessaire pour l&#39;assemblage. Variez les longueurs de toutes les conduites en fonction des besoins et des contraintes d&#39;espace spécifiques. </li><li> Assemblez les tuyaux et les raccords comme prévu précédemment en utilisant l&#39;amorce PVC et de ciment, voir ci-dessous (3.3.2 et 3.3.3). <ol><li> Poncer les bords de la pipe avec du papier de verre coupées jusqu&#39;à ce qu&#39;ils soient lisses à l&#39;un tactiled il n&#39;y a pas de débris. </li><li> Premier l&#39;extérieur du tuyau et l&#39;intérieur de la ferrure avec l&#39;amorce PVC acheté avec l&#39;écouvillon inclus à partir de l&#39;amorce PVC. </li><li> Collez le tuyau et le raccord ensemble par l&#39;application de la colle à PVC à l&#39;extérieur de la conduite en utilisant l&#39;écouvillon inclus. Répétez l&#39;opération pour toutes les mesures qui doivent cimentation de PVC. </li><li> Placer le godet 18 gallons d&#39;un côté de la construction du plateau. </li><li> Installer un connecteur de cloison du côté du réservoir. </li><li> Fixez un tuyau en PVC ¾ &quot;à la cloison. </li><li> Branchez l&#39;autre extrémité du ¾ &quot;pipe à l&#39;entrée de la pompe en utilisant un ¾&quot; filetée faire raccord. </li><li> Connecter les filtres à la pompe à l&#39;aide de deux coudes de 90 degrés et ¾ &quot;tuyau en PVC. <ol><li> Utilisez un filtre de papier suivi d&#39;un filtre à charbon actif pour filtrer les débris et réduire les niveaux de chlore et de produits chimiques dans l&#39;eau. </li></ol></li><li> Connectez la lampe UV à l&#39;filtres according aux instructions du fabricant. </li></ol></li><li> Fixez la pompe et système de filtration à la face supérieure de la première étagère de l&#39;armoire en utilisant les liens de fil. Variez l&#39;ordre de l&#39;assemblage et fixation selon la taille et la disposition de la tablette / support choisi. </li><li> Ajouter une BioBall par gallon d&#39;eau au réservoir d&#39;eau, une fois la pompe et le système de filtre sont assemblés et fixés à l&#39;étagère. </li></ol> 4. Construire la ligne principale d&#39;alimentation (figures 1B et 2). <ol><li> Mesurer et couper un morceau de ¾ &quot;tuyau en PVC pour connecter la lampe UV à l&#39;alimentation principale </li><li> Utilisez un &quot;té en PVC pour commencer la ligne d&#39;alimentation principale en connectant le tee au ¾&quot; ¾ PVC échappement provenant du filtre UV. . <ol><li> Ajouter une soupape de marche-arrêt vers le bas du tee PVC pour servir de drain pour l&#39;entretien du système de rinçage. </li><li> Lancer la ligne principale d&#39;alimentation par le haut de la ¾ &quot;té en PVC en mesurant et en coupe ¾&quot; PVCtube à la longueur désirée pour fournir des tuyaux d&#39;alimentation de chacune des étagères sur le rack. </li><li> Installez ¾ &quot;tees de PVC pour les lignes horizontales qui entreront chaque étagère de l&#39;armoire. </li><li> Éventuellement (et suggéré), ajouter un ¾ &quot;on-off valve pour chaque ligne d&#39;alimentation du plateau pour permettre l&#39;isolement de chaque étagère et une conception modulaire du système. </li></ol></li><li> Utilisez un tuyau en PVC ¾ &quot;comme un réducteur de pression en ligne sur l&#39;étagère du haut. Si cette ligne se exécute sur l&#39;arrière de la tablette, utiliser cette étagère pour le stockage. Fin de la réduction de pression ligne dans la ligne d&#39;échappement principale (figure 1B) avec un ¾ &quot;vanne on-off. </li><li> Une fois les étapes ci-dessus ont été réalisés, le premier et cimenter le tuyau en PVC ensemble comme déjà fait lors de la construction du système de réservoir et filtration. </li></ol> 5. Construire la ligne principale d&#39;échappement (figures 1C et 2). <ol><li> Exécuter la longueur verticale de la conduite principale d&#39;échappement de l&#39;autre côté de la crémaillère fepuis la ligne d&#39;alimentation principale. Utilisez un tuyau 2 &quot;en PVC pour cette ligne verticale pour recevoir la ligne d&#39;alimentation principale sur les lignes du haut et du drainage des réservoirs de poissons de chaque étagère. </li><li> Couper les morceaux de 2 &quot;tuyau en PVC pour la ligne d&#39;échappement principale verticale et utiliser deux&quot; t-shirts en PVC pour recevoir le réservoir de poissons conduites de vidange de chaque étagère. Fin de la ligne d&#39;échappement principale dans le réservoir de stockage d&#39;expulser l&#39;eau recyclée sur un filtre éponge. </li><li> Couper une ouverture sur toute la longueur des deux &quot;morceaux de tuyaux en PVC d&#39;un pouce et joindre à la ligne d&#39;échappement principale verticale avec tees PVC pour les lignes horizontales de vidange des réservoirs de poissons. </li><li> Boucher les deux &quot;lignes de drain horizontal avec deux« plafond de PVC. </li><li> Premier et cimenter les pièces en utilisant les accessoires nécessaires (connecteurs, mamelons ou coudes). </li></ol> 6. Construire les réservoirs de poissons (figure 3). <ol><li> Avec la perceuse, couper un «trou à l&#39;arrière de l&#39;aquarium 1&quot; 1 1/8 dessous du bord supérieur avec une scie de trou. </li><li&gt; glisser une ¾ &quot;PVC adaptateur fileté mâle (avec une bague 18 # O-joint) au travers du trou (de l&#39;intérieur de la cuve à l&#39;extérieur). </li><li> Fixez et serrez un 90 degrés 3/4 &quot;fileté coude PVC à l&#39;adaptateur mâle pour créer le tuyau d&#39;échappement de réservoir de poissons. Ajouter un morceau de maille fine avant de visser l&#39;écrou de retenue pour empêcher le poisson zèbre en passant par le système d&#39;échappement. </li><li> Placez le réservoir de poissons avec son fin du tuyau d&#39;échappement dans l&#39;espace ouvert de la ligne de drainage derrière les chars </li></ol> 7. Démarrage du système de l&#39;eau <ol><li> Dans plusieurs récipients, tels que celui utilisé pour le réservoir, mesurer trois fois la quantité d&#39;eau contenue dans le système. Laissez cette eau reposer à température ambiante pendant conditionné pendant 48 heures. </li><li> Ajouter de l&#39;eau au système et le laisser circuler pendant un minimum de trois jours. <ol><li> Changez l&#39;eau dans le système vigoureusement durant les 3 premiers jours de la circulation afin de débarrasser le système de contaminants that résident dans les tuyaux. </li></ol></li><li> Ajouter le poisson zèbre démarrage et laisser le cycle de l&#39;azote de se établir. Normaliser pH à une politique cohérente 7. Gardez sept poisson zèbre par 1,8 L, et de les nourrir deux fois par jour en semaine et une fois par jour le week-end comme suggéré par Linbo 4 et 6 McNabb. </li><li> Surveiller le nitrate (&lt;75 mg / L), le nitrite (&lt;10 mg / L), et d&#39;ammonium (0 mg / L) par jour pendant la première semaine après l&#39;ajout de poissons, et des niveaux deux fois par semaine, une fois escomptés ont été maintenus avec des changements réguliers de l&#39;eau . </li></ol>

<ol>
	<li>Detrich, H., Westerfield, M., Zon, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Zebrafish: Cellular and Developmental Biology, Part A.. , (2010).</li><li>Grim, J. M., 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=Hemogen+(hemgn),+a+transcription+factor+that+regulates+erythropoiesis+and+skeletogenesis+in+zebrafish.">Hemogen (hemgn), a transcription factor that regulates erythropoiesis and skeletogenesis in zebrafish.</a> , (2013).</li><li>Kim, S., Carlson, R., Zafreen, L., Rajpurohit, S. K., Jagadeeswaran, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular,+easy-to-assemble,+low-cost+facility.">Modular, easy-to-assemble, low-cost facility.</a> Zebrafish. 6 (3), 269-274 (2009).</li><li>Linbo, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+(Danio+rerio)+husbandry+and+colony+maintenance+at+the+Northwest+Fisheries+Science+Center.">Zebrafish (Danio rerio) husbandry and colony maintenance at the Northwest Fisheries Science Center.</a> U.S. Dept. Commer., NOAA Tech. Memo. 62, (2009).</li><li>Avdesh, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regular+care+and+maintenance+of+a+zebrafish+(Danio+rerio)+laboratory:+an+introduction.">Regular care and maintenance of a zebrafish (Danio rerio) laboratory: an introduction.</a> J Vis Exp. 69, 4196 (2012).</li><li>McNabb, A., Scott, K., Ochsenstein, E., Seufert, K., Carl, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Don't+be+afraid+to+set+up+your+fish+facility.">Don't be afraid to set up your fish facility.</a> Zebrafish. 9 (3), 120-125 (2012).</li><li>Garcia, R. L., Sanders, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+cleaning+and+disinfection+procedures+in+a+zebrafish+rerio)+facility.">Efficacy of cleaning and disinfection procedures in a zebrafish rerio) facility.</a> J Am Assoc Lab Anim Sci. 50 (6), 895-900 (2011).</li><li>Rivas-Boyero, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+characterization+of+a+nociceptin+receptor+from+zebrafish+(Danio+rerio).">Pharmacological characterization of a nociceptin receptor from zebrafish (Danio rerio).</a> J Mol Endocrinol. 46 (2), 111-123 (2011).</li><li>Sanchez-Simon, F. M., Zhang, X. X., Loh, H. H., Law, P. Y., Rodriguez, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphine+regulates+dopaminergic+neuron+differentiation+via+miR-133b.">Morphine regulates dopaminergic neuron differentiation via miR-133b.</a> Mol Pharmacol. 78 (5), 935-942 (2010).</li><li>Velasco, E. M., Law, P. Y., Rodr&#237;guez, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mu+opioid+from+the+zebrafish+exhibits+functional+characteristics+as+those+of+mammalian+mu+opioid+receptor.">Mu opioid from the zebrafish exhibits functional characteristics as those of mammalian mu opioid receptor.</a> Zebrafish. 6 (3), 259-268 (2009).</li></ol>

Les auteurs déclarent aucun conflit d&#39;intérêt pour le présent document.

Le but principal de cet article est de démontrer que la construction d&#39;une installation fonctionnelle poisson zèbre ne est pas compliqué et peut être construit par les chercheurs de poissons débutants ayant une connaissance minimale de la construction. En outre, cet article montre que les matériaux pour cette installation poisson zèbre fonctionnelle peuvent être acquises facilement et à moindre coût dans les magasins de fournitures pour animaux locaux ou les quincailleries. Une fois que tous les matériaux...

In vivo modèles sont essentiels dans la conduite de la recherche biomédicale globale car ils permettent à la recherche in vitro à traduire au niveau du système et organismal. Par exemple, la recherche de petits mammifères, à savoir les rongeurs, a été avéré nécessaire dans le développement de médicaments et à l&#39;amélioration des médicaments disponibles. Cependant, la complexité (financièrement et logistiquement) pourrait rendre pratiquement impossible à construire et à entretenir les installations de rongeurs d&#39;expérimentation propose aux petites institutions universitaires de recherche. Néanmoins, la recherche in vivo peut être effectuée en utilisant d&#39;autres espèces animales qui nécessitent une logistique moins élaborés. Poisson zèbre (Danio rerio) ont été utilisés avec succès comme un modèle in vivo pour étudier la génétique, le développement, la physiopathologie et pharmacologie 1,2,10,11,12. Les installations requises pour accueillir le poisson zèbre pour l&#39;expérimentation sont relativement simples et beaucoup moins cher par rapport aux rinstallations Odent. Kim et al. 3 ont récemment décrit en détail un système de logement poisson zèbre 80 du réservoir qui peut être construite pour environ $ 1500, qui comprend les deux parties et les coûts de main-d&#39;œuvre. Ceci est en contraste frappant avec la presque $ 8,500 qui serait nécessaire pour acheter un système de réservoir 80 ready-made de tout nombre de fournisseurs commerciaux. Toutefois, la description donnée par Kim et al. peut être compliqué et difficile à suivre pour les novices de la recherche de poisson. Nous croyons qu&#39;une installation poisson zèbre peut être construit de manière simplifiée pour un investissement de $ 1500. La mise en place d&#39;une installation in vivo à des petits établissements universitaires fournit leurs facultés un outil supplémentaire avec laquelle de produire une recherche plus puissant et complet. Les données in vitro soutenues par résultats in vivo généralement produire la recherche biomédicale plus complète que les données obtenues en utilisant seulement des approches in vitro. Par conséquent, lamise en œuvre d&#39;un centre de recherche in vivo améliorera considérablement le succès de l&#39;institution dans ses efforts de recherche globaux (de l&#39;exposition des étudiants à la recherche, publications, communications, recherche de financement externe, etc.). La construction d&#39;une installation de poisson zèbre comme celle décrite dans cette étude est idéale pour les établissements universitaires de recherche avec des moyens limités (ou institutions sans in vivo installations de recherche) au cours de constriction fois financiers.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Item</td>
			<td>Brand</td>
			<td>Quantity</td>
			<td></td>
		</tr>
		<tr>
			<td>74-in H x 48-in W x 18-in D 5-shelf Steel Freestanding Shelving Unit</td>
			<td>Style Selections</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>18-Gallon General Bucket/Tote</td>
			<td>Centrex Plastics: LLC&#160;Rugged Tote</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>6-Outlet Power Strip With 15-Ft. Cord</td>
			<td>Master Electrician</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>9-Watt UV Water Garden Clarifier FiIter and UV lamp</td>
			<td>Smartpond&#160;</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>Whole House Water Replacement Filter</td>
			<td>Whirlpool</td>
			<td>6</td>
			<td></td>
		</tr>
		<tr>
			<td>Opaque Whole-House Pre-Filtration Housing</td>
			<td>Whirlpool</td>
			<td>2</td>
			<td></td>
		</tr>
		<tr>
			<td>Thread Seal Tape</td>
			<td>William H Harvey</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>8-fl oz LO-VOC PVC Cement</td>
			<td>Oatey&#160;</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>8-fl oz LO-VOC Primer</td>
			<td>Oatey&#160;</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>1/4-in x 100-ft Vinyl Drip Irrigation Distribution Tubing</td>
			<td>Mister Landscaper&#160;</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>1/4-in Barbed Drip Irrigation On/Off Valve</td>
			<td>Mister Landscaper</td>
			<td>33</td>
			<td></td>
		</tr>
		<tr>
			<td>9-Port NPT Irrigation Manifold with Filter</td>
			<td>&#160;Mister Landscaper</td>
			<td>6</td>
			<td></td>
		</tr>
		<tr>
			<td>Drop-In Filter with House&#160;</td>
			<td>Whirlpool</td>
			<td>6</td>
			<td></td>
		</tr>
		<tr>
			<td>2-in Dia 90-Degree PVC Elbow</td>
			<td>LASCO</td>
			<td>3</td>
			<td></td>
		</tr>
		<tr>
			<td>2-in Dia 90-Degree Tee</td>
			<td>LASCO</td>
			<td>3</td>
			<td></td>
		</tr>
		<tr>
			<td>3/4-in Dia 90-Degree Slip Elbow</td>
			<td>LASCO</td>
			<td>15</td>
			<td></td>
		</tr>
		<tr>
			<td>3/4-in Dia PVC Adapter</td>
			<td>LASCO</td>
			<td>7</td>
			<td></td>
		</tr>
		<tr>
			<td>3/4-in Dia PVC Tee</td>
			<td>LASCO</td>
			<td>12</td>
			<td></td>
		</tr>
		<tr>
			<td>2-in Dia PVC Cap</td>
			<td>LASCO</td>
			<td>3</td>
			<td></td>
		</tr>
		<tr>
			<td>1-in Dia x 3/4-in Dia PVC Adapter</td>
			<td>LASCO</td>
			<td>2</td>
			<td></td>
		</tr>
		<tr>
			<td>3/4-in PVC Socket In-Line Ball Valve</td>
			<td>American Valve</td>
			<td>5</td>
			<td></td>
		</tr>
		<tr>
			<td>1/2-in Dia x 3/4-in Dia PVC Adapter</td>
			<td>Lasco</td>
			<td>8</td>
			<td></td>
		</tr>
		<tr>
			<td>3/4-in Dia PVC Cap</td>
			<td>Lasco</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>2-In x 5-Ft Pvc Pipe</td>
			<td>Charlotte Pipe</td>
			<td>4</td>
			<td></td>
		</tr>
		<tr>
			<td>&#190;-In x 10-Ft Pvc pipe</td>
			<td>Charlotte Pipe</td>
			<td>3</td>
			<td></td>
		</tr>
		<tr>
			<td>11/16x1-7/80 PVC ring</td>
			<td>SXT</td>
			<td>10</td>
			<td></td>
		</tr>
		<tr>
			<td>&#189;&#8221; PVC male adapter</td>
			<td>SXT</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>&#190;&#8221; PVC coupling&#160;</td>
			<td>SXT</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>&#189;&#8221; PVC female adapter&#160;</td>
			<td>SXT</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>link</td>
			<td></td>
		</tr>
		<tr>
			<td>1.8 L Dual Beta Keeper</td>
			<td>Top Fin</td>
			<td>27</td>
			<td></td>
		</tr>
		<tr>
			<td>&#190;&#8221; PVC male adapter (w/ #18 O-ring)</td>
			<td>SXT</td>
			<td>28</td>
			<td></td>
		</tr>
		<tr>
			<td>90-degree &#190;&#8221; threaded PVC elbow</td>
			<td>SXT</td>
			<td>27</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Mesh</td>
			<td>N/A</td>
			<td>1 yard</td>
			<td></td>
		</tr>
		<tr>
			<td>Bulkhead Connector</td>
			<td>N/A</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>Hacksaw</td>
			<td>N/A</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>Drill</td>
			<td>N/A</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>Hole saw</td>
			<td>N/A</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>Adjustable Wrench</td>
			<td>N/A</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>Pliers&#160;</td>
			<td>N/A</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tape Measure</td>
			<td>N/A</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>Safety Glasses</td>
			<td>N/A</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>Work Gloves</td>
			<td>N/A</td>
			<td>1</td>
			<td></td>
		</tr>
	</tbody>
</table>

construction of an affordable and easy-to-build zebrafish facility

In vivo la recherche biomédicale est essentiel pour traduire dans les résultats in vitro dans les avancées cliniques. Les petits établissements universitaires avec des ressources limitées trouver qu&#39;il est pratiquement impossible de construire et d&#39;entretenir les installations typiques de rongeurs pour la recherche. La recherche du poisson zèbre a été démontré pour être une alternative intéressante pour la recherche in vivo en pharmacologie, la physiologie, le développement et les études génétiques. Cet article montre qu&#39;une installation poisson zèbre fonctionnel peut être construit d&#39;une manière facile et abordable. Nous démontrons qu&#39;une telle installation pourrait être construit dans environ un jour travailler avec des outils et de l&#39;expertise minimes. Le coût de la 27 1,8 L installation de poisson zèbre de réservoir de poissons construit dans cette étude était d&#39;environ $ 1500. Nous estimons que le maintien d&#39;une colonie initiale de poisson travail 150 pour trois mois est de 1000 $. Ce projet consistait étudiants, qui ont été introduites à aquaculturing de poisson zèbre pour la recherche propose.

La construction du système de pompe et fltres Une fois le rack est assemblé et mis en place la première étape est de construire le système de pompe et le filtre. Les filtres sont reliés à la pompe du système et sont situés au-dessus du niveau bas de la première étagère, ce qui permet un entretien facile du filtre et le remplacement. De même, la lampe UV est relié à un tuyau en PVC au filtre et maintenu en place avec attaches en plastique pour permettre le remplacement facile de la lampe UV...

Watch this Scientific Journal Video about Construction of an Affordable and Easy-to-Build Zebrafish Facility at JoVE.com

Construction of an Affordable and Easy-to-Build Zebrafish Facility

A Zebrafish facility suitable for breeding and in vivo research is built in an easy and affordable manner. Maintenance is minimal. This facility is ideal for student involvement and independent research.

Construction d&#39;un Construire facile à l&#39;installation de poisson zèbre et abordable

In vivo biomedical research is pivotal to translate in vitro findings into clinical advances. Small academic institutions with limited resources find it ...

construction-of-an-affordable-and-easy-to-build-zebrafish-facility

Research

JoVE Journal

Biology

16.2K vues.  Presbyterian College School of Pharmacy. A Zebrafish facility suitable for breeding and in vivo research is built in an easy and affordable manner. Maintenance is minimal. This facility is ideal for student involvement and independent research.

Vidéo: Construction of an Affordable and Easy-to-Build Zebrafish Facility

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

L&#39;imagerie in vivo et des traitements thérapeutiques dans un modèle de souris orthotopique de cancer des ovaires

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Recherche

Biologie

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharides1, 2. The modified monosaccharides, processed by the glycan biosynthetic machinery, are incorporated into cell surface glycoconjugates. The bioorthogonal azide or alkyne tags then allow covalent conjugation with fluorescent probes for visualization, or with affinity probes for enrichment and glycoproteomic analysis. This protocol describes the procedures typically used for noninvasive imaging of fucosylated glycans in zebrafish embryos, including: 1) microinjection of one-cell stage embryos with GDP-5-alkynylfucose (GDP-FucAl), 2) labeling fucosylated glycans in the enveloping layer of zebrafish embryos with azide-conjugated fluorophores via biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and 3) imaging by confocal microscopy3. The method described here can be readily extended to visualize other classes of glycans, e.g. glycans containing sialic acid4 and N-acetylgalactosamine5, 6, in developing zebrafish and in other living organisms.

A click-chemistry based method that allows for the rapid, noninvasive, and robust labeling of alkyne-tagged glycans in zebrafish embryos is described. Fucosylated glycans in the enveloping layer of zebrafish embryos in the late gastrulation stage were imaged in this study.

Glycanes imagerie dans embryons de poissons zèbres par marquage métabolique et Chimie Cliquez Bioorthogonal

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

En électroporation in vivo de morpholinos dans la rétine de poisson zèbre adulte

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

En électroporation in vivo de morpholinos dans la régénération des adultes Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and behavioural research. As a vertebrate, zebrafish share considerable genetic sequence similarity with humans and are being used as an animal model for various human disease conditions. The advantages of zebrafish in comparison to other common vertebrate models include high fecundity, low maintenance cost, transparent embryos, and rapid development. Due to the spur of interest in zebrafish research, the need to establish and maintain a productive zebrafish housing facility is also increasing. Although literature is available for the maintenance of a zebrafish laboratory, a concise video protocol is lacking. This video illustrates the protocol for regular housing, feeding, breeding and raising of zebrafish larvae. This process will help researchers to understand the natural behaviour and optimal conditions of zebrafish husbandry and hence troubleshoot experimental issues that originate from the fish husbandry conditions. This protocol will be of immense help to researchers planning to establish a zebrafish laboratory, and also to graduate students who are intending to use zebrafish as an animal model.

Regular Care and Maintenance of a Zebrafish Danio rerio Laboratory: An Introduction

This protocol outlines regular maintenance and care to maintain optimal conditions for zebrafish husbandry. The video illustrates the protocol for system maintenance, regular housing, feeding, breeding, and raising of zebrafish larvae.

Soins et entretien réguliers d&#39;un poisson zèbre (<em&gt; Danio rerio</em&gt;) Laboratoire: An Introduction

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and ...

Using an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The presented automatic video tracking system accomplishes this by using a mirror system and a calibration procedure that corrects for the considerable error introduced by the transition of light from water to air. With this system it is possible to record both single and groups of adult zebrafish. Before use, the system has to be calibrated. The system consists of three modules: Recording, Path Reconstruction, and Data Processing. The step-by-step protocols for calibration and using the three modules are presented. Depending on the experimental setup, the system can be used for testing neophobia, white aversion, social cohesion, motor impairments, novel object exploration etc. It is especially promising as a first-step tool to study the effects of drugs or mutations on basic behavioral patterns. The system provides information about vertical and horizontal distribution of the zebrafish, about the xyz-components of kinematic parameters (such as locomotion, velocity, acceleration, and turning angle) and it provides the data necessary to calculate parameters for social cohesions when testing shoals.

The use of a 3D automatic video system that can track individual and groups of zebrafish is described. As application example we explore the effects of the NMDA-receptor antagonist MK-801 on shoals of zebrafish.

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

Le poisson zèbre rapide et efficace génotypage utilisant la PCR avec haute résolution Melt analyse

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

An Assay for Lateral Line Regeneration in Adult Zebrafish

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.17&#160;that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish rather than the embryo/larvae, we developed a quantitative lateral line regenerative assay that can be applied to adult zebrafish disease models. The assay involved resolution at the 1) neuromast and 2) individual hair cell levels.

Un test pour ligne latérale de régénération à l&#39;âge adulte de poisson zèbre

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a &#8216;targeting&#8217; vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term &#8216;subcloning plus insertion&#8217;, which can be used to generate custom gene targeting vectors rapidly.

Sous-clonage plus insertion (SPI) - Un Recombineering nouveau procédé pour la construction rapide de ciblage de gène Vecteurs

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Néphrotoxine microinjection dans Zebrafish au Modèle lésion rénale aiguë

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...