Die Autoren möchten die Office of Research der Presbyterian College School of Pharmacy für die finanzielle Unterstützung dieses Projektes durch die kleine Apotheke Auszeichnungen für Forschung &amp; Collaboration (SPARC) Zuschlags danken. Die Autoren danken Herrn John Smink, Leiter der Wassertiere Research Laboratory, Clemson University, Clemson, SC für seine technische Unterstützung Manuskript überarbeitet.

HINWEIS: Diese Protokolle an die Richtlinien der IACUC der Presbyterian College. 1. Erwerben Materialien In Tabelle 1. 2. Setzen Sie die 5 Lager Chrome Nach den Anweisungen des Herstellers. Dies wird verwendet, um die Einrichtung Haus. 3. Konstruieren Sie den Wasserbehälter und Filtersystem (1A und 2). <ol><li> Mit 1A als ein Führer, das Layout, die 18 Gallonen-Eimer, Pumpe, Filter und UV-Lampe zum endgültigen Aufstellort und Größe zu bestimmen. </li><li> Nachdem Teile angelegt, zu messen und schneiden Sie die ¾ &quot;PVC-Rohr auf die Länge für die Montage benötigt. Variieren die Längen aller Rohre nach bestimmten Raumbedarf und Einschränkungen. </li><li> Setzen Sie die Rohre und Formstücke wie zuvor mit dem PVC-Primer und Zement gelegt, siehe unten (3.3.2 und 3.3.3). <ol><li> Sand die Schnittkanten des Rohres mit Sandpapier, bis sie glatt anfühlt ein sindd gibt es keine Ablagerungen. </li><li> Prime die Außenseite des Rohres und das Innere der Armatur mit dem gekauften PVC Primer mit dem mitgelieferten Tupfer aus der PVC-Grundierung. </li><li> Zementieren Sie die Rohr und Fitting zusammen durch Anwendung der PVC-Kleber auf die Außenseite des Rohres mit dem mitgelieferten Tupfer. Wiederholen Sie für alle Schritte, die PVC Verklebung benötigen. </li><li> Platzieren Sie die 18-Gallonen-Eimer auf einer Seite des Regalkonstruktion. </li><li> Installieren eines Einbaubuchse in der Seite des Vorratstanks. </li><li> Bringen Sie einen ¾ &quot;PVC-Rohr mit dem Schott. </li><li> Schließen Sie das andere Ende des ¾ &quot;Rohr zum Pumpeneinlaß mit einem ¾&quot; Gewinde machen Fitting. </li><li> Schließen Sie die Filter an die Pumpe mit zwei 90-Grad-Bogen und ¾ &quot;PVC-Rohr. <ol><li> Verwenden Sie einen Papierfilter, gefolgt von einem Aktivkohle-Filter, um im Wasser herauszufiltern Schutt und reduzieren Chlor und chemischen Ebenen. </li></ol></li><li> Schließen Sie die UV-Lampe auf den Filter according den Herstelleranweisungen. </li></ol></li><li> Befestigen Sie die Pumpe und Filtersystem zur Oberseite des ersten Regal des Racks mit Drahtbinder. Variieren Sie die Reihenfolge der Montage und Befestigung nach der Größe und Layout der gewählten Regal / Rack. </li><li> 1 BioBalls pro Liter Wasser in den Wasserbehälter, wenn die Pumpe und die Filteranlage zusammengebaut und an dem Regal angebracht ist. </li></ol> 4. Bauen Sie die Hauptversorgungsleitung (1B und 2). <ol><li> Messen und schneiden Sie ein Stück ¾ &quot;PVC-Rohr, um die UV-Lampe an das Stromnetz anschließen </li><li> Verwenden Sie einen ¾ &quot;PVC-T, um die Hauptversorgungsleitung zu starten, indem Sie den Abschlag auf den ¾&quot; PVC Auspuff aus dem UV-Filter. . <ol><li> Fügen Sie ein Schaltventil, um den Boden der PVC-T als Abfluss für Wartung Spülen der Anlage dienen. </li><li> Starten Sie die Hauptversorgungsleitung von der Spitze des ¾ &quot;PVC-stück von Mess- und Schneide ¾&quot; PVCRohr auf die gewünschte Länge zu Zufuhrleitungen für jedes der Fächer an der Zahnstange vorzusehen. </li><li> Installieren ¾ &quot;PVC-T-Stücke für die horizontalen Linien, die jedes Regal des Racks geben wird. </li><li> Gegebenenfalls (und vorgeschlagen), einen ¾ &quot;Ein-Aus-Ventil für jeden Regal Zuleitung für die Isolierung von jedem Regal und ein modularer Aufbau des Systems ermöglicht. </li></ol></li><li> Verwenden Sie einen ¾ &quot;PVC-Rohr als Druckminderleitung auf dem obersten Regal. Wenn diese Linie verläuft auf der Rückseite des Regals, verwenden Sie dieses Regal für die Lagerung. Beenden Sie den Druckminderer Zeile in der Hauptabgasleitung (1B) mit einem ¾ &quot;Schaltventil. </li><li> Wenn die oben genannten Schritte durchgeführt wurden, grundieren und Zement die PVC-Rohr zusammen wie bisher bei der Konstruktion des Behälters und Filtersystem. </li></ol> 5. Bauen Sie die Hauptabgasleitung (1C und 2). <ol><li> Führen der vertikalen Länge der Hauptabgasleitung auf der anderen Seite der Zahnstange fROM Die Hauptversorgungsleitung. Verwenden Sie ein 2 &quot;PVC-Rohr für diese vertikale Linie, um die Hauptversorgungsleitung an den oberen und Ableitungen aus den Aquarien von jedem Regal erhalten. </li><li> Schneiden Sie die Stücke von 2 &quot;PVC-Rohr für die vertikale Hauptabgasleitung und mit 2&quot; PVC-T-Stücke, um die Fischbehälter Abflussleitungen von jedem Regal erhalten. Beenden Sie die Hauptabgasleitung im Vorratstank, um das Recyclingwasser über einen Schwammfilter zu vertreiben. </li><li> Schneiden Sie ein ein Zoll Öffnen der gesamten Länge der 2 &quot;PVC-Rohrstücke und heften sich an vertikalen Hauptabgasleitung mit PVC-stücke für die horizontalen Abflussleitungen aus den Aquarien. </li><li> Verschließen Sie die 2 &quot;horizontale Abflussleitungen mit einem 2&quot; PVC-Kappe. </li><li> Prime und festigen die Stücke mit den notwendigen Armaturen (Steckverbinder, Brustwarzen oder Ellenbogen). </li></ol> 6. Bauen Sie die Aquarien (Abbildung 3). <ol><li> Mit dem Bohrer, schneiden Sie ein 1 1/8 &quot;Loch an der Rückseite des Aquariums 1&quot; unterhalb der Oberkante mit einer Lochsäge. </li><li&gt; Schieben Sie einen Gewinde ¾ &quot;PVC-Stecker-Adapter (mit einem # 18 O-Ring befestigt) durch das Loch (von der Innenseite des Tanks nach außen). </li><li> Anbringen und festziehen 90 Grad 3/4 &quot;Gewinde PVC Ellbogen zum männlichen Adapter, um das Aquarium Auspuffrohr zu schaffen. Fügen Sie ein Stück feines Netz vor dem Einschrauben der Haltemutter an Zebrafischen gehen durch das Abgassystem zu verhindern. </li><li> Stellen Sie das Aquarium mit dem Auspuffrohr endet in den offenen Raum der Entleerung der Schlange hinter den Tanks </li></ol> 7. Starten des Wassersystems <ol><li> In mehreren Behältern, wie dem für den Behälter verwendet wird, messen die dreifache Menge an Wasser innerhalb des Systems enthalten sind. Lassen Sie dieses Wasser bei Raumtemperatur stehen zur Konditionierung für 48 Stunden. </li><li> Wasser hinzu, bis das System und lassen Sie es für mindestens 3 Tage zu zirkulieren. <ol><li> Wechseln Sie das Wasser in der Anlage kräftig in den ersten 3 Tagen nach der Kreislauf, um das System von Verunreinigungen zu befreien that befinden sich innerhalb der Rohre. </li></ol></li><li> In Starterzebrafisch und lassen Sie den Stickstoffkreislauf zu etablieren. Normalisieren pH in einen konsistenten 7. Halten sieben Zebrafisch pro 1,8 l, und füttern sie zweimal täglich während der Woche und einmal täglich an den Wochenenden wie Linbo 4 und McNabb 6 vorgeschlagen. </li><li> Überwachen Sie die Nitrat (&lt;75 mg / l), Nitrit (&lt;10 mg / l) und Ammonium (0 mg / l) täglich während der ersten Woche nach der Zugabe von Fisch und zweimal wöchentlich einmal gewünschten Spiegel wurden mit regelmäßigen Wasserwechsel beibehalten . </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>

Die Autoren erklären, von Interesse für dieses Papier keine Konflikte.

Der Hauptzweck dieses Artikels ist es zu zeigen, dass der Bau eines Funktionszebrafisch-Anlage ist nicht kompliziert und kann von Anfänger Fisch Forscher mit minimalem Konstruktionswissen aufgebaut werden. Darüber hinaus zeigt dieser Artikel, dass die Materialien für diese Funktionszebrafisch-Anlage kann leicht und kostengünstig aus lokalen Haustierfachhandel oder Baumarkt erworben werden. Nachdem alle Materialien und Werkzeuge sind die Konstruktion des Systems erfasst und die Aquarien dauert etwa einen Arbeitstag. ...

In-vivo-Modelle sind wesentlich umfassender und biomedizinischen Forschung, da sie eine in-vitro-Forschung auf dem System und organismischen Ebene übersetzt werden. Zum Beispiel Forschung mit kleinen Säugetieren, insbesondere Nagern, wurde es als notwendig für die Entwicklung von Arzneimitteln und zur Verbesserung des zur Verfügung stehenden Medikamente erwiesen. Allerdings könnte die Komplexität (finanziell und logistisch) machen es praktisch unmöglich ist, aufzubauen und zu pflegen Nagetier Einrichtungen für Experimente schlägt bei kleinen Forschungs akademischen Institutionen. Dennoch kann in vivo Forschung unter Verwendung anderer Tierarten, die weniger aufwendige Logistik erfordert. Zebrafisch (Danio rerio) wurden erfolgreich als in vivo Modell zur Genetik, Entwicklung, Pathophysiologie und Pharmakologie 1,2,10,11,12 studieren. Im Vergleich zu r Die zur Zebrafisch für Experimente beherbergen Einrichtungen sind relativ einfach und deutlich günstigerOdent Einrichtungen. Kim et al. 3 wurden kürzlich im Detail eine 80 Tankzebrafisch-Gehäuse-System, das für ca. $ 1500, die beide Teile und Arbeitskosten umfasst gebaut werden können beschrieben. Dies steht in krassem Gegensatz zu den fast 8500 $, die erforderlich wäre, um eine fertige, 80 Tanksystem aus einer beliebigen Anzahl von kommerziellen Anbietern zu kaufen. Jedoch ist die Beschreibung durch Kim et al. kann kompliziert und schwierig für Anfänger Fischforschung folgen. Wir glauben, dass ein Zebrafisch-Anlage kann in vereinfachter Weise für eine Investition von $ 1500 gebaut werden. Die Einrichtung einer In-vivo-Anlage in kleinen akademischen Einrichtungen bietet ihre Fähigkeiten ein zusätzliches Instrument, um stärker und gründlicher Forschung zu produzieren. In durch in vivo-Ergebnisse unterstützt vitro-Daten in der Regel produzieren umfassen biomedizinischen Forschung als Daten unter Verwendung von In-vitro-Ansätze. Daher ist dieImplementierung einer In-vivo-Forschungsanlage wird entscheidend zur Steigerung der Institution Erfolg in der Gesamtforschungsvorhaben (Student Engagement in Forschung, Veröffentlichungen, Kommunikation, Verfolgung von Drittmitteln, etc.). Der Bau eines Zebrafisch-Anlage, wie die in dieser Studie beschrieben ist während gende finanziellen Zeiten ideal für Forschung akademischen Einrichtungen mit begrenzten Möglichkeiten (oder Institutionen ohne in vivo Forschungseinrichtungen).

<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 biomedizinischen Forschung ist von zentraler Bedeutung, um in vitro Erkenntnisse in die klinische Fortschritte zu übersetzen. Kleine akademischen Institutionen mit beschränkten Ressourcen finden es praktisch unmöglich ist, aufzubauen und zu pflegen typischen Nagetiereinrichtungen für die Forschung. Zebrafisch-Untersuchung wurde nachgewiesen, dass eine wertvolle Alternative zur in vivo Erforschung der Pharmakologie, Physiologie, Entwicklung und genetische Untersuchungen sein. Dieser Artikel beschreibt, dass eine funktionelle Zebrafisch-Anlage kann auf eine einfache und kostengünstige Art und Weise aufgebaut werden. Wir zeigen, dass eine solche Einrichtung könnte in etwa einen Arbeitstag mit minimalem Werkzeuge und Know-how aufgebaut werden. Die Kosten für das 27 1,8 l Aquarium Zebrafisch-Anlage in dieser Studie aufgebaut war ungefähr $ 1.500. Wir gehen davon aus, dass die Aufrechterhaltung eines ersten Arbeits 150 Fischkolonie für 3 Monate ist $ 1.000. Dieses Projekt beteiligten Studenten, die eingeführt wurden, um der Zebrafisch aquaculturing für Forschung schlägt.

Der Bau der Pumpe und Flters Systems Wenn das Rack montiert und den ersten Schritt gesetzt ist, um die Pumpe und Filtersystem zu bauen. Die Filter sind mit der Systempumpe verbunden sind und über der unteren Ebene des ersten Abschnitts, die eine einfache Wartung des Filters und Austausch ermöglicht entfernt. In ähnlicher Weise wird die UV-Lampe mit PVC-Rohr mit dem Filter verbunden und an Ort und Stelle mit Kunststoffkabelbinder gehalten, um für den einfachen Austausch der UV-Lampe nach Bedarf <stron...

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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.

Bau eines erschwinglichen und einfach zu bauen Zebrafisch Einrichtung

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 Aufrufe.  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.

Serie: 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.

In-vivo-Imaging und therapeutische Behandlungen in einem orthotopen Mausmodell des Eierstockkrebs

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

Forschung

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.

Imaging Glykane in Zebrafisch-Embryonen durch metabolische Markierung und Bioorthogonale Click-Chemie

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.

In-vivo-Elektroporation von Morpholinos in die Adult Zebrafisch Retina

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.

In-vivo-Elektroporation von Morpholinos in das Regenerations Adult Zebrafisch Leitwerk

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.

Regelmäßige Pflege und Wartung eines Zebrafisch (<em&gt; Danio rerio</em&gt;) Labor: Eine Einführung

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.

Schnelle und effiziente Verwendung von Zebrafisch-Genotypisierung PCR mit Hochauflösende Schmelzanalyse

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.

Ein Test für Laterallinie Regeneration im erwachsenen Zebrafisch

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.

Subklonierung Plus-Insertion (SPI) - A Novel Recombineering Verfahren zur schnellen Bau der Gene Targeting Vektoren

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.

Nephrotoxin Mikroinjektion in Zebrabärbling akuten Nierenschädigung zu Modell

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 ...