Yazarlar Araştırma ve İşbirliği (SPARC) Ödülü Küçük Eczane Ödülleri aracılığıyla bu projenin mali destek Eczacılık Presbyterian Koleji Okulu Araştırma Ofisi teşekkür etmek istiyorum. Yazarlar Bay John Smink, Su Hayvancılık Araştırma Laboratuvarı Müdürü, Clemson Üniversitesi, Clemson, onun teknik yardım yazması revizyonu için SC teşekkür ederiz.

NOT: Bu protokoller Presbyterian College IACUC yönergeleri izleyin. Tablo 1 Listelenen 1. Edinme Malzemeler. 2. Üreticinin Talimatına göre 5 Raf Chrome&#39;u birleştirin. Bu Tesisi evine kullanılacaktır. 3. Su haznesini ve filtrasyon sistemi (Şekil 1A ve 2) Construct. <ol><li> Bir kılavuz olarak Şekil 1A kullanarak, nihai montaj konumunu ve boyutunu belirlemek için 18 galon kova, pompa, filtre ve UV lambası düzeni. </li><li> Parçalar ortaya koydu sonra, ölçmek ve montaj için gerekli uzunluğu ¾ &quot;PVC boru kesti. Özel alan ihtiyaçları ve kısıtlamalara göre tüm boru uzunluklarını Vary. </li><li> PVC astar ve çimento kullanarak daha önce düzenlendiği şekilde boru ve ek parçaları birleştirin, aşağıda (3.3.2 ve 3.3.3) bkz. <ol><li> Kum zımpara ile boru kesme kenarları dokunmatik An pürüzsüz olana kadard hiçbir enkaz yoktur. </li><li> Başbakan boru dış ve PVC astar gelen dahil çubukla alınan PVC astar ile bağlantı içinde. </li><li> Dahil çubukla kullanarak borunun dışına PVC çimento uygulayarak birlikte boru ve uydurma çimento. PVC çimentolama gereken tüm adımları tekrarlayın. </li><li> Raf yapımında bir tarafına 18 galonluk kova yerleştirin. </li><li> Rezervuar tankının yan bir bölme konektörü takın. </li><li> Bölmeye ¾ &quot;PVC boru takın. </li><li> ¾ &quot;Bir ¾ kullanarak pompa girişine boru&quot; diğer ucunu takın uydurma yapmak dişli. </li><li> İki 90 derecelik dirsek ve ¾ &quot;PVC boru kullanılarak pompa için filtreleri bağlayın. <ol><li> Sudaki klor ve kimyasal seviyeleri enkaz süzmek ve azaltmak için bir aktif karbon filtreleri ardından bir kağıt filtre kullanın. </li></ol></li><li> Filtreler accordi UV lambası bağlayınüreticinin talimatlarına ng. </li></ol></li><li> Tel bağları kullanarak rafa ilk rafın üst tarafına pompa ve filtre sistemi takın. Seçilen raf / raf boyutu ve düzeni göre montaj ve bağlantı sırasını Vary. </li><li> Pompa ve filtre sistemi monte ve raf bağlı kez su deposuna su galon başına 1 BioBall ekleyin. </li></ol> 4. ana besleme hattı (Şekil 1B ve 2) Construct. <ol><li> Tedbir ve ana kaynağı UV lamba bağlamak için PVC boru &quot;¾ bir parça kesti </li><li> UV filtresi gelen PVC egzoz ¾ &quot;için tee bağlayarak ana besleme hattı başlatmak için PVC tee&quot; ¾ kullanın. . <ol><li> Sistemin bakımı yıkama için bir boşaltma olarak hizmet etmek üzere, PVC tee altına bir açma-kapama valfi ekleyin. </li><li> PVC ¾ &quot;ölçme ve kesme PVC tee&quot; ¾ üst ana besleme hattı başlayınİstenilen uzunlukta boru raf raf her besleme boruları sağlamak. </li><li> Install ¾ &quot;raf her raf girecek yatay çizgiler PVC tee. </li><li> İsteğe bağlı olarak (ve önerilen), bir ekleme ¾ &quot;açma-kapama valfı, her raf besleme hattı için her bir raf izolasyonu ve sistemin modüler tasarımı için izin verir. </li></ol></li><li> Üst rafta çizgi basınç düşürme gibi ¾ &quot;PVC boru kullanın. Bu hat raf arkasında çalışıyorsa, depolama için bu raf kullanın. Ana egzoz hattı, bir ¾ ile (Şekil 1B) &quot;açma-kapama vanası çizgiyi basınç azaltma End. </li><li> Yukarıdaki adımlar, başbakan tamamlanmış ve rezervuar ve filtrasyon sistemi inşa sırasında önceden yapıldığı gibi birlikte PVC boru çimento edildikten sonra. </li></ol> 5. Ana egzoz hattı (Şekil 1C ve 2) Construct. <ol><li> Raf f diğer tarafında ana egzoz hattının düşey uzunluğunu çalıştırınana besleme hattı rom. Her raftan balık tankları üst ve drenaj hatlarında ana besleme hattı almak için bu dikey hat için 2 &quot;PVC boru kullanın. </li><li> &quot;Dikey ana egzoz hattı için PVC boru ve 2 kullanın&quot; 2 parçaları Kes her raftan balık tankı boşaltma hatları almak için PVC tee. Bir sünger filtre üzerinde dönüştürülmüş su çıkarmak için stok tankı ana egzoz hattı End. </li><li> 2 &quot;PVC boru parçaları tüm uzunluğu açılış bir inç kesin ve PVC balık tankları yatay drenaj hatları için tee dikey ana egzoz hattına takın. </li><li> PVC kapağı 2 &quot;, 2 yatay drenaj hatları&quot; Cap. </li><li> Başbakan ve gerekli parçaları (konektörler, meme veya dirsekler) kullanarak parçaları çimento. </li></ol> 6. balık tankları (Şekil 3) Construct. <ol><li> Matkap ile bir delik testere ile üst kenarının altına &quot;balık tankı 1 arkasındaki delik&quot; 1 1/8 kesti. </li><li&gt; (dış tankın içinden) delikten (bağlı bir 18. O halkası ile) bir dişli ¾ &quot;PVC erkek adaptörü kaydırın. </li><li> Takın ve 90 derecesi 3/4 sıkın &#39;balık tankı egzoz borusunu oluşturmak için erkek adaptöre PVC dirsek dişli. Egzoz sistemi geçiyor zebrafish önlemek için tutma somunu vidalama önce ince örgü bir parça ekleyin. </li><li> Tanklar arkasındaki boşaltma hattı açık uzaya egzoz borusu biten balık tankı yerleştirin </li></ol> 7. suyu sistemi Başlangıç <ol><li> Bu hazne için kullanılan gibi çeşitli kaplar olarak, sistem içinde bulunan su için miktarın üç katı ölçer. Bu su 48 saat süre ile klima oda sıcaklığında bekletin. </li><li> Sisteme su ilave edin ve 3 gün, en az deveran etmesi. <ol><li> Kirletici tha sistemi kurtulmak için dolaşım ilk 3 gün boyunca şiddetle sistemde su değiştirint borular içinde bulunur. </li></ol></li><li> Marş zebrafish ekleyin ve azot döngüsü kurulan almak için izin verir. Tutarlı 7. pH Normale 1.8 L başına yedi zebrafish tutun ve Linbo 4 ve 6 McNabb tarafından önerilen sonları günde bir kez hafta içi günde iki kez onları beslemek ve. </li><li> Nitrat Monitör (&lt;75 mg / L), nitrit (&lt;10 mg / L), ve amonyum balık ekledikten sonra ilk hafta boyunca her gün (0 mg / L), ve iki haftada bir kez istenilen seviyeleri düzenli su değişimleri ile muhafaza edilmiştir . </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>

Yazarlar bu kağıt için herhangi bir çıkar çatışmalarını beyan ederim.

Bu makalenin temel amacı fonksiyonel Zebra balığı tesisin yapımı karmaşık değildir ve minimal inşaat bilgisine sahip acemi balık araştırmacılar tarafından inşa edilebilir olduğunu göstermektir. Ayrıca, bu makalede, bu fonksiyonel Zebra balığı tesisi için malzemeler yerel evcil hayvan besleme mağazalarda veya donanım mağazalarından kolayca ve uygun maliyetle elde edilebilir olduğunu göstermektedir. Tüm malzemeler ve aletler sisteminin inşasını elde edilmiş ve balık tankları yaklaşık ...

İn vivo modeller bunlar sistem ve organizma düzeyinde tercüme edilecek in vitro araştırma izin beri kapsamlı biyomedikal araştırma esastır. Örneğin, küçük memeliler, yani kemirgenler, araştırma ilaçların geliştirilmesinde ve mevcut ilaçların geliştirilmesi için gerekli olduğu kanıtlanmıştır. Ancak, karmaşıklık (mali ve lojistik) neredeyse imkansız oluşturmak ve deneme küçük araştırma akademik kurumlarda teklif için kemirgen tesisleri korumak için yapmak olabilir. Bununla birlikte, in vivo araştırmalar daha az karmaşık, lojistik gerektiren diğer hayvan türlerini kullanarak gerçekleştirilebilir. Balığı (Danio rerio) başarılı bir şekilde genetik, geliştirme, patofizyolojisi ve farmakoloji 1,2,10,11,12 üzerinde çalışmak için in vivo bir modeli olarak kullanılmıştır. deney için zebrafish ev için gerekli imkanları r ile karşılaştırıldığında nispeten basit ve daha az pahalıOdent tesisler. Kim ve ark. 3 son zamanlarda detaylı parça ve işçilik maliyetlerini hem de içeren yaklaşık 1500 $ için inşa edilebilir 80 tankı Zebra balığı konut sistemi tarif var. Bu ticari tedarikçi herhangi bir sayıda bir hazır, 80 tankı sistemi satın almak için gerekli olacağını yaklaşık 8500 $ tezat duruyor. Ancak, Kim ve arkadaşları tarafından verilen açıklama. Balık araştırma acemiler için takip etmek karmaşık ve zor olabilir. Biz bir Zebra balığı tesis 1.500 $ bir yatırım için basitleştirilmiş bir şekilde inşa edilebilir inanıyoruz. küçük akademik kurumlarda in vivo tesisin kurulması okudukları fakülte daha güçlü ve kapsamlı araştırma üretmek için ek bir araç sağlar. in vivo sonuçları ile desteklenmektedir in vitro veriler, genellikle in vitro yaklaşımlar sadece kullanılarak elde edilen verilerin daha kapsamlı biyomedikal araştırmalar üretmek. Bu nedenle,in vivo araştırma tesisi uygulanması önemli ölçüde genel araştırma çalışmalarında kurum başarısını artıracaktır (dış finansman, vb araştırma, yayınlar, iletişim, peşinde öğrenci maruz kalma.). Bu çalışmada açıklanan gibi bir Zebra balığı tesisin yapımı finansal kez daraltıcı sırasında (in vivo araştırma tesisleri olmadan veya kurumlar) sınırlı imkanlarla araştırma, akademik kurumlar için idealdir.

<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

İn vivo biyomedikal araştırma klinik ilerlemeler in vitro bulgular çevirmek için çok önemlidir. Sınırlı kaynaklarla Küçük akademik kurumlar neredeyse imkansız oluşturmak ve araştırma için tipik kemirgen tesisleri korumak için bulabilirsiniz. Balığı araştırma farmakoloji, fizyoloji, geliştirme ve genetik çalışmalarda in vivo araştırmalar için önemli bir alternatif olduğu kanıtlanmıştır. Bu makalede, fonksiyonel Zebra balığı tesis kolay ve uygun fiyatlı bir şekilde inşa edilebileceğini göstermektedir. Biz böyle bir tesis asgari araç ve uzmanlığı ile ilgili bir iş günü içinde inşa edilebileceğini göstermektedir. Bu çalışmada inşa 27 1.8 L akvaryum Zebra balığı tesisin maliyeti yaklaşık 1500 $ oldu. Biz 3 ay boyunca bir ilk çalışma 150 balık kolonisinin bakım 1.000 $ olduğunu tahmin. Araştırma önermektedir için getirilmiştir Bu proje katılan öğrenciler, zebrafish aquaculturing için.

Pompa ve Flters Sistemi İnşaatı Raf monte ve ilk adımı kurulduktan sonra pompa ve filtre sistemi inşa etmektir. Filtreler sistem pompasının bağlı ve kolay filtre bakım ve değişimi için izin veren ilk raf, alt seviyesinden bulunmaktadır. Benzer bir şekilde, UV lambası filtresi PVC boru ile bağlı olan ve gerektiğinde (Şekil 4A) ve UV lambası kolayca değiştirilmesine olanak tanımak amacıyla, plastik tel bağlar ile yerinde tutulur. Hemen bir kağıt filtre, aktif k...

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.

Bir Ekonomik ve Kolay oluşturun Zebra balığı Tesisi İnşaatı

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

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Research

JoVE Journal

Biology

16.2K Görüntüleme.  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.

Video: 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 Görüntüleme ve Yumurtalık Kanseri Ortotopik Fare Modeli Tedavi Tedaviler

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

Biyoloji

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.

Metabolik Etiketleme ve Bioorthogonal tıklayın Kimya Zebra balığı Embriyolar Görüntüleme glukanlardır

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.

Morpholinos in vivo Elektroporasyon Yetişkin Zebra balığı 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.

Yenileyici Yetişkin Zebra balığı Tail Fin içine Morpholinos in vivo olarak Elektroporasyon

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.

Bir Zebra balığı Düzenli Bakım ve Onarım (<em&gt; Br.rerio</em&gt;) Laboratuvar: 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.

Yüksek çözünürlüklü Melt Analizi ile PCR kullanarak hızlı ve verimli Zebra Genotiplemesi

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.

Yetişkin Zebra balığı Lateral Hat Rejenerasyon için bir Deneyi

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.

Alt klonlama Artı Ekleme (SPI) - Gen Hedefleme Vektörler Rapid İnşaat için Yeni Bir Yöntem Recombineering

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.

Zebra balığı Nephrotoxin Mikroenjeksiyon Akut Böbrek Yaralanması Model

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 ...