저자는 연구 및 공동 작업 (SPARC) 수상의 작은 약국 상을 통해이 프로젝트의 금융 지원을 위해 약국의 장로교 대학 학교의 연구의 사무실에게 감사의 말씀을 전합니다. 저자는 씨 존 Smink, 수생 동물 연구소의 관리자 클렘 슨 대학 (Clemson University), 클렘 슨, 자신의 기술 지원 원고 개정 SC 감사합니다.

참고 :이 프로토콜은 장로교 대학의 IACUC의 지침을 따르십시오. 표 1에 획득 1. 재료. 2. 제조업체의 지침에 따라 5 선반 크롬을 조립합니다. 이 시설을 수용하는 데 사용됩니다. 3. 물 용기와 여과 시스템 (도 1a 및도 2)을 구축. <ol><li> 가이드로 그림 1A를 사용하여, 최종 설치 위치와 크기를 결정하기 위해 18 갤런 물통, 펌프, 필터 및 UV 램프의 레이아웃. </li><li> 부품이 배치되면, 측정 및 조립에 필요한 길이로 ¾ &quot;PVC 파이프를 잘라. 특정 공간 요구 사항 및 제한 사항에 따라 모든 파이프의 길이가 다릅니다. </li><li> PVC 프라이머와 시멘트를 사용하여 이전 배치로 파이프 및 피팅을 조립, 아래 (3.3.2과 3.3.3)를 참조하십시오. <ol><li> 모래 사포 파이프의 절단면들은 터치에 매끄러운까지D 이물질이 없습니다. </li><li> 프라임 파이프의 외부에서 프라이머 PVC 포함 면봉 구입 PVC 프라이머 피팅의 내부. </li><li> 포함 면봉을 사용하여 파이프의 외부로 PVC 시멘트를 적용하여 함께 파이프 피팅 시멘트. PVC 점착을 필요로하는 모든 단계를 반복합니다. </li><li> 선반 건설의 한면에 18 갤런 양동이를 놓습니다. </li><li> 리저버 탱크의 측면에 격벽 커넥터를 설치. </li><li> 격벽에 ¾ &quot;PVC 파이프를 연결합니다. </li><li> ¾ &quot;¾을 사용하여 펌프 흡입구에 관&quot;의 다른 쪽 끝을 연결 제조사 스레드. </li><li> 두 개의 90도 팔꿈치 및 ¾ &quot;PVC 파이프를 사용하여 펌프에 필터를 연결합니다. <ol><li> 물에서 염소 및 화학 수준 이물질을 필터링하고 감소시키기 위해 하나의 활성탄 필터 뒤에 하나의 종이 필터를 사용한다. </li></ol></li><li> 필터 accordi에 UV 램프를 연결제조업체의 지침을 ng를. </li></ol></li><li> 와이어 타이를 사용 랙 제 선반의 상부 측에 펌프 및 필터 시스템을 첨부. 선택된 선반 / 랙의 크기 및 레이아웃에 따라 조립 및 첨부 파일의 순서를 다릅니다. </li><li> 펌프와 필터 시스템 조립 및 선반에 부착하면 물이 저수지에 물 갤런 당 1 biobal​​l를 추가합니다. </li></ol> 4. 주요 공급 라인 (그림 1B 및 2)를 구축합니다. <ol><li> 측정하고 주요 공급 UV 램프를 연결하는 PVC 파이프 &quot;¾의 조각을 잘라 </li><li> UV 필터에서 오는 PVC 배기 &quot;¾에 티를 연결하여 주요 공급 라인을 시작합니다 PVC 티&quot;¾을 사용합니다. . <ol><li> 시스템의 유지 보수 및 세척을위한 드레인로 사용하기 위해 PVC 티의 바닥에 온 - 오프 밸브를 추가합니다. </li><li> PVC를 &quot;측정 ¾ 절단함으로써 PVC 티&quot;¾의 상단에서 메인 공급선 시작원하는 길이로 파이프 랙 선반들 각각에 대한 공급 파이프를 제공한다. </li><li> 설치 ¾ &quot;랙의 각 선반을 입력합니다 수평 라인 PVC 티를. </li><li> 선택적으로 (그리고 제안)을 추가 ¾ &quot;온 - 오프 밸브 각 선반 공급선 각 선반의 단리 및 시스템의 모듈 설계를 허용하도록. </li></ol></li><li> 상단 선반에 줄을 감압로 ¾ &quot;PVC 파이프를 사용합니다. 이 라인은 선반의 후면에서 실행되는 경우, 저장이 선반을 사용합니다. 주 배기 라인 ¾와 (그림 1B) &quot;온 - 오프 밸브 라인을 감압을 종료합니다. </li><li> 위의 단계는 프라임 완료하고 저장 및 여과 시스템을 구성 할 때 이전에 수행으로 함께 PVC 파이프 시멘트되고 나면. </li></ol> 5. 주 배기 라인 (그림 1C 2)를 구축합니다. <ol><li> 랙 (F)의 다른쪽에 메인 배기 라인의 수직 길이를 실행주요 공급 라인을 ROM. 각 선반에서 물고기 탱크의 상단과 드레인 라인에서 주요 공급 라인을 수신 할이 수직 라인 2 &quot;PVC 파이프를 사용합니다. </li><li> &quot;수직 주 배기 라인 PVC 파이프와 2 사용&quot;2 조각을 잘라 각각의 선반에서 물고기 탱크 드레인 라인을받을 PVC 티를. 스폰지 필터를 통해 재활용 된 물을 배출하기 위해 저장 탱크의 주요 배기 라인을 종료합니다. </li><li> 2 &quot;PVC 파이프 조각의 전체 길이를 열어 1 인치를 잘라 PVC는 물고기 탱크에서 수평 드레인 라인 티셔츠와 수직 주 배기 라인에 연결합니다. </li><li> PVC 캡 2 &quot;2 수평 드레인 라인을&quot;모자. </li><li> 프라임 필요한 피팅 (커넥터 또는 니플 팔꿈치)를 사용하여 시멘트 조각. </li></ol> 6. 물고기 탱크 (그림 3)를 구축합니다. <ol><li> 드릴, 구멍 톱 위쪽 가장자리 아래의 &quot;물고기 탱크 (1)의 뒤쪽에 구멍&quot;1 1/8를 잘라. </li><l난&gt; (외부로 탱크의 내부에서) 구멍을 통해 (첨부 # 18 O 링) 스레드 ¾ &quot;PVC 남성 어댑터를 밀어 넣습니다. </li><li> 부착하고 90도 3/4를 조 &#39;물고기 탱크 배기 파이프를 만들기 위해 남성 어댑터에 PVC 팔꿈치를 스레드. 배기계 거치지 지브라 피쉬 않도록 유지 너트를 나사 결합하기 전에 미세한 메쉬의 조각을 추가한다. </li><li> 탱크 뒤에 배수 라인의 열린 공간으로의 배기 파이프 결말 물고기 탱크를 배치 </li></ol> 7. 물 시스템 시작 <ol><li> 이러한 저장조에 사용한 것과 여러 용기에있어서, 시스템 내에 포함 된 물이 3 배 양을 측정한다. 이 물이 48 시간 동안 조절 실온에 서 보자. </li><li> 시스템에 물을 추가하고 3 일의 최소 순환 할 수 있습니다. <ol><li> 오염물 그쪽의 시스템을 제거하기 위하여 순환 초기 삼일 걸쳐 격렬 시스템에서 물을 변경t은 파이프 내에 상주. </li></ol></li><li> 스타터 제브라 피쉬를 추가하고 질소 순환이 설정 얻을 수 있습니다. 일관된 7로 pH를 정상화 1.8 L 당 일곱 제브라 피쉬를 유지하고 Linbo 4 McNabb (6)에 의해 제안 주말 동안 하루에 한 번 주 일 동안 하루에 두 번을 공급합니다. </li><li> 질산 모니터 (&lt;75 ㎎ / L), 아질산염 (&lt;10 ㎎ / L), 암모늄 물고기를 추가 한 후 첫 주 동안 매일 (0 ㎎ / L), 두 번 매주 한 번 원하는 수준은 일반 물 변화와 유지되었다 . </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>

저자는이 문서에 대한 관심의 충돌을 선언합니다.

이 문서의 주요 목적은 기능 제브라 피쉬 시설의 건설이 복잡하지 않고 최소한의 건설 지식 초보자 물고기 연구자들에 의해 만들어 질 수 있음을 입증하는 것입니다. 또한,이 문서에서는이 기능 제브라 피쉬 시설에 대한 자료는 지역의 애완 동물 공급 상점 또는 하드웨어 상점에서 쉽게 저렴하게 획득 할 수 있음을 보여줍니다. 모든 재료 및 도구는 시스템의 구성을 획득되었으며 어항 약 1 근무?...

생체 내 모델은 시스템 및 유기체 수준에서 번역 할 시험관 연구에서 수 있기 때문에 종합적인 생물 의학 연구 수행에 필수적이다. 예를 들어, 작은 포유류, 설치류, 즉, 함께 연구 약물의 개발 및 사용 가능한 약물의 개선에 필요한 입증되었다. 그러나, 복잡성 (재정적 물류는)이 사실상 불가능 구축하고 실험이 작은 연구 교육 기관에서 제안에 대한 설치류 시설을 유지 보수 할 수 있도록 다음과 같은 수 있습니다. 그럼에도 불구하고, 생체 내 연구는 덜 정교한 물류 필요한 다른 동물 종을 사용하여 수행 될 수있다. 제브라 피쉬 (다니오 레 리오 (rerio))을 성공적으로 유전학, 개발, 병태 생리 및 약리학 1,2,10,11,12을 연구하는 생체 내 모델로 사용되어왔다. 실험에 대한 제브라 피쉬를 수용하는 데 필요한 시설은 R에 비해 상대적으로 간단하고 훨씬 적은 비용이odent의 시설과 서비스를 제공합니다. Kim 등. (3)는 최근 상세히 부품 및 노동 비용을 모두 포함하는 약 $ 1,500에 대한 구축 할 수있다 (80) 탱크 제브라 피쉬의 주택 시스템을 설명했다. 이것은 상용 공급 업체의 수에 상관없이 기성품, 80 탱크 시스템을 구입해야 할 것이라고 거의 $ 8,500 극명한 대조를 이룬다. 그러나, 김 등의 알에 의해 주어진 설명. 물고기 연구의 초보자 따라하기 복잡하고 어려울 수 있습니다. 우리는 제브라 피쉬 설비가 1,500 달러 투자에 대한 단순화 된 방식으로 구성 할 수 있다고 생각합니다. 작은 교육 기관에서 생체 시설의 설립은 자신의 능력을 더 강력하고 철저한 연구를 생산하는와 추가 도구를 제공합니다. 생체 내 결과에서 지원하는 체외 데이터 일반적으로 체외 방식 만 사용하여 얻은 데이터보다 더 포괄적 인 생물 의학 연구를 생성합니다. 이 때문에,생체 내 연구 시설의 구현은 크게 전반적인 연구 노력에 기관의 성공을 향상시킬 것입니다 (외부 자금 조달 등의 연구, 출판, 통신, 추구 학생 노출을.). 이 연구에서 설명 된 것과 같은 Zebrafish의 시설의 건설은 금융 번이 수축하는 동안 (생체 내 연구 시설없이 또는 기관) 제한된 시설과 연구 교육 기관에 이상적입니다.

<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

생체 내에서 생물 의학 연구는 임상 발전에 시험관 연구 결과에 번역 중요한 것입니다. 제한된 자원을 가진 작은 교육 기관은 사실상 불가능 구축 및 연구를위한 일반적인 설치류 시설을 유지하기 위해 찾을 수 있습니다. Zebrafish의 연구는 약리학, 생리학, 개발 및 유전 연구에서 생체 연구를위한 중요한 대안이 될 입증되었습니다. 이 문서는 기능 제브라 피쉬 설비가 간단하고 저렴한 방법으로 구축 할 수 있다는 것을 보여줍니다. 우리는 설비가 최소한의 도구와 전문 지식에 대한 하나의 작업 일에 내장 될 수 있음을 보여줍니다. 본 연구에서 구축 한 27 1.8 L 수조 제브라 피쉬 시설의 비용은 약 1,500달러했다. 우리는 3 개월의 초기 작업 (150) 물고기 식민지의 유지 보수는 1,000 달러 것으로 추정된다. 연구는 제안을 위해 도입 된이 프로젝트 참여 학생들은 제브라 피쉬의 aquaculturing합니다.

펌프 및 Flters 시스템 구축 랙 조립 및 첫 번째 단계를 설정되면 펌프와 필터 시스템을 구축하는 것입니다. 필터 시스템에 연결되어 펌프 손쉽게 필터 유지 보수 및 교체 할 제 선반의 하부 레벨 위에 위치된다. 유사하게, UV 램프 필터 PVC 파이프를 연결하고 필요한 (도 4a)와 같은 UV 램프를 쉽게 교체 할 수 있도록 플라스틱 와이어 타이 제자리에 유지된다. 즉시 종이 필터, ?...

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.

저렴하고 간편한 구축 Zebrafish의 시설의 건설

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 조회수.  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.

비디오: 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.

생체내 이미징 및 난소암의 Orthotopic 마우스 모델에서 치료 트리 트먼트

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

연구

생물학

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.

신진 대사 라벨링 및 Bioorthogonal 클릭 화학에 의해 Zebrafish 태아의 영상 Glycans

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.

Morpholinos의 생체내의 Electroporation에서 성인 Zebrafish의 망막에

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.

생성중 성인 Zebrafish 테일 핀으로 Morpholinos의 생체내 Electroporation에

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.

Zebrafish의 일반 관리 및 유지 관리 (<em&gt; Danio rerio</em&gt;) 실험실 : 소개

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.

기록 개인과 성인 Zebrafish의 떼에 자동 3D 추적 시스템을 사용하여

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.

고해상도 용융 분석과 PCR을 이용하여 신속하고 효율적인 Zebrafish의 유전자형

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.

성인 Zebrafish의의 측면 라인 재생을위한 분석

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.

서브 클로닝 플러스 삽입 (SPI) - 유전자 타겟팅 벡터의 신속한 건설 소설 Recombineering 방법

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.

Zebrafish의에서 콩팥 세포 독소 미세 주입은 급성 신장 손상을 모델링하기

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 ...