록펠러 대학 비교 생명 과학 센터는이 프로젝트에 대한 지원을 제공했습니다. 우리는 기술 지원을 쟈넬 Monnas 감사합니다.

1. 마취 용액을 제조 <ol><li> 원액이나 분말 형태에서 하나의 시스템 물 150 ㎎ / MS-222 L의 용액을 준비합니다. </li><li> 산도 측정기를 사용하여 pH를 확인합니다. </li><li> pH가 7-7.5 사이까지 중탄산 나트륨 버퍼가 필요. </li><li> 복구 시간을 안으로 복구하는 물고기는 일반적으로보다 1 분입니다에 대한 MS-222에서 무료로 시스템 물 탱크를 준비합니다. </li></ol> 2. 투여 장치를 준비 <ol><li> 입에서 물고기의 장 전구에 대략 길이보다 조금 더에 분명 22-G 주입 튜브를 잘라. 처음에는, 우리는 필요한 길이에 대한 최선의 추정치를 얻을하는 데 사용되는 각 새 그룹에 대해 하나의 물고기를 마취하는 것이 좋습니다. </li><li> 그것은 안전하고 분리의 위험이 없을 때까지 ½ 인치 22-G 바늘 위에 컷 명확한 주입 튜브를 밀어 넣습니다. ½ 22-C 바늘을 사용할 수없는 경우, 응용 프로그램에 더 이상 바늘을 잘라roximately 그 길이입니다. </li><li> 완료되면, 카테터 주입 튜브 1 바늘의 끝에서 cm, 또는 물고기가 작은 경우 짧은 대해 확장해야합니다. </li><li> 1-CC luerlok 주사기에 22 G 바늘 허브를 연결합니다. </li></ol> 3. 위관을위한 준비 <ol><li> 메스 블레이드와 스폰지로 홈을 잘라. </li><li> MS-222에서 무료로 시스템 물에 스폰지를 적시십시오. </li><li> 평평한 표면에 적신 스폰지를 놓습니다. </li><li> 22 G 카테터 튜브와 1-CC 주사기로 투여에 해당하는 5 μL 솔루션을 그립니다. </li></ol> 4. 마취 투여 <ol><li> 빠른 절차를하기 전에 적어도 24 시간 동안 물고기. </li><li> 그것의 바로 잡기 반사를 잃고, 그리고 꼬리 지느러미 핀치에 응답하지 않지만, 아가미의 움직임을 유지 할 때까지 MS-222 솔루션에있는 물고기를 놓습니다. 이는 일반적으로 약 2-3 분 걸립니다. </li><li> 마취 용액에서 물고기를 제거하고에 배치머리를 약간 스폰지에서 튀어 나온 스폰지의 홈하지만, 아가미 스폰지에 의해 덮여. </li><li> 수직 위치에 스폰지를 이동합니다. </li><li> 22 G 카테터 튜브를 사용하여 제브라 피쉬의 입을 엽니 다. </li><li> 팁 아가미 (약 cm 또는 튜브의 길이) 과거 때까지 부드럽게 튜브를 삽입합니다. 주입 튜브 강제 할 필요는 없습니다. 저항 튜브 아가미 아치 또는 심장을 타격 할 수 있습니다 제안합니다. </li><li> 저항이있을 경우, 부드럽게, 철회 위치를 변경하고 다시 시도하십시오. </li><li> 천천히 물질을 주입. </li><li> 주입하는 동안 솔루션은 아가미 나 입을 통해 종료되지 않습니다 있는지 확인하십시오. </li><li> 복구 탱크로 스폰지 곳에서 물고기를 제거합니다. </li><li> 복구는 일반적으로 이하 1 분에 발생 똑바로 수영과 균형을 유지 물고기로 표시됩니다. </li><li> 적극적으로 M 추방 물고기를 시각화하여 그림과 같이 역류에 대한 물고기를 모니터링입하거나, 아가미 운동에서 aterial. </li><li> 그들이 회복되면 물고기들은 정기적으로 탱크로 반환 할 수 있습니다. </li></ol>

<ol>
	<li>Royes, J. -. A. B., Chapman, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Preparing Your Own Fish Feeds. , (2009).</li><li>Gomez-Gil, B., Cabanillas-Ramos, J., Paez-Brambila, A., Roque, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardization+of+the+bioencapsulation+of+enrofloxacin+and+oxytetracycline+in+Artemia+fransciscana+Kellogg.">Standardization of the bioencapsulation of enrofloxacin and oxytetracycline in Artemia fransciscana Kellogg.</a> Aquaculture. 196, 1-12 (1906).</li><li>Langdon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microparticle+types+for+delivering+nutrients+to+marine+fish+larvae.">Microparticle types for delivering nutrients to marine fish larvae.</a> Aquaculture. 227, 259-275 (2003).</li><li>Langdon, C., Nordgreen, A., Hawkyard, M., Hamre, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+wax+spray+beads+for+delivery+of+low-molecular+weight,+water+soluble+nutrients+and+antibiotics+to+Artemia.">Evaluation of wax spray beads for delivery of low-molecular weight, water soluble nutrients and antibiotics to Artemia.</a> Aquaculture. 284, 151-158 (2008).</li><li>Zang, L., Morikane, D., Shimada, Y., Tanaka, T., Nishimura, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Protocol+for+the+Oral+Administration+of+Test+Chemicals+to+Adult+Zebrafish.">A Novel Protocol for the Oral Administration of Test Chemicals to Adult Zebrafish.</a> Zebrafish. 8, 203-210 (2011).</li><li>Goldsmith, J. R., Jobin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Think+Small:+Zebrafish+as+a+Model+System+of+Human+pathology.">Think Small: Zebrafish as a Model System of Human pathology.</a> Journal of Biomedicine and Biotechnology. , (2012).</li><li>Tysnes, K. R., Jorgensen, A., Poppe, T., Midtlyng, P. J., Robertson, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preliminary+expermients+on+use+of+zebrafish+as+a+laboratory+model+for+Giardia+duodenalis+infection.">Preliminary expermients on use of zebrafish as a laboratory model for Giardia duodenalis infection.</a> Acta Parasitologica. 57, 1-6 (2012).</li><li>Marie, B., Huet, H., 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=Effects+of+a+toxic+cyanobacterial+bloom+(Planktothris+agardhii)+on+fish:+Insights+from+histopathological+and+quantitative+proteomic+assessments+following+the+oral+exposure+of+medaka+fish+(Oryzias+latipes).">Effects of a toxic cyanobacterial bloom (Planktothris agardhii) on fish: Insights from histopathological and quantitative proteomic assessments following the oral exposure of medaka fish (Oryzias latipes).</a> Aquatic Toxicology. 114-115, 39-48 (2012).</li><li>Canadian Council on Animal Care. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> CCAC guidelines on: the care and use of fish in research, teaching and testing. , (2005).</li><li>Harper, C., Lawrence, C. <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 Laboratory Zebrafish. , (2011).</li><li>De Tolla, L. J., Srinivas, S., 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=Guidelines+for+the+Care+and+Use+of+Fish+in+Research.">Guidelines for the Care and Use of Fish in Research.</a> ILAR Journal. 37 (4), (1995).</li><li>Topic Popovic, N., Strunjak-Perovic, I., 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=Tricaine+methane-sulfonate+(MS-222)+application+in+fish+anaesthesia.">Tricaine methane-sulfonate (MS-222) application in fish anaesthesia.</a> Journal of Applied Ichthyology. 28, 553-564 (2012).</li><li>Field, H. A., Kelley, K. A., Martell, L., Goldstein, A. M., Serluca, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+gastrointestinal+physiology+using+a+novel+intestinal+transit+assay+in+zebrafish.">Analysis of gastrointestinal physiology using a novel intestinal transit assay in zebrafish.</a> Neurogastroenterology &amp; Motility. 21, 304-312 (2009).</li></ol>

저자는 그들이 더 경쟁 재정적 이익이 없다는 것을 선언합니다.

의미 이 기술은 개선 성인 제브라 피쉬를 gavaging에 대한 두 가지 이전에 설명한 기술에 비해 몇 가지 장점을 제공합니다. 첫째, 물고기를 억제하는 스폰지의 사용은 두 사람이 필요로 설명한 기술을 반대 한 사람이 절차를 수행 할 수 있습니다. 7 둘째, 카테터 주입 튜브 인해 차 외상을 최소화 할 수있는 IV 카테터보다 더 유연 유사 무딘 팁 주사기를 사용하여 천공한다. ...

제브라 피쉬에 구두 화합물을 관리하기위한 현재의 방법은 관리 화합물 공급 위에 상단 코팅, 젤라틴 다이어트 1로 혼합, 소금물 새우 2에 bioencapsulated, 지질 캡슐 다이어트 3, 왁스 스프레이 구슬 4에 글루텐 기반의 다이어트를 통해 혼합 (가) 있습니다. 5 제한 경구 투여의 방법들은 높은 침출 속도와 각 물고기에 의해 불완전하거나 예측할 수없는 복합 소비를 이용하실 수 있습니다. 전염성 질병 연구에, 물고기에 투여 감염 복용량 연구의 성공을위한 중요한 수 있습니다 알고 있기 때문에 이러한 변수는 문제가 있습니다. 또한, 전작 수조에 투여 특정 화합물은 연구 중에있을 수 있습니다 창자 효과가 발생하기 전에 아가미를 제브라 피쉬에 독성 병변을 일으킬 것으로 나타났습니다. 6 위관 알래스카와 제품의 정확한 수량을 관리하는 다른 실험실 동물 종에 사용되는 표준 방법입니다생물 의학 및 제약 연구에 연구를위한 nown 농도. 최근에 문헌에 기술 된 제브라 피쉬를 gavaging하는 방법이 있습니다. 설명 하나의 기술 솔루션의 5 μL를 전달하기 위해 2-20 μL 피펫에 부착 된 24 G 카테터 칼집을 사용하여 두 사람 방법입니다. 설명 7 방법은 첫 번째와 두 번째의 사망률 8.7 %의 속도와 39 %를했다 각각 재판, 사망의 대부분은 임신하는 여성에 기인 하였다. 두 번째 투여 기술은 5 medakas에 대한 솔루션의 μL (Oryzias의 latipes). 사망률 8 정보가 제공되지 않았으며 투여 설명되지 않았습니다 수행하는 정확한 과정을 관리하는 데 사용 무딘 팁 투여 주사기를 설명했다. 위로 물고기의 체중의 1 % 동물 관리 및 실험실 제브라 피쉬에 캐나다위원회에 따르면, 위관을 통해 관리 할 수 있습니다. 9,10 우리의 목표는 COM 정확한 양을 전달하는 반복, 안전하고 효율적인 방법을 개발하는 것이 었습니다파운드 구두 남녀 성인 제브라 피쉬에와 재생의 모든 단계합니다. 이 절차는 화합물의 정확한 경구 투여가 요구되는 모든 연구에 적용 할 수있다.

<table border="1">
<tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>A sponge</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>1-cc luer lock syringe</td>
 <td>Becton Dickinson &amp; Co</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>22-G Micro-Renathane Implantation tubing (cut)</td>
 <td>Braintree Scientific, Inc.</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>1 22-G needle (cut, if needed)</td>
 <td>Becton Dickinson &amp; Co</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>pH meter</td>
 <td>Hanna probe</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>1 scalpel blade</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>[header]</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent/Material</td>
 </tr>
 <tr>
 <td>MS-222 (Finquel)</td>
 <td>Argent Laboratories</td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

gavaging adult zebrafish

제브라 피쉬는 생물 의학 연구에서 생체 내 모델에서 중요 해지고있다. 효과적인 방법이 개발 및 과학 연구를위한 솔루션 화합물 또는 대리인을 제공하기 위해 활용해야합니다. 성인 zebrafish의 구두로 화합물을 관리하기위한 현재의 방법은 물고기의 자발적 소비의 변화로 인해 부정확합니다. 위관 절차는 생물 의학 연구에 연구 제브라하는 병원체의 정확한 양을 제공하기 위해 개발되었다. 생후 6 개월 성인 제브라 피쉬는 150 ㎎ / L로 마취 MS-222을 버퍼링 컷 22-G 바늘 팁에 부착 된 유연한 카테터 주입 튜브를 사용하여 용액 5 μL와 gavaged. 튜브의 끝이 아가미 (약 1cm)을지나 연장까지 유연한 튜브는 제브라 피쉬의 구강으로 낮아졌다. 이 솔루션은 천천히 창자에 주입 하였다. 이 방법은 물고기가 평온 무사하게 회복과 함께, 시간의 88 % 효과가 있었다. 이한 사람이 한 시간 20-30 물고기를 투여 수있는 절차도 효율적입니다. 이 방법은 정확하게 성인 zebrafish에있는 전염성 질병 연구, 또는 다른 화합물의 연구에 에이전트를 관리하는 데 사용할 수 있습니다.

임신하는 여성을 포함한 물고기의 남녀가 성공적으로 (그림 1) gavaged 하였다. 성공적인 절차는 아가미 나 구강을 종료 볼 수없는 액체를 분 미만 소요됩니다. 튜브 카테터 제거에 볼 수없는 피, 힘없이 쉽게 들어갑니다. 이 절차는 약 10 분 물고기 당 30 ~ 45 초, 평균 3-4 물고기를 투여 할 필요 빠르다. 제브라 피쉬는 모든 솔루션의 5 μL로 gavaged 할 수 없습니다. 10 μL ...

Watch this Scientific Journal Video about Gavaging Adult Zebrafish at JoVE.com

Gavaging Adult Zebrafish

동물 모델로 제브라 피쉬의 사용 증가 화합물 및 솔루션의 알려진 양의 전달을위한 효과적인 방법의 개발이 필요합니다. 아래에 설명 된 위관 절차는 성인 zebrafish의를 안전하고 효율적으로, 안정적으로 솔루션 정확한 양의 경구 제공 할 수 있습니다.

성인 Zebrafish의를 Gavaging

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

Research

JoVE Journal

Biology

20.7K 조회수.  The Rockefeller University. 동물 모델로 제브라 피쉬의 사용 증가 화합물 및 솔루션의 알려진 양의 전달을위한 효과적인 방법의 개발이 필요합니다. 아래에 설명 된 위관 절차는 성인 zebrafish의를 안전하고 효율적으로, 안정적으로 솔루션 정확한 양의 경구 제공 할 수 있습니다.

비디오: Gavaging Adult Zebrafish

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

성인 Zebrafish의 전체 신장 마로우의 이식

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

연구

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Retro-orbital Injection in Adult Zebrafish

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is often the most effective and noninvasive form of delivery of an agent of interest. In the zebrafish (Danio rerio), IV injection of drugs has long been a challenge because of the small vessel diameter. This has also proved a significant hurdle for the injection of cells during hematopoeitic stem cell transplantation. Historically, injections into the bloodstream were done directly through the heart. However, this intra-cardiac procedure has a very high mortality rate as the heart is often punctured during injection leaving the fish prone to infection, massive blood loss or fatal organ damage. Drawing on our experience with the mouse, we have developed a new injection procedure in the zebrafish in which the injection site is behind the eye and into the retro-orbital venous sinus. This retro-orbital (RO) injection technique has been successfully employed in both the injection of drugs in the adult fish as well as transplantation of whole kidney marrow cells. RO injection has a much lower mortality rate than traditional intra-cardiac injection. Fish that are injected retro-orbitally tend to bleed less following injection and are at a much lower risk of injury to a major organ like the heart. Further, when performed properly, injected cells and/or drugs quickly enter the bloodstream allowing compounds to exert their effect on the whole fish and kidney cells to easily home to their niche. Thus, this new injection technique minimizes mortality while allowing efficient delivery of material into the bloodstream of adult fish. Here we exemplify this technique by retro-orbital injection of Tg(globin:GFP) cells into adult casper fish as well as injection of a red fluorescent dye (dextran, Texas Red ) into adult casper fish. We then visualize successful injections by whole animal fluorescence microscopy.

Here we show how to do retro-orbital injection in adult zebrafish.

성인 Zebrafish에서 레트로 - 궤도 분사

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is ...

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

성인 Zebrafish에서 기관의 해부

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

성인 Zebrafish에 Intraperitoneal 사출

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases1. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease2. Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells3. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.

Cell transplantation is an essential technique for studying tissue regeneration and for developing cell-based therapies of disease. We demonstrate here a microsurgical technique that permits the transplantation of genetically labeled cells directly into the kidney of adult zebrafish fish.

직접 성인 Zebrafish의 신장에 세포의 이식

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

성인 Zebrafish의 신장의 해부

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

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 ...

Blood Collection for Biochemical Analysis in Adult Zebrafish

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies1. 
There are relatively few publications related to adult zebrafish physiology of organs and systems2, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking3. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues4 who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta5. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year6. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish7. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al.8. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. 
Recently, Gupta et al.9 described how to dissect adult zebrafish organs, Kinkle et al.10 described how to perform intraperitoneal injections, and Pugach et al.11 described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish.
The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.

This paper presents a technique for collection of blood from the dorsal aorta of Zebrafish. It also provides instructions for obtaining serum for use in biochemical analyses, such as tests for determining cholesterol and triglyceride levels.

성인 Zebrafish의 생물 화학 분석을위한 혈액 수집

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of ...

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.