Rockefeller Üniversitesi Karşılaştırmalı Biyobilim Merkezi bu projeye destek sağladı. Teknik yardım için Janelle Monnas teşekkür ederim.

1. Anestezik sıvısının hazırlanması <ol><li> , Bir stok çözeltisinden veya toz şeklinde ya da ikinci sistem su içinde 150 mg / MS-222, L içinde bir çözeltisi hazırlandı. </li><li> Bir pH metre kullanarak pH doğrulayın. </li><li> PH 7-7.5 arasında olana kadar sodyum bikarbonat tampon olarak gerekli. </li><li> Iyileşme süresi içeri kurtarmak için balık genellikle 1 dakikadan daha az olduğu için MS-222 ücretsiz sistem su tankı hazırlayın. </li></ol> 2. Gavaj Aparatı hazırlanması <ol><li> Ağzından balık bağırsak ampul yaklaşık uzunluğu biraz daha fazla açık 22-G implantasyon boru kesin. Başlangıçta, gerekli uzunluğu en iyi tahmin almak için kullanılan her yeni grup için bir balık anestezi öneririz. </li><li> Bu güvenli ve ayırma riski yoktur kadar ½ inç 22-G iğne üzerine kesme açık implantasyon boru itin. Bir ½ 22-C iğne mevcut değilse, uygulama için daha uzun bir iğne kestiroximately bu uzunluğu. </li><li> Tamamlandığında, kateter implantasyon boru 1 iğne ucundan cm, ya da balık küçük ise daha kısa uzanmalıdır. </li><li> 1-cc luerlok şırıngaya 22-G iğne hub takın. </li></ol> 3. Gavaj için hazırlanıyor <ol><li> Bir neşter bıçak ile bir süngeri bir oluk kesin. </li><li> MS-222 ücretsiz sistemi su sünger bekletin. </li><li> Düz bir yüzeye nemlendirilmiş sünger yerleştirin. </li><li> 22-G kateter tüp ve 1-cc şırınga içine sonda için uygun 5 ul çözüm hazırlayın. </li></ol> 4. Anestezi ve Gavaj <ol><li> Hızlı işlem öncesi en az 24 saat balık. </li><li> Onun düzeltilmesi refleks kaybeder ve bir kuyruk yüzgeci tutam yanıt vermiyor, ama operküler hareket tutar kadar MS-222 çözüm balık yerleştirin. Bu genellikle 2-3 dakika sürer. </li><li> Anestezi çözüm balık çıkarın ve içine yerleştirinBaş hafifçe sünger çıkıntılı ile sünger oluk, ama solungaçları sünger ile kaplı. </li><li> Dikey pozisyona sünger taşıyın. </li><li> 22-G kateter boru kullanarak Zebra balığı ağzına açın. </li><li> Ucu solungaçları (yaklaşık 1 cm ya da boru uzunluğu) Son kadar yavaşça boru takın. Implantasyon boru zorunda olması gerekmez. Direnç tüp solungaç kemer veya kalp isabet olabilir göstermektedir. </li><li> Direnç varsa, yavaşça, geri yeniden konumlandırmak ve yeniden deneyin. </li><li> Yavaş yavaş malzeme enjekte edilir. </li><li> Enjekte ederken, çözüm solungaçları veya ağız yoluyla çıkmak olmadığından emin olun. </li><li> Alım deposunun içine sünger ve yerden balık çıkarın. </li><li> Kurtarma tipik olarak en az 1 dakika içinde meydana gelir ve dik yüzme ve dengenin muhafaza balık tarafından belirtilir. </li><li> Aktif m kovma balık görselleştirme tarafından gösterildiği gibi yetersizliği için balık Monitörağzında, ya da hiç operküler hareketinden aterial. </li><li> Onlar iyileşti kez balık düzenli tanka iade edilebilir. </li></ol>

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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. 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Yazarlar hiçbir rakip mali çıkarlarının olmadığını beyan ederim.

Önem Bu teknik bir gelişmedir ve yetişkin zebrafish gavaging için iki daha önce açıklanan teknikleri üzerinde çeşitli avantajlar sağlamaktadır. İlk olarak, balık tutmaya bir sünger kullanımı iki kişi gerektiren bir önce açıklanan teknik aksine bir kişi, yordamı gerçekleştirmek için izin verir. 7 İkincisi, kateter implantasyon tüp nedeniyle ikincil travma en aza indirmek yardımcı olabilir, IV kateterler daha esnektir benzer künt uçlu şırınga kullanar...

Zebra balığı için sözlü bileşikler yönetmek için mevcut yöntemler yönetmek bileşikler besleme üzerine üst kaplı, jelatin diyetler 1 karışık, tuzlu su karidesi 2 içine bioencapsulated, lipid kapsüllü diyetler 3, mum sprey boncuk 4 ve gluten tabanlı diyetler ile karışık içerir. 5 Sınırlamalar oral bu yöntemler yüksek yıkama oranları ve bireysel balık tarafından eksik veya öngörülemeyen bileşik tüketimi içerir. Bulaşıcı hastalık çalışmalarında, balık uygulanan enfektif doz çalışmasında başarı için kritik olabilir bilerek çünkü bu değişkenler sorunludur. Ayrıca, önceki çalışma su banyoları uygulanan bazı bileşikler çalışılan olabilir bağırsak etkileri ortaya çıkmadan önce solungaçları Zebra balığı için toksik lezyon neden olduğunu göstermiştir. 6 Sonda ak ile bir ürünün kesin miktarda yönetmek için diğer laboratuvar hayvan türlerinde kullanılan standart bir yöntemdirbiyomedikal ve ilaç araştırma çalışma için nown konsantrasyon. Ancak son zamanlarda literatürde tarif edilmiştir zebrabalıkları gavaging için yöntemler vardır. Açıklanan bir teknik çözüm 5 ul teslim etmek için bir 2-20 ul pipet bağlanmış bir 24 G kateter kılıf kullanılarak, iki kişilik bir yöntemdir. Tarif 7 yöntem, birinci ve ikinci bir mortalite oranı% 8.7 ve% 39 oldu sırasıyla deneme; mortalitenin en gebe kadın bağlandı. İkinci sonda tekniği 5 medakas çözüm ul (Oryzias latipes). Mortalite 8 bilgiler bulunmuyordu ve sonda tarif değildi yerine tam süreci yönetmek için kullanılan künt uçlu sonda şırınga nitelendirdi. Kadar balığın vücut ağırlığının% 1 Hayvan Bakım ve Laboratuar Zebra balığı, günü Kanada Konseyi&#39;ne göre sonda ile verilebilir. 9,10 Amacımız com hassas cilt teslim bir tekrarlanabilir, güvenli ve etkili bir yöntem geliştirmektikilo sözlü her iki cinsten yetişkin zebrafish ve üreme her aşamasında. Bu prosedür, bir bileşiğin doğru oral dozlama gerekli olduğu herhangi bir çalışma için geçerli olacaktır.

<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

Zebra balığı biyomedikal araştırma in vivo modelde önemli bir hale gelmiştir. Etkili yöntemler geliştirilmiş ve bilimsel araştırma için çözümler bileşikler veya acenteleri sunmak için kullanılmalıdır. Yetişkin zebrafish için sözlü bileşikler yönetmek için mevcut yöntemler balık gönüllü tüketimi değişkenlik nedeniyle yanlış. Bir sonda prosedürü biyomedikal araştırmalarda çalışma için Zebra balığı için enfeksiyöz ajanların hassas miktarlarda sunmak için geliştirilmiştir. Yaş 6 ay içinde Yetişkin Zebra balığı 150 mg / L ile anestezi edildi MS-222 tamponlu ve bir kesim 22-G iğne ucu bağlı esnek kateter implantasyonu boru kullanarak çözüm 5 ul zorla ağız yolundan verilmiştir. Borunun ucu solungaçları (yaklaşık 1 cm) uzamasına kadar esnek boru zebrabalıkları ağız boşluğu içine düşürülmüştür. Bu çözelti daha sonra yavaş yavaş bağırsak içine enjekte edildi. Bu yöntem, balık sorunsuz kurtarma ile, zamanın% 88 etkili olmuştur. Bubir kişi bir saat içinde 20-30 balık sonda gibi prosedürü de etkilidir. Bu yöntem, tam olarak yetişkin zebrabalıkları bulaşıcı hastalıklar çalışmalar ya da diğer bileşikler çalışmaları için ajanlar yönetmek için kullanılabilir.

Gebe kadın dahil olmak üzere balık, her iki cinste başarıyla (Şekil 1) zorla ağız yolundan verilmiştir edildi. Başarılı bir prosedür solungaçlar veya ağız çıkarken görülen herhangi bir sıvı ile bir dakikadan az sürer. Boru kateter çıkarıldıktan görülen kan ile, kuvvet olmadan, kolayca girer. Prosedür yaklaşık 10 dakika balık başına 30-45 saniye bir ortalama ile, 3 ile 4 balık sonda gerektiren, hızlıdır. Balığı herhangi bir çözümü...

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

Gavaging Adult Zebrafish

Bir hayvan modeli olarak zebrabalıkları artan kullanımı bileşikler ve çözeltiler bilinen miktarlarda teslimat için etkili yöntemlerin geliştirilmesini gerektirmektedir. Aşağıda açıklanan sonda prosedür yetişkin zebrafish için güvenli ve verimli bir şekilde, güvenilir bir çözüm hassas hacimleri sözlü teslimat için izin verir.

Yetişkin Zebra balığı 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 Görüntüleme.  The Rockefeller University. Bir hayvan modeli olarak zebrabalıkları artan kullanımı bileşikler ve çözeltiler bilinen miktarlarda teslimat için etkili yöntemlerin geliştirilmesini gerektirmektedir. Aşağıda açıklanan sonda prosedür yetişkin zebrafish için güvenli ve verimli bir şekilde, güvenilir bir çözüm hassas hacimleri sözlü teslimat için izin verir.

Video: 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.

Yetişkin Zebra balığı Tüm Böbrek İliği Nakli

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

Biyoloji

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.

Yetişkin Zebra balığı retro-orbital Enjeksiyon

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.

Yetişkin Zebra balığı Organları diseksiyonu

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.

Yetişkin Zebra balığı içine intraperitoneal enjeksiyon

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.

Doğrudan Yetişkin Zebra balığı Böbrek Hücre Nakli

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.

Yetişkin Zebra balığı Böbrek diseksiyonu

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.

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 ...

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.

Yetişkin Zebra balığı Biyokimyasal Analiz için Kan Alma

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.