Nous remercions CE Liao de l&#39;aide pour suture, et R. et L. Ethier Gyr pour les soins de poisson zèbre. La DJA a été soutenu par l&#39;Institut Harvard Stem Cell, l&#39;American Society of Nephrology, et les NIH / NIDDK (P50DK074030). CQD a été soutenue par le Massachusetts General Hospital et le Fonds pour la découverte médicale.

<html> 1. Conditionnement du poisson bénéficiaire non-transgénique. <ol><li> Sélectionnez le plus gros poisson mâle dans le système de transplantation. </li><li> Placer le poisson dans 0,02% tricaïne (pH 7) pendant environ 3 minutes ou jusqu&#39;à ce qu&#39;il s&#39;arrête natation, mais pas aussi longtemps que le cœur cesse de battre. </li><li> Mettez le poisson anesthésié sur une serviette en papier humide sur une paillasse. </li><li> Utilisez une aiguille de seringue d&#39;insuline à injecter 40 ug de gentamicine dans 20 uL d&#39;eau dans la cavité péritonéale du poisson. Pour les études de régénération rénales, cette étape est importante pour induire un environnement favorable à la prise de greffe renforcée. </li><li> Mettez le poisson dans l&#39;eau pour la récupération du système. </li><li> Le poisson peut être docile et très sensible aux stimuli externes tels que les vibrations. Après 3 heures de récupération, de traitement du poisson avec 25 gris de l&#39;irradiation gamma. </li><li> Mettez le poisson dans le système pendant 3 jours sans nourriture avant la transplantation. </li></ol>_title &quot;&gt; 2. Préparation de cellules donneuses génétiquement progénitrices marqué avec un marqueur fluorescent (EGFP ou mCherry). <ol><li> Juste avant l&#39;intervention chirurgicale, préparer les cellules du donneur à transplanter et de les garder sur la glace. </li><li> Pour les études rénales, nous utilisons l&#39;ensemble des cellules de moelle rénaux de la Tg (cdh17: EGFP) lignée transgénique, qui contiennent des cellules souches hématopoïétiques et rénales. Ces cellules progénitrices sont génétiquement marqués et seulement exprimer EGFP s&#39;ils se greffer et se différencient en cellules épithéliales rénales. D&#39;autres types de cellules souches marquées peuvent également être utilisés, tels que Tg (scl: EGFP) sur les cellules souches hématopoïétiques ou Tg (fli1a: EGFP) pour les greffes de progéniteurs angioblastes. </li><li> Euthanasier 3 poissons adultes transgénique en les plaçant dans 0,2% tricaïne pendant 5 minutes. </li><li> Utilisez une lame de rasoir stérile pour enlever la tête et la queue, et la dissection des ciseaux pour faire une incision médiane le long de la face ventrale. </li><li> En vertu d&#39;un stéréoscope,supprimer tous les organes internes dans la cavité du corps, y compris la vessie natatoire avec des pincettes. Veillez à ne pas retirer le rein, qui est un organe plat et pigmentée attaché à la paroi dorsale de la cavité. </li><li> Ensuite, disséquer les reins de la paroi dorsale et le placer dans un tube à centrifuger de 1,5 ml contenant 300 ul de solution saline de tampon phosphate froid additionné de 2% de sérum de veau fœtal (1X PBS / 2% FCS). </li><li> Utilisez un pilon pour cisailler les reins jusqu&#39;à ce qu&#39;elle devienne homogène. </li><li> Placer une crépine 40 pM cellule à l&#39;intérieur d&#39;un tube Falcon de 50 ml et transférer la solution de cellules au-dessus de la crépine cellule, ce qui permet aux cellules libres à filtrer dans le tube Falcon. </li><li> Utilisez le pilon en douceur les frottis de grands morceaux de tissus qui sont laissés sur le tamis de continuer à briser les cellules. </li><li> Laisser la solution de cellules dans le tube Falcon et laver le filtre avec 1,2 ml de PBS 1X / 2% FCS pour recueillir des cellules résiduelles provenant de l&#39;épurateur. </li><li> Transférerla solution de cellules dans un nouveau tube de 1,5 ml et centrifuger à 4 ° C à 300 rcf pendant 5 minutes. </li><li> Laver le culot cellulaire avec 1,5 ml de PBS 1X / 2% de FCS et le passer à travers une passoire pour filtrer les nouveaux amas de cellules collantes et centrifuger à nouveau. </li><li> Remettre en suspension le culot cellulaire dans 200 ul de PBS 1X de / 2% SVF et transférer dans un tube PCR. </li><li> Centrifuger le tube de PCR (4 ° C, 300 rcf, 1 min) et retirer la plupart de surnageant. </li><li> Reprendre les cellules dans le surnageant résiduel pour obtenir un volume de 2-5 uL finale et le stocker sur glace. </li><li> Finale rendement cellulaire devrait être d&#39;environ 2 millions de cellules par les reins. </li><li> Compter le nombre de cellules avec un hémocytomètre et ajuster la concentration de 5 x 10 5 cellules par uL. </li></ol> 3. Chargez la solution de cellules dans une seringue Hamilton. <ol><li> Rincer la seringue Hamilton avec de l&#39;eau stérile 3 fois. </li><li> Rincer la seringue avec de l&#39;éthanol à 75% 3 fois. </li><li> Rincer la SyriESN nouveau avec 1X PBS / 2% FCS 3 fois. </li><li> Puis charger la seringue avec 1 ul de solution de cellules contiennent environ 5 x 10 5 cellules juste avant l&#39;injection. </li></ol> 4. La transplantation de cellules souches directement dans le rein. <ol><li> Anesthésier le poisson receveur conditionné dans 0,02% tricaïne pendant environ 3 minutes, ou jusqu&#39;à ce que le poisson a cessé de bouger, mais pas trop longtemps que le cœur cesse de battre. </li><li> Sous un stéréomicroscope, mettez le poisson sur une serviette en papier avec la tête vers la gauche, jusqu&#39;à la face ventrale, afin de sécher sur le côté transplanté. </li><li> Ensuite, enroulez le poisson pour exposer le côté séché. </li><li> L&#39;aide de pinces stériles, enlever les écailles dans la région immédiatement postérieure aux branchies. </li><li> Faire une incision de 1 cm latérale dans ce domaine sur la ligne médiane avec un scalpel tout en stabilisant le tissu avec des pincettes. </li><li> Utiliser les pinces, exposer le tissu tout en continuant à faire l&#39;incision assez profondepour exposer le rein antérieur. </li><li> S&#39;il ya beaucoup de saignement qui empêche la visualisation du rein, euthenize le poisson et recommencer avec un autre poisson destinataire. </li><li> Tout en soulevant le tissu ouverte pour exposer la tête du rein, injecter 1 ul de la solution de cellules directement dans le rein tête. </li><li> Puis, en même temps que l&#39;aiguille de la seringue est en cours de récupération, de libérer la pince de telle sorte que le tissu retombe en place pour empêcher une fuite des cellules injectées. </li><li> Tenir une aiguille de suture avec un entraînement d&#39;aiguille et la percer à travers le muscle de chaque côté de l&#39;incision. </li><li> Attachez 2 noeuds et couper le fil de suture excès. Seule une suture simple est nécessaire pour une incision de 1 cm. </li><li> Placer le poisson dans de l&#39;eau contenant des poissons 10 parties par million de tricaïne de 5 heures pour produire un poisson avec un effet calmant analgésique. </li><li> Placer le poisson dans le système de récupération des tétées normales. </li><li> Après 7-18 jours de reprise, nous disséquer et d&#39;analyser les poissons engraftment de cellules progénitrices rénales par fluorescence EGFP en direct. </li></ol> 5. Les résultats représentatifs: La stratégie globale de la transplantation est illustré à la figure 1. Pour nos études rénales, le rein est disséqué et analysé par fluorescence EGFP. La prise de greffe et la différenciation des cellules progénitrices rénales est détectée dès sept après la greffe jours. Cela est évident par la présence de plusieurs groupes de EGFP + cellules épithéliales rénales (Figure 2). À 18 ans après la greffe jours, nous détectons une moyenne de 24 EGFP + néphrons, ce qui indique que les cellules du donneur ont formé du tissu nouveau rein (Figure 3). Pour démontrer greffe à long terme, nous avons disséqué un poisson receveur après la greffe 59 jours (figure 4). Ce poisson a eu beaucoup de néphrons au site d&#39;injection (rouge tête de flèche), en plus de plusieurs néphrons sur des sites distants (flèches blanches), ce qui suggère que les cellules progénitrices rénales sont des oiseaux migrateurs. Cette technique est plus rapide d&#39;une plus efficace par rapport à quand les cellules sont injectées dans la circulation par le cœur, ce qui prend environ trois mois pour détecter la prise de greffe. <img src="/files/ftp_upload/2725/2725fig1.jpg" alt="Figure 1"/> Figure 1. Diagramme d&#39;ensemble de la transplantation. Poisson receveur non-transgénique sont irradiés pour supprimer le rejet immunitaire des cellules transplantées. Trois jours plus tard, une incision est pratiquée sur le flanc du poisson et des donneurs de cellules souches sont injectées dans le rein tête exposée. L&#39;incision est refermée avec une suture et les poissons sont placés dans le système de récupération. Reins des poissons destinataire sont disséqués à des temps plus tardifs et analysés pour la prise de greffe et de l&#39;activité géniteur. <img src="/files/ftp_upload/2725/2725fig2.jpg" alt="Figure 2"/> Figure 2. La prise de greffe de cellules souches après sept jours. La prise de greffe de cellules souches rénales est détectée sur le côté injectédu rein tête à sept heures après la transplantation jours, tel que déterminé par la présence d&#39;amas de EGFP + cellules épithéliales rénales (en médaillon - vue agrandie). <img src="/files/ftp_upload/2725/2725fig3.jpg" alt="Figure 3"/> Figure 3. Prise de greffe de cellules souches après 18 jours Après 18 jours après la transplantation, nous avons détecté une moyenne de 24 + EGFP donateurs issus de néphrons (insert - vue agrandie de chaque EGFP + néphrons).. <img src="/files/ftp_upload/2725/2725fig4.jpg" alt="Figure 4"/> Figure 4. La prise de greffe et la migration des cellules progénitrices après 59 jours. Nous avons continué à détecter la prise de greffe de cellules souches longtemps après la transplantation, même après 59 jours (tête de flèche rouge). A ce stade, plus tard, des bailleurs de fonds issus de néphrons sont détectés sur des sites en dehors du site d&#39;injection (têtes de flèches blanches), conformément à la migration des cellules progénitrices.

<ol>
	<li>Hopkins, C., Li, J., Rae, F., Little, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+options+for+kidney+disease.">Stem cell options for kidney disease.</a> The Journal of Pathology. 217, 265-281 (2009).</li><li>Brittijn, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+development+and+regeneration:+new+tools+for+biomedical+research.">Zebrafish development and regeneration: new tools for biomedical research.</a> Int J Dev Biol. 53, 835-850 (2009).</li><li>Traver, D. <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+lethal+irradiation+in+zebrafish+and+rescue+by+hematopoietic+cell+transplantation.">Effects of lethal irradiation in zebrafish and rescue by hematopoietic cell transplantation.</a> Blood. 104, 1298-1305 (2004).</li></ol>

Les techniques de transplantation de cellules sont cruciales pour les études de régénération et le dosage sont l&#39;étalon-or pour mesurer l&#39;activité des cellules souches. Ici, nous présentons une méthode de transplantation de cellules directement dans le rein du poisson zèbre adulte pour déterminer tige et l&#39;activité des cellules progénitrices. Cette procédure consiste à faire une incision à travers le muscle pour exposer le rein tête, suivie de l&#39;injection de cellules directement dans le rein. L&#39;incision est fermée par un fil de suture unique et le poisson à commande ont un taux de survie 90-100%. Dans nos expériences, greffées des cellules progénitrices rénales sont détectés dans 80-100% des poissons transplantés par sept après la transplantation jours. Une limitation de cette technique est que l&#39;incision est proche de l&#39;aorte dorsale, qui peut être facilement rompue par le scalpel. Cependant, une fois maîtrisé, le taux de saignement peut être réduite à moins de un pour cent. Notre méthode pourrait être adapté pour fournir d&#39;autres types cellulaires (sang, du stroma, ou vasculaire) ainsi que de petites moléles au rein. Le développement de cette technique aidera les études préalables de régénération et de permettre à la tige et le potentiel de cellules souches à évaluer in vivo.

<table border="1"><tbody><tr><th> Nom du réactif </th><th> Entreprise </th><th> Numéro de catalogue </th></tr><tr><td> Aiguille de la seringue d&#39;insuline (28 gauge) </td><td> Becton Dickinson </td><td> 329461 </td></tr><tr><td> Cellule filtre (40 uM) </td><td> Becton Dickinson </td><td> 352340 </td></tr><tr><td> Tricaïne </td><td> Sigma </td><td> A5040 </td></tr><tr><td> Seringue Hamilton (26 gauge) </td><td> Sigma </td><td> 20734 </td></tr><tr><td> Ethilon suture (taille 9-0) </td><td> Ethicon </td><td> 2819G </td></tr></tbody></table>

transplantation of cells directly into the kidney of adult zebrafish

La médecine régénératrice basée sur la transplantation de cellules souches ou progénitrices dans les tissus endommagés a le potentiel de traiter un large éventail de maladies chroniques 1. Cependant, la plupart des organes ne sont pas facilement accessibles, ce qui nécessite de développer des méthodes chirurgicales pour avoir accès à ces structures. Dans cet article, vidéo, nous décrivons une méthode de transplantation de cellules directement dans le rein de l&#39;adulte poisson zèbre, un modèle populaire pour étudier la régénération et la maladie 2. Poissons destinataire sont pré-conditionnées par irradiation pour supprimer le rejet immunitaire des cellules injectées 3. Nous montrent comment la tête du rein peut être exposée par une incision dans le flanc latéral du poisson, suivi de l&#39;injection des cellules directement dans l&#39;organe. Utilisation entières marquées par fluorescence des cellules de moelle rein comprenant une population mixte de précurseurs hématopoïétiques et rénales, nous montrons que les progéniteurs néphron peut greffer et se différencier en tissu rénal nouvelle - la gancienne norme de toute thérapie à base de cellules régénératrices. Cette technique peut être adapté pour fournir souches purifiées ou des cellules progénitrices et / ou de petites molécules au rein, ainsi que d&#39;autres organes internes et améliore encore le poisson zèbre comme modèle polyvalent pour étudier la médecine régénérative.

Watch this Scientific Journal Video about Transplantation of Cells Directly into the Kidney of Adult Zebrafish at JoVE.com

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

La transplantation de cellules est une technique essentielle pour l&#39;étude de la régénération des tissus et pour le développement des thérapies cellulaires de la maladie. Nous démontrons ici une technique microchirurgicale qui permet la transplantation de cellules génétiquement marquées directement dans le rein de poisson zèbre adulte.

La transplantation de cellules directement dans le rein de poisson zèbre adulte

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

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14.9K vues.  Massachusetts General Hospital. La transplantation de cellules est une technique essentielle pour l'étude de la régénération des tissus et pour le développement des thérapies cellulaires de la maladie. Nous démontrons ici une technique microchirurgicale qui permet la transplantation de cellules génétiquement marquées directement dans le rein de poisson zèbre adulte.

Vidéo: Transplantation of Cells Directly into the Kidney of 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.

La transplantation de moelle rein entier chez le poisson zèbre pour adultes

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

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Biologie

Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice. We found that it was easier to concentrate the islets or cells into pellets in the final delivery tubing (PE50) used to transplant the cells under the kidney capsule. This technique provides both speed and ease while reducing any undue stress to the cells or to the mouse.

Loading: Settled, hand picked, islets or pelleted cells are carefully aspirated off the bottom of a 1.5 mL microcentrifuge tube using a p200 pipetteman and a straight, thin-wall pipette tip. A length of PE50 tubing is attached to the pipette tip using a small silicone adapter tubing. Cells are allowed to settle, in the tip, and then are transferred to the PE50 tubing by slowly dialing the pipetteman. Once the cells are near the end of the PE50 tubing, a kink is made and the silicone adaptor tubing is placed over the kink. The PE50 tubing is transferred to a 15 mL conical containing a cut 5 mL pipet, and the PE50 tubing is taped over the side of the 5 mL pipet to prevent curling during centrifuging. Cells are allowed to reach 1,000 rpm and stopped.
Transplantation: Recipient mice are anesthetized, shaved, and cleaned. A small incision is made on the left flank of the mouse and the kidney is exposed. The kidney, fat, and tissue are kept moist with normal saline swab. The distal end of the PE50 is attached to a Hamilton screw drive syringe, containing a pipette tip, using the silicone adaptor tubing. A small nick is made on the right flank side of the kidney, not too large nor too deep. The beveled end of the PE50 tubing, nearest the cells, is carefully placed under the capsule, the tubing is moved around gently to make space while swabbing normal saline; a dry capsule can tear easily. A small air bubble is delivered under the capsule by slowly dialing the syringe screw drive. Islets are then slowly delivered behind the air bubble. Once the islets have been delivered kidney homeostasis is maintained and the knick is cauterized with low heat. The kidney is placed back into the cavity and the peritoneum and skin are sutured and stapled. Mice are immediately treated with Flunixin and Buprenorphine s.q. and placed in a cage on a heating pad.

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of mice. Cells are concentrated into pellets in the final tubing used for transplanting the cells under the kidney capsule. The ease of this technique reduces stress to the cells and the mouse.

Transplantation d&#39;îlots pancréatiques dans la capsule du rein de souris diabétiques

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice.  We found that it was easier ...

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

Objectif transplantation chez le poisson zèbre et son application dans l&#39;analyse de mutants Eye

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

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.

Dissection d&#39;organes sur le poisson zèbre pour adultes

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.

Injection intrapéritonéale en Zebrafish adultes

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, ...

Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as tumor-propagating cells, or tumor-initiating cells. It is thought that targeting these self-renewing cells for destruction will block tumor progression and stop relapse, greatly improving patient prognosis1. The most common way to determine the frequency of self-renewing cells within a tumor is a limiting dilution cell transplantation assay, in which tumor cells are transplanted into recipient animals at increasing doses; the proportion of animals that develop tumors is used the calculate the number of self-renewing cells within the original tumor sample2, 3. Ideally, a large number of animals would be used in each limiting dilution experiment to accurately determine the frequency of tumor-propagating cells. However, large scale experiments involving mice are costly, and most limiting dilution assays use only 10-15 mice per experiment. 
Zebrafish have gained prominence as a cancer model, in large part due to their ease of genetic manipulation and the economy by which large scale experiments can be performed. Additionally, the cancer types modeled in zebrafish have been found to closely mimic their counterpart human disease4. While it is possible to transplant tumor cells from one fish to another by sub-lethal irradiation of recipient animals, the regeneration of the immune system after 21 days often causes tumor regression5. The recent creation of syngeneic zebrafish has greatly facilitated tumor transplantation studies 6-8. Because these animals are genetically identical, transplanted tumor cells engraft robustly into recipient fish, and tumor growth can be monitored over long periods of time. Syngeneic zebrafish are ideal for limiting dilution transplantation assays in that tumor cells do not have to adapt to growth in a foreign microenvironment, which may underestimate self-renewing cell frequency9, 10. Additionally, one-cell transplants have been successfully completed using syngeneic zebrafish8 and several hundred animals can be easily and economically transplanted at one time, both of which serve to provide a more accurate estimate of self-renewing cell frequency.
Here, a method is presented for creating primary, fluorescently-labeled T-cell acute lymphoblastic leukemia (T-ALL) in syngeneic zebrafish, and transplanting these tumors at limiting dilution into adult fish to determine self-renewing cell frequency. While leukemia is provided as an example, this protocol is suitable to determine the frequency of tumor-propagating cells using any cancer model in the zebrafish.

Limiting dilution cell transplantation assays are used to determine the frequency of tumor-propagating cells. This protocol describes a method for generating syngeneic zebrafish that develop fluorescently-labeled leukemia and details how to isolate and transplant these leukemia cells at limiting dilution into the peritoneal cavity of adult zebrafish.

Quantifier la fréquence des tumeurs se propageant Cellules utilisant Limiter la greffe de cellules syngéniques de dilution dans Zebrafish

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as ...

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.

Dissection du rein Zebrafish adultes

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.

En électroporation in vivo de morpholinos dans la rétine de poisson zèbre adulte

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.

En électroporation in vivo de morpholinos dans la régénération des adultes 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 ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

Gavage poisson zèbre pour adultes

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