The authors would like to thank Dr. Shaun Coughlin for hosting the filming of this procedure in his laboratory, and for general support. R.A. was supported by the NIH (F32HL110489) and the Sarnoff Cardiovascular Research Foundation. S.R. was supported by a Research Fellowship of the Deutsche Forschungsgemeinschaft (DFG) and the American Heart Association (AHA). D.Y.R.S was supported by the NIH (RO1HL54737), the Packard Foundation, and the Max Planck Society.

REMARQUE: Assurez-vous toujours que l&#39;approbation IACUC ou comité d&#39;éthique est en place avant de commencer toute procédure expérimentale utilisant le poisson zèbre. 1. Préparer les réactifs et configuration <ol><li> Préparer les solutions suivantes. Trouver les recettes pour l&#39;ensemble de ces solutions dans les manuels de poisson zèbre standards 10. <ul><li> 500 ml d&#39;eau Egg </li><li> 200 ml de 0,03% tricaïne à Egg eau </li><li> 1x 100 ml de tampon phosphate salin (PBS) </li><li> RBC tampon de lyse (facultatif) </li><li> tampon de destination de choix (fixateur, Trizol, PBS, etc.) dans un tube de 1,5 ml sur la glace REMARQUE: fixateur et Trizol sont toxiques si elles entrent en contact avec la peau. Porter des gants lors de la manipulation de ces substances. </li></ul></li><li> Préparer la configuration de dissection: <ol><li> Placez la source de lumière à col de cygne sur le microscope de dissection. Placez le couvercle d&#39;une boîte de Pétri de 9 cm sur la scène de dissection microscope, et verser une fine couche de 1x PBS dans(le couvercle est utilisé parce que le plat lui-même est trop profonde pour une dissection facile). </li><li> Placez la boîte de Pétri lui-même (pas le couvercle) sur le comptoir près du microscope à dissection et versez environ 15 ml de PBS 1x en elle. Utilisez cette solution pour laver le cœur après dissection. Vous pouvez également utiliser tampon de lyse RBC. </li><li> Lieu lame de rasoir, deux paires de pinces, microciseaux (facultatif), et une pipette de transfert à côté du microscope. Obtenir un conteneur de glace si l&#39;euthanasie d&#39;un refroidissement rapide est choisie. </li><li> Verser 200 ml de 0,03% tricaïne à Egg eau dans un bécher de 250 ml en verre si l&#39;euthanasie par tricaïne est choisi. Préparer un petit sac de risque biologique pour l&#39;élimination des carcasses de poisson zèbre. </li></ol></li></ol> 2. Préparer le poisson zèbre <ol><li> Pour les poissons fixe, apporter du poisson fixe, rincées dans du PBS, à la configuration du microscope. <ol><li> Euthanasier tout le poisson zèbre adulte par un refroidissement rapide avec de la glace pour&gt; 10 min. </li><li> Utilisez une pince à faire un trou dans la peau sur le péritoine(Du coeur) pour faciliter la pénétration du fixateur, puis mettez-les dans 4% de paraformaldehyde dans Fix Buffer 10 pendant 2 heures à la température ambiante ou O / N à 4 ° C. </li></ol></li><li> Sinon, pour le poisson frais, le lieu poisson pour être euthanasié dans un petit réservoir de retenue et apporter à la configuration du microscope. Selon le protocole de poissons de l&#39;installation individuelle et les utilisations en aval pour les cœurs de poissons disséqués, anesthésier les poissons avec de tricaïne ou euthanasier par un refroidissement rapide avant la décapitation. <ol><li> Pour utiliser de la glace, ramasser un poisson avec le filet de pêche et les placer dans de l&#39;eau glacée jusqu&#39;à ce que le poisson se arrête de bouger, environ 5 min. </li><li> Sinon, pour utiliser tricaïne, ramasser un poisson avec le filet de pêche et le lieu dans le bécher de la solution jusqu&#39;à l&#39;arrêt des mouvements maillants. </li></ol></li><li> Prendre rapidement le poisson euthanasié par sa nageoire caudale et le poser sur son côté dans le plat couvercle Petri qui a été rempli de PBS 1x. Utilisez la pince pour soulever une nageoire pectorale avec une main, tout en utilisant le razor lame pour décapiter le poisson avec l&#39;autre main, juste en arrière de l&#39;attachement de la nageoire pectorale (figure 1A). Effectuez cette étape soit en regardant par le microscope ou simplement en visualisant directement le poisson sur la platine du microscope. REMARQUE: poissons fraîchement euthanasiés seront toujours saigner après la décapitation. </li></ol><img alt="Figure 1" src="/files/ftp_upload/52248/52248fig1highres.jpg" /> Figure 1. poisson zèbre dissection cardiaque adulte utilise des repères anatomiques de poisson zèbre. (A) à décapiter le poisson, soulevez la nageoire pectorale avec une pince et d&#39;utiliser une lame de rasoir propre forte le long de la ligne pointillée rouge comme indiqué. (B) Pour stabiliser la tête du poisson, placer une dent de la pince dans la bouche de poisson tout l&#39;autre dents se trouve à travers l&#39;œil, puis tournez la tête du poisson afin que la surface ventrale est en place et les deux dents de la pince sont stables contre le fond de la boîte de Pétri. (C) Utilisez le forc gratuitementeps pour couper l&#39;attachement de l&#39;opercule (flèche). (D) de levage cela, l&#39;aorte dorsale est visible comme une structure blanche avec une bande rose dénotant sang luminale (flèche). <a href="https://www.jove.com/files/ftp_upload/52248/52248fig1large.jpg" target="_blank">Se il vous plaît cliquer ici pour voir une version plus grande de cette figure.</a> 3. Disséquer le Cœur <ol><li> Visualisez la tête de poisson sous le microscope. Avec une pince, stabiliser la tête du poisson en plaçant une dent de la pince dans la bouche de poisson, à travers le cerveau, tandis que l&#39;autre dent est en dehors de la tête, dans l&#39;œil (figure 1B). Vérifiez que les deux dents de la pince sont stables contre le fond de la boîte de Pétri. Tenir la tête de poisson de cette façon, sa surface ventrale doit être orientée vers le haut. </li><li> Avec l&#39;autre main, utilisez les secondes pinces pour couper le attache ventral de l&#39;opercule au corps (flèche, Figure 1C). Levage cette légèrement avec la pince, le dOrsal aorte apparaîtra sous forme d&#39;une structure blanche avec une bande rouge de sang dans la lumière aortique (flèche, figure 1D). Couper l&#39;aorte dorsale avec la pince en pinçant l&#39;aorte et tirant vers le haut; alternativement, utiliser microciseaux pour couper l&#39;aorte dorsale. </li><li> Maintenant, utilisez les secondes pince pour saisir la nageoire pectorale de sa base, y compris le cartilage du corps à la base de la nageoire, et soulevez cette option. Répétez avec l&#39;autre nageoire pectorale. Parfois, le cœur sort avec les nageoires pectorales, afin d&#39;examiner ces pièces pour se assurer que le cœur ne est pas fixé avant de les jeter. NOTE: A ce stade, le cœur doit être visible, encore intacte et relié à la tête de poisson restant (bien que parfois il se détache avec les nageoires pectorales). Le cœur peut être identifié par sa forme, sa couleur rose, et son être entouré par péricarde pigmentée. Parce que l&#39;euthanasie du poisson est effectué rapidement, coeurs de poissons fraîchement euthanasiés peuvent encore être battu slovers l &#39;ouest. </li><li> Lâchez la tête du poisson avec les premiers forceps, afin que les deux mains tiennent pince et sont gratuits. Utilisez les deux pinces pour taquiner le coeur loin du péricarde. Saisissez le cœur par le reste de l&#39;aorte dorsale tandis que le péricarde restante est retirée. NOTE: Que fixe ou frais, le cœur est robuste pour tirer douce tandis que le tissu conjonctif environnant est plus friable. Par conséquent, ne importe quel tissu péricardique entourant peut être disséquée avec le cœur reste intacte. </li><li> Jeter la carcasse de restes de poisson dans le sac pour matières contaminées. </li></ol> 4. Préparer le coeur pour des applications en aval <ol><li> En utilisant des pinces, saisir le cœur par le bord de la aorte dorsale / bulbe artériel et placez le coeur dans le plat frais de 1x PBS. Vous pouvez également utiliser une pipette de transfert. Déplacez le coeur avant et en arrière dans le PBS environ 10x pour laver autant de sang que possible. </li><li> Pour les applications où il est important d&#39;éliminer toute possibLe globules, laver dans une boîte de Petri de 9 cm avec 15 ml de tampon de lyse RBC au lieu de PBS, en utilisant le même mouvement de va-et-vient. Si la préservation de la structure du coeur ne est pas importante pour l&#39;application en aval (par exemple, ce qui rend l&#39;ARN), utiliser la pince pour ouvrir ou microciseaux la cavité cardiaque et de faciliter le rinçage loin des cellules sanguines. </li><li> Pour les applications dans lesquelles cavités cardiaques différents sont désirés, utiliser une pince à disséquer ou microciseaux le bulbe artériel, l&#39;atrium et le ventricule à l&#39;écart l&#39;une de l&#39;autre. </li><li> Transférer le cœur, ou des chambres séparées, à la mémoire tampon de destination sur de la glace. NOTE: destinations communs comprennent des tampons pour la dissociation cellulaire, 4% de paraformaldéhyde ou Trizol. </li></ol>

<ol>
	<li>Arnaout, R., 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=Zebrafish+model+for+human+long+QT+syndrome.">Zebrafish model for human long QT syndrome.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (27), 11316-11321 (2007).</li><li>Chi, N. C., 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=Genetic+and+physiologic+dissection+of+the+vertebrate+cardiac+conduction+system.">Genetic and physiologic dissection of the vertebrate cardiac conduction system.</a> PLoS Biology. 6 (5), 109 (2008).</li><li>Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+to+the+heart+of+regeneration+in+zebrafish.">Getting to the heart of regeneration in zebrafish.</a> Seminars in Cell &amp; Developmental Biology. 18 (1), 36-45 (2007).</li><li>Fang, Y., 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=Translational+profiling+of+cardiomyocytes+identifies+an+early+Jak1/Stat3+injury+response+required+for+zebrafish+heart+regeneration.">Translational profiling of cardiomyocytes identifies an early Jak1/Stat3 injury response required for zebrafish heart regeneration.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (33), 13416-13421 (2013).</li><li>Manoli, M., Driever, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-activated+cell+sorting+(FACS)+of+fluorescently+tagged+cells+from+zebrafish+larvae+for+RNA+isolation.">Fluorescence-activated cell sorting (FACS) of fluorescently tagged cells from zebrafish larvae for RNA isolation.</a> Cold Spring Harbor Protocols. 2012 (8), (2012).</li><li>Cannon, J. E., 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=Global+analysis+of+the+haematopoietic+and+endothelial+transcriptome+during+zebrafish+development.">Global analysis of the haematopoietic and endothelial transcriptome during zebrafish development.</a> Mechanisms of Development. 130 (2-3), 122-131 (2013).</li><li>Wang, Z., Gerstein, M., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq:+a+revolutionary+tool+for+transcriptomics.">RNA-Seq: a revolutionary tool for transcriptomics.</a> Nature Reviews Genetics. 10 (1), 57-63 (2009).</li><li>Gupta, T., Mullins, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+organs+from+the+adult+zebrafish.">Dissection of organs from the adult zebrafish.</a> Journal of Visualized Experiments. 37, (2010).</li><li>Singleman, C., Holtzman, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+dissection+in+larval,+juvenile+and+adult+zebrafish,+Danio+rerio.">Heart dissection in larval, juvenile and adult zebrafish, Danio rerio.</a> Journal of Visualized Experiments. 55, (2011).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book: a guide for the laboratory use of zebrafish (Brachydanio rerio. , (1993).</li></ol>

The authors have no disclosures.

Bien que les méthodes pour disséquer le cœur de poisson zèbre adulte ont été décrits, ces méthodes étaient de temps et souvent causé des dommages au cœur lors de la dissection. Pour effectuer des expériences où un grand nombre de coeurs adultes peuvent être nécessaires, et / ou lorsque éviter la dégradation du tissu cardiaque est important pour les applications en aval, le temps nécessaire en utilisant des techniques de dissection traditionnels est prohibitif. De même, l&#39;obtention reproductible en...

Zebrafish are an excellent model for studying heart development and human disease1,2. Specific advantages include the translucent nature of zebrafish embryos, the availability of many genetic mutants and transgenic reporter lines, and the availability of genome editing technologies. In addition to their advantages for studying early heart development, zebrafish are an ideal system for studying vertebrate heart regeneration3.
More recently, adult zebrafish are playing an important part in bioinformatics approaches to studying cardiovascular development and disease, due to their relatively large clutch size and relatively quick and inexpensive breeding compared to other vertebrate models. Promising techniques include ribosome profiling, RNA-Seq, and cell dissociation and FACS sorting4-7. However, for these techniques the quality of the data can depend on obtaining a large number of samples in a rapid, efficient, and reproducible manner, before gene regulatory, metabolic, transcriptional, and other changes occur.
Dissection of adult zebrafish organs has been described in the past8,9. However, previous approaches to dissection of the heart were slow, ran the risk of damaging the heart during dissection, required special tools, and/or required fixation of the zebrafish prior to dissection; for these reasons, past approaches to zebrafish adult heart dissection were not optimized for high-throughput applications and/or applications requiring fresh tissue.
Here, we describe a method for adult zebrafish heart dissection that is simple, fast, efficient, and reproducible, while preserving cardiac morphology. This method does not include cutting into the pericardial space and therefore does not risk damaging the heart during dissection. Instead, this method relies on anatomical landmarks of the zebrafish, and therefore, it is highly reproducible. This dissection method is also versatile in that it can be used on fresh or fixed fish, and on zebrafish as young as one month old. Finally, this method results in minimal suffering to the zebrafish because after anesthesia and/or rapid cooling, the fish is additionally decapitated and pithed in the course of the dissection procedure.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >small tank for transporting fish</td>
			<td >Aquaneering</td>
			<td >ZHCT100</td>
			<td></td>
		</tr>
		<tr >
			<td >fish net</td>
			<td >Petsmart</td>
			<td >36-16731</td>
			<td></td>
		</tr>
		<tr >
			<td >250mL glass beaker</td>
			<td >Kimble</td>
			<td >14005-250</td>
			<td></td>
		</tr>
		<tr >
			<td >9cm polystyrene petri dish</td>
			<td >Nunc</td>
			<td >172958</td>
			<td></td>
		</tr>
		<tr >
			<td >razor blade</td>
			<td >Personna American Safety Razor Company</td>
			<td >94-120-71</td>
			<td></td>
		</tr>
		<tr >
			<td >two Dumont #5SF forceps</td>
			<td >Fine Science Tools</td>
			<td >11252-00</td>
			<td></td>
		</tr>
		<tr >
			<td >dissecting microscope</td>
			<td >Olympus</td>
			<td >SZX16</td>
			<td></td>
		</tr>
		<tr >
			<td >Tricaine</td>
			<td >Sigma</td>
			<td >A-5040</td>
			<td></td>
		</tr>
		<tr >
			<td >plastic transfer pipette</td>
			<td >Thermo Scientific</td>
			<td >202-20S</td>
			<td></td>
		</tr>
		<tr >
			<td >gooseneck light source</td>
			<td >Dolan-Jenner Industries, Inc</td>
			<td >Fiber-Lite 180 Illuminator, 181 Dual Gooseneck System</td>
			<td></td>
		</tr>
		<tr >
			<td >fluorescent light source</td>
			<td >Lumen Dynamics</td>
			<td >X-Cite 120Q</td>
			<td>optional</td>
		</tr>
		<tr >
			<td >micro-scissors</td>
			<td >Biomedical Research Instruments, Inc</td>
			<td >11-1000</td>
			<td>optional</td>
		</tr>
		<tr >
			<td >RBC Lysis Buffer</td>
			<td >eBioscience</td>
			<td >00-4333-57</td>
			<td>optional</td>
		</tr>
	</tbody>
</table>

recovery of adult zebrafish hearts for high-throughput applications

Utilisation du système modèle pour étudier le développement du poisson zèbre, la régénération et la maladie est l&#39;expansion vers l&#39;utilisation de cœurs adultes pour la dissociation cellulaire et la purification de l&#39;ARN, l&#39;ADN et les protéines. Toutes ces applications exigent la reprise rapide d&#39;un grand nombre de cœurs de poisson zèbre pour éviter gène régulateur, métabolique, et d&#39;autres changements qui commencent après la mort. Coeurs poisson zèbre adultes sont également nécessaires pour étudier la structure de coeur pour une variété de mutants et pour étudier la régénération cardiaque. Cependant, la dissection traditionnelle cardiaque poisson zèbre est lente et difficile et nécessite des outils spécialisés, ce qui rend la dissection grande échelle des cœurs de poisson zèbre adultes fastidieux. Les méthodes traditionnelles abritent également le risque d&#39;endommager le coeur lors de la dissection. Ici, nous décrivons une méthode pour la dissection des cœurs de poisson zèbre adultes qui est rapide, reproductible, et préserve l&#39;architecture cardiaque. En outre, cette méthode ne nécessite pas d&#39;outils spécialisés, est indolore pour le poisson-zèbre,peut être effectuée sur des échantillons frais ou fixes, et peut être effectuée sur le poisson zèbre dès l&#39;âge de un mois. L&#39;approche décrite élargit l&#39;utilisation de poisson zèbre adulte pour la recherche cardiovasculaire.

En utilisant cette méthode, un poisson zèbre cœur adulte peut être disséqué en moins de 1 min, par rapport à plus de 5 min selon la méthode traditionnelle 8. Coeurs disséqués en utilisant cette méthode sont fiable intacte (figure 2A), alors que les méthodes traditionnelles nécessitent huit coupe aveuglément dans le péricarde et donc souvent causer des dommages ou la perte de l&#39;atrium ou bulbe artériel (figure 2B). Coeurs disséqués conservent l...

Watch this Scientific Journal Video about Recovery of Adult Zebrafish Hearts for High-throughput Applications at JoVE.com

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Recouvrement des adultes coeurs de poisson zèbre à haut débit pour des applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

recovery-of-adult-zebrafish-hearts-for-high-throughput-applications

Research

JoVE Journal

Biology

12.2K vues.  University of California San Francisco. Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Vidéo: Recovery of Adult Zebrafish Hearts for High-throughput Applications

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

Une méthode à haut débit pour la cryoconservation du sperme de poisson zèbre et de In Vitro La fertilisation

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

Recherche

Biologie

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, ...

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.

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 ...

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

Assay saccharification haut débit pour les matériaux lignocellulosiques

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

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 ...

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.

Collecte de sang pour analyse biochimique chez le poisson zèbre adulte

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 ...

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 ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

Un petit écran Molecule Manuel approchant à haut débit utilisant embryons de poissons zèbres

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Haut-débit criblage de modulateurs chimique des gènes post-transcriptionnelle réglementées

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...