Ce travail a été soutenu par la Deutsche Forschungsgemeinschaft (DFG), nous tenons à remercier Ayele Tsedeke Taddese pour le support technique.

Une. Préparation des aiguilles de verre (Figure 1) <ol><li> Utiliser des capillaires en verre d&#39;un diamètre de 0,75 mm pour préparer des aiguilles d&#39;injection (Figure 1A). </li><li> Placer le capillaire de verre dans un extracteur d&#39;aiguille et retirez l&#39;aiguille avec le paramètre suivant: valeur du cycle de chauffage: 463; tirant valeur du cycle: 230; vitesse: 150 ms; temps: 150 ms (figure 1B). </li><li> Briser le capillaire en verre tiré avec des pincettes horloger pour produire une aiguille 20 um de diamètre sous un stéréoscope avec un micromètre oculaire. </li><li> Utilisez un tour avec un rouet de caoutchouc mouillée pour aiguiser l&#39;aiguille et produire un biseau 20 um (figures 1C et 1D). Remarque: Sharp, aiguilles biseautées améliorer la facilité d&#39;insertion dans le ventricule et de minimiser ainsi les dommages aux tissus et autorisent le muscle perforé pour resceller après le retrait de l&#39;aiguille (figures 1E-G). </li></ol> 2. Preparation de la solution morpholino <ol><li> Préparer le morpholino en dissolvant le morpholino lyophilisée en 1x tampon phosphate salin (PBS) à une concentration finale de 7,5 mM. [Voir les instructions du fabricant pour plus de détails (de table Matériaux)]. </li><li> Préparer la solution d&#39;injection de morpholino par mélange d&#39;une solution de 2,5 pi morpholino d&#39;actions (7,5 mM) avec Endo-Porter solution stock de 2,8 pi (1 mM) (Voir le tableau des matériaux) pour une concentration finale de morpholino 3,5 mM et 0,5 mM Endo-Porter. </li></ol> 3. Préparation du morpholino injection (figure 2) <ol><li> Chargez l&#39;aiguille de verre biseauté avec 5 pi de cette solution à l&#39;aide d&#39;une micropipette avec une pointe de pipette microloader de 10 pi. </li><li> Insérer l&#39;aiguille de verre dans le porte-aiguille de la micro-manipulateur relié à la pompe de pico pneumatique (figures 2A-C). </li><li> Placez le porte-aiguille à côté du microscope afin que l&#39;aiguille seulement les besoinspour être déplacée dans une direction plane de l&#39;insérer dans le ventricule cardiaque de la chenille (figure 2A). </li><li> Ajustez l&#39;angle pour les injections à environ 45 °. </li><li> Définissez les valeurs de micro-injecteur comme suit: pression de maintien: 20 livres par pouce carré (psi); pression d&#39;éjection: 15 psi; 100 msec gamme de déclenchement, la valeur de période de 1,9 (correspondant à 10,9 ms). </li><li> Faire fondre l&#39;agarose dans du PBS 1x en utilisant un four micro-ondes standard pour produire 20 ml d&#39;un 1,5% (p / v) de gel. Verser gel fondu dans une boîte de 10 cm de Pétri. Placez une forme d&#39;injection à gorge dans la gélose chaud de sorte qu&#39;une fois durcie, la gélose aura sillons dans lesquels la larve sous sédation sera placé 11 (figures 2D et 2E). </li></ol> 4. Injections (Figure 3) <ol><li> Anesthésier larve dans 100 ml d&#39;eau de l&#39;aquarium avec 20 mg / L tricaïne (L-éthyl-m-amino-benzoate-méthane sulfonate) jusqu&#39;à ce qu&#39;ils cessent de répondre au toucher. </li><li> Utilisez une pipett Pasteur en plastiquee pour transférer soigneusement la larve sédation dans une rainure du moule agarose humide de sorte que la face ventrale est confronté contre la paroi verticale de la rainure d&#39;agarose (figure 3A). </li><li> Placer la plaque d&#39;agarose sous la loupe binoculaire de telle sorte que le ventricule n&#39;est pas tourné vers l&#39;aiguille d&#39;injection (figure 3A). </li><li> Abaissez l&#39;aiguille pour l&#39;insérer dans le ventricule cardiaque. Insérez l&#39;aiguille seulement 1-2 pm dans le ventricule en prenant soin de ne pas insérer l&#39;aiguille trop profondément (figures 3B et 3C). </li><li> Une fois que l&#39;aiguille est insérée, injecter la solution de morpholino dans le ventricule avec 4-6 impulsions, chaque 3 nl délivrant de la solution, avec des intervalles d&#39;attente pour permettre la compensation du coeur (Figures 3D et 3E). </li><li> Après l&#39;injection, retirez l&#39;aiguille (parallèle au plan de l&#39;insertion), puis transférer avec soin la larve à l&#39;aide d&#39;une pipette Pasteur en plastique rempli de E3 milieu back dans une boîte de Petri contenant un milieu de E3 frais. </li><li> Placer l&#39;aiguille dans une boîte de Petri contenant du PBS 1x pour empêcher le séchage de l&#39;aiguille lors du transfert de la larve injecté. </li><li> Répétez les étapes 4.1 à 4.6 tous les 12-24 heures pour la durée de l&#39;expérience. Remarque: La répétition des injections assure la fixation et le maintien de la morpholino dans les cellules. </li></ol> 5. Analyses des injections (figure 4) <ol><li> Anesthésier le poisson dans 100 ml d&#39;eau de l&#39;aquarium avec 20 mg / L tricaine jusqu&#39;à ce qu&#39;ils cessent de répondre au toucher. </li><li> Placez la larve latéralement sur un plat humide de 1,5% (p / v) d&#39;agarose plaque recouverte de milieu E3. </li><li> larves de l&#39;image en utilisant un stéréomicroscope clair et l&#39;imagerie de fluorescence. Remarque: Comme les larves sont transparentes, le morpholino-fluorescéine marqué devrait être visible que le vert fluorescent dans les vaisseaux sanguins dans tout l&#39;animal immédiatement après l&#39;injection. Cette fluorescence va encore se disperser dans les tissus vascularisés par15 min (Figures 4A-4C). </li></ol> 6. Évaluation de la médecine régénérative excroissance <ol><li> Évaluer les images capturées à l&#39;aide Fidji Image J logiciel libre (http://fiji.sc/Fiji). Pour enquêter sur la régénération, utilisez l&#39;outil de ligne de traçage pour déterminer le montant de la croissance régénératrice qui a eu lieu sur le contrôle et la larve de morpholino-injecté. </li><li> Pour calibrer les images, ouvrez l&#39;un des fichiers d&#39;image d&#39;origine à Fidji en faisant glisser le fichier dans la fenêtre principale de Fidji. </li><li> Pour définir l&#39;échelle de toutes les images suivantes, allez dans &quot;Analyser&quot; dans le menu principal. </li><li> Dans le menu déroulant, cliquez sur &quot;Définir échelle&quot;. </li><li> Dans cette fenêtre, activez l&#39;option «Global» en cliquant sur la case à cocher. </li><li> Maintenant, ouvrez l&#39;image pour mesurer la quantité de la croissance régénératrice encore par glisser-déposer (voir l&#39;étape 6.1). </li><li> Choisissez l&#39;option &quot;sélections à main levée&quot; outil dans la barre d&#39;outils. </li><li> Entourer la zone à mesurer (Figures 4J-4L). </li><li> Appuyez sur Ctrl + M. Cela va ouvrir une nouvelle fenêtre «Résultats» montrant la valeur de la zone encerclée en pixels carrés. </li></ol>

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Les auteurs n&#39;ont rien à révéler.

L&#39;injection intraventriculaire fournit une méthode d&#39;évaluation rapide et fiable pour tester la fonction des gènes à des stades ultérieurs du développement ou de l&#39;homéostasie du corps sans affecter la fonction des gènes au cours de l&#39;embryogenèse. Pour assurer le succès de cette technique, il faut être conscient de quatre points essentiels: 1) la taille de l&#39;aiguille, 2) le séchage de l&#39;aiguille, 3) réduire au minimum le volume, et 4) la durée d&#39;exposition minimale à la soluti...

Régénération des organes et des appendices est d&#39;une importance fondamentale pour la survie et remise en forme; Toutefois, plusieurs vertébrés, y compris l&#39;homme ont limité capacités régénératrices. Bien que plusieurs modèles animaux existent qui ont une vaste capacité de régénération, inverser techniques génétiques pour évaluer la fonction des gènes au cours de l&#39;orgue et appendice régénération reste très limitée ou inexistante. Par conséquent, de nouvelles approches sont nécessaires pour disséquer la biologie moléculaire de la régénération dans ces organismes modèles. Le poisson zèbre est un modèle bien établi pour la compréhension de la biologie cellulaire et moléculaire de l&#39;orgue et appendice régénération 1, non seulement en raison de son importante capacité de régénérer plusieurs organes, de tissus et des appendices, mais aussi parce que plusieurs lignes de poissons transgéniques existent pour suivre les cellules et surexpriment le gène construit 2, 3. Cependant, l&#39;inhibition du gène chez le poisson zèbre larves se limite essentiellement à la overexpression de constructions dominants négatifs, qui ne sont pas disponibles pour tous les gènes d&#39;intérêt ou dont le produit transgène peut acquérir un gain de fonction des effets qui ne reflètent pas l&#39;activité endogène du gène. Ainsi, une méthode alternative pour éliminer spécifiquement l&#39;expression des gènes par KO ou effet de choc est nécessaire pour surmonter ces problèmes. Gène spécifique ciblant l&#39;aide TALENS existe comme un moyen de génétique inverse pour Knockout la fonction des gènes; Toutefois, cette stratégie de knock-out est très souvent limitée à des évaluations fonctionnelles au cours de l&#39;embryogenèse précoce, parce que les exigences initiales du gène empêchent la progression du développement embryonnaire. Ainsi, l&#39;étude des phénomènes ultérieurs tels que la régénération ou l&#39;homéostasie des organes après le développement à l&#39;aide TALENS est exclue 4, 5. Par conséquent, une stratégie d&#39;élimination de gène de remplacement est nécessaire que la fonction du gène cible après le développement précoce d&#39;évaluer les besoins de gènes dans les organes et structures complètement formés. </p&gt; Morpholino injection a été montré pour être efficace dans le ciblage de gènes dans quelques organes adultes et l&#39;adulte régénération nageoire 6-8, mais ces méthodes nécessitent l&#39;électroporation et de nombreux organes internes sont difficiles à électroporation soit en raison de leur emplacement ou de leur sensibilité à l&#39;électrique perturbation. En outre, certains tissus de la larve sont difficiles à injecter directement, car l&#39;injection directe peut perturber leur intégrité structurelle ou parce que leur taille est limitant. La nageoire caudale de la chenille est une telle structure, car une injection directe dans le finfold n&#39;est pas possible. Ainsi, une alternative à l&#39;électroporation et l&#39;injection directe a été nécessaire pour cibler des gènes dans des tissus qui sont soit trop petite pour injecter ou ne peut pas être soumis à une électroporation. Afin de cibler et d&#39;inhiber la fonction de gènes spécifiques au cours de la régénération de la nageoire caudale larvaire, nous avons modifié les technologies existantes permettant morpholino l&#39;unÉVALUATION de la fonction des gènes lors de la régénération de la nageoire caudale dans larve de fin de mise en scène. Cette méthode emploie livraison intraventriculaire 9 de marqué à la fluorescéine morpholinos avec Endo-Porter réactif de transfection 10. Une fois dans le ventricule, le mélange morpholino-Endo-Porter se propage rapidement à travers la larve via le système vasculaire et pénètre dans les tissus qui ont été auparavant impossible à cibler. Cette méthode d&#39;injection peut être modifié pour cibler des gènes dans des tissus spécifiques et très probablement peut être appliquée dans d&#39;autres modèles animaux qui n&#39;ont pas actuellement de méthodes de génétique inverse pour inhiber la fonction des gènes. Ainsi, il offre une méthode rapide et facile avec le potentiel pour une large utilisation de la gamme d&#39;étudier immédiatement la fonction des gènes au cours de l&#39;homéostasie des organes général et la régénération au stade larvaire.

<table border="0" cellpadding="0" cellspacing="0" style="width:672px;" width="673">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:171px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Reagent/Material</td>
			<td style="width:171px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Morpholino with 3&#180;fluorescein tag</td>
			<td style="width:171px;">Gene Tools Inc.&#160;</td>
			<td style="width:119px;">Customized</td>
			<td style="width:167px;">More information on www.genetools.com</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Endo-Porter</td>
			<td style="width:171px;">Gene Tools Inc.&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">More information on www.genetools.com</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Agarose</td>
			<td style="width:171px;">Serva</td>
			<td style="width:119px;">120623</td>
			<td style="width:167px;">For pouring plates to place the fish during injections and imaging</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tricane (L-Ethyl-m-amino-benzate-methane sulfonate)</td>
			<td style="width:171px;">Applichem</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">For anesthesia</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">E3 medium (5 mM NaCl; 0.17 mM KCl; 0.33 mM CaCl2; 0.33 mM MgSo4)</td>
			<td style="width:171px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">For larval husbandry</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Petri Dishes</td>
			<td style="width:171px;">Sarstedt</td>
			<td style="width:119px;">821.472</td>
			<td style="width:167px;">For placing the fish during injections and imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#160;Equipment</td>
			<td style="width:171px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Thin Wall Glass Capillaries</td>
			<td style="width:171px;">World Precision Instruments, Inc.&#160;</td>
			<td style="width:119px;">TW100F-3</td>
			<td style="width:167px;">For preparing injection needles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microloader pipette tips</td>
			<td style="width:171px;">Eppendorf</td>
			<td style="width:119px;">5,242,956,003</td>
			<td style="width:167px;">For loading the needle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pasteur Pipettes (3 ml/1 ml)</td>
			<td style="width:171px;">Brand</td>
			<td style="width:119px;">747760/ 74775</td>
			<td style="width:167px;">For transfering larva</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Flaming / Brown&#160; needle puller&#160;</td>
			<td style="width:171px;">Sutter</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">More information on www.sutter.com</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Watchmaker (Dumont) Tweezers (size 5)</td>
			<td style="width:171px;">World Precision Instruments, Inc.&#160;</td>
			<td style="width:119px;">501985</td>
			<td style="width:167px;">To brake the needle before sharpening</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dissecting microscope</td>
			<td style="width:171px;">Olympus, Leica, Zeiss</td>
			<td style="width:119px;">Varies with the manufacturer</td>
			<td style="width:167px;">Part of the whole injection setup</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Fluorescence camera</td>
			<td style="width:171px;">Olympus, Leica, Zeiss</td>
			<td style="width:119px;">Varies with the manufacturer</td>
			<td style="width:167px;">To image the fluorescence after injeciton</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PicoNozzle kit (microinjector holder)</td>
			<td style="width:171px;">World Precision Instruments</td>
			<td style="width:119px;">5430-12</td>
			<td style="width:167px;">For microinjections</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pneumatic PicoPump (microinjector)</td>
			<td style="width:171px;">World Precision Instruments</td>
			<td style="width:119px;">SYS-PV820</td>
			<td style="width:167px;">For microinjections</td>
		</tr>
	</tbody>
</table>

reverse genetic morpholino approach using cardiac ventricular injection to transfect multiple difficult-to-target tissues in the zebrafish larva

Le poisson zèbre est un modèle important de comprendre la biologie cellulaire et moléculaire de l&#39;orgue et appendice régénération. Cependant, les stratégies moléculaires pour employer la génétique inverse ne sont pas encore suffisamment développées pour évaluer la fonction d&#39;un gène dans la régénération ou l&#39;homéostasie tissulaire au cours de stades larvaires après l&#39;embryogenèse poisson-zèbre, et de plusieurs tissus dans la larve de poisson zèbre est difficile à cibler. Injections intraventriculaires de morpholinos spécifiques d&#39;un gène offrent une méthode alternative pour l&#39;incapacité actuelle à une cible, sur le plan génomique de poisson zèbre gènes d&#39;une manière contrôlée dans le temps à ces stades. Cette méthode permet une dispersion complète et l&#39;incorporation ultérieure de la morpholino dans divers tissus du corps, y compris les structures qui étaient auparavant impossible à atteindre telles que celles de la nageoire caudale larvaire, une structure souvent utilisée pour la recherche de manière non invasive la régénération des tissus. Plusieurs gènes activés au cours des larves régénération finfold sont également présentationt dans la régénération de tissus de vertébrés adulte, si la larve est un modèle utile pour comprendre la régénération chez les adultes. Ce procédé de dispersion morpholino permet l&#39;identification rapide et facile des gènes requis pour la régénération des tissus larvaires ainsi que d&#39;autres phénomènes physiologiques régulant l&#39;homéostasie tissulaire après l&#39;embryogenèse. Par conséquent, ce mode de livraison fournit une stratégie actuellement nécessaires pour le contrôle temporel de l&#39;évaluation de la fonction des gènes après l&#39;embryogenèse.

L&#39;aiguille d&#39;injection biseauté forte est facilement placé dans le ventricule cardiaque de la larve de poisson zèbre lorsqu&#39;il est sollicité dorsale (figure 3A). Le cœur continue pompage et la circulation sanguine est maintenue en dépit de la présence de l&#39;aiguille (Figures 3B et 3C). Injections prudents ne perturbent pas la morphologie du ventricule ou les contractions cardiaques (figure 3D) malgré l&#39;injection du morpholino ...

Watch this Scientific Journal Video about Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva at JoVE.com

Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva

Un procédé de génétique inverse adapté pour un poisson zèbre pour déterminer la fonction des gènes au cours des stades ultérieurs de développement et de l&#39;homéostasie physiologique tel que la régénération des tissus en utilisant des injections intraventriculaires de morpholinos spécifiques d&#39;un gène.

Inverser approche Morpholino génétique utilisant cardiaque ventriculaire injection à Transfecter tissus difficiles à cibles multiples dans le poisson zèbre Larve

The zebrafish is an important model to understand the cell and molecular biology of organ and appendage regeneration. However, molecular strategies to ...

reverse-genetic-morpholino-approach-using-cardiac-ventricular

Research

JoVE Journal

Biology

10.5K vues.  Technische Universität Dresden.  Un procédé de génétique inverse adapté pour un poisson zèbre pour déterminer la fonction des gènes au cours des stades ultérieurs de développement et de l'homéostasie physiologique tel que la régénération des tissus en utilisant des injections intraventriculaires de morpholinos spécifiques d'un gène. 

Vidéo: Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.

Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.

Microinjection d&#39;ARNm et oligonucléotides antisens morpholino dans embryons de poissons zèbres.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression ...

Recherche

Biologie

Zebrafish Brain Ventricle Injection

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved cavities within the brain that are filled with cerebrospinal fluid.  In zebrafish, after neural tube formation, the neuroepithelium undergoes a series of constrictions and folds while it fills with fluid resulting in brain ventricle formation.  In order to understand the process of ventricle formation, and the neuroepithelial shape changes that occur at the same time, we needed a way to visualize the ventricle space in comparison to the brain tissue.  However, the nature of transparent zebrafish embryos makes it difficult to differentiate the tissue from the ventricle space.  Therefore, we developed a brain ventricle injection technique where the ventricle space is filled with a fluorescent dye and imaged by brightfield and fluorescent microscopy.  The brightfield and the fluorescent images are then processed and superimposed in Photoshop.  This technique allows for visualization of the ventricle space with the fluorescent dye, in comparison to the shape of the neuroepithelium in the brightfield image.  Brain ventricle injection in zebrafish can be employed from 18 hours post fertilization through early larval stages.  We have used this technique extensively in our studies of brain ventricle formation and morphogenesis as well as in characterizing brain morphogenesis mutants (1-3).

After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.

Injection Zebrafish ventricule cérébral

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved ...

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.

Rétro-orbitaire d&#39;injection chez le poisson zèbre pour adultes

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

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Utilisation du système d&#39;électroporation Gene Pulser MXcell à transfecter des cellules primaires à haut rendement

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage.
GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreERT). CreERT contains a modified estrogen receptor ligand binding domain which renders CreERT sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreERT, which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP.
Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreERT release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreERT.
We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders.
This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreERT;mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

Une approche pratique de la cartographie inductible destin génétique: un guide visuel à Mark et Track Cellules In Vivo</em

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing ...

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In the zebrafish model, effective reverse genetic techniques have been developed using microinjection of gene-specific antisense morpholinos. Morpholinos target an mRNA through specific base-pairing and block gene function transiently by inhibiting translation or splicing for several days during embryogenesis (knockdown). However, in vertebrates such as mouse or zebrafish, some gene functions can be obscured by these approaches due to the presence of another gene that compensates for the loss. This is especially true for gene families containing sister genes that are co-expressed in the same developing tissues. In zebrafish, functional compensation can be tested in a relatively high-throughput manner, by co-injection of morpholinos that target knockdown of both genes simultaneously. Likewise, using morpholinos, a genetic interaction between any two genes can be demonstrated by knockdown of both genes together at sub-threshold levels. For example, morpholinos can be titrated such that neither individual knockdown generates a phenotype. If, under these conditions, co-injection of both morpholinos causes a phenotype, a genetic interaction is shown. Here we demonstrate how to show functional redundancy in the context of two related GATA transcription factors. GATA factors are essential for specification of cardiac progenitors, but this is revealed only by the loss of both Gata5 and Gata6. We show how to carry out microinjection experiments, validate the morpholinos, and evaluate the compensated phenotype for cardiogenesis.

Gene function can be obscured in loss-of-function experiments if there is compensation by another gene. The zebrafish model provides a relatively high-throughput means to reveal such functional redundancy in living embryos.

Une approche de génétique inverse pour tester redondance fonctionnelle pendant l&#39;embryogenèse

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In ...

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, ...

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 ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Néphrotoxine microinjection dans Zebrafish au Modèle lésion rénale aiguë

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A Multi-hole Cryovial Eliminates Freezing Artifacts when Muscle Tissues are Directly Immersed in Liquid Nitrogen

Studies on skeletal muscle physiology face the technical challenge of appropriately processing the specimens to obtain sections with clearly visible cytoplasmic compartments. Another hurdle is the tight apposition of myofibers to the surrounding tissues. Because the process of tissue fixation and paraffin embedding leads to the shrinkage of muscle fibers, freezing is an optimal means of hardening muscle tissue for sectioning. However, a commonly encountered issue, the formation of ice crystals, occurs during the preparation of frozen sections because of the high water content of muscle. The protocol presented here first describes a simple and efficient method for properly freezing muscle tissues by immersing them in liquid nitrogen. The problem with using liquid nitrogen alone is that it causes the formation of a nitrogen gas barrier next to the tissue, which acts as an insulator and inhibits the cooling of the tissues. To avoid this "vapor blanket" effect, a new cryovial was designed to increase the speed of liquid flow around the tissue surface. This was achieved by punching a total of 14 inlet holes in the wall of the vial. According to bubble dynamics, a higher rate of liquid flow results in smaller bubbles and fewer chances to form a gas barrier. When liquid nitrogen flows into the cryovial through the inlet holes, the flow velocity around the tissue is fast enough to eliminate the gas barrier. Compared to the method of freezing muscle tissues using pre-chilled isopentane, this protocol is simpler and more efficient and can be used to freeze muscle in a throughput manner. Furthermore, this method is optimal for institutions that do not have access to isopentane, which is extremely flammable at room temperature.

This protocol describes a procedure to freeze muscle tissues by plunging them directly into liquid nitrogen. This protocol also highlights a new cryovial that can avoid the &#34;blanket effect&#34; of nitrogen gas when liquid nitrogen contacts the tissue surface of a specimen.

Un multi-trous Cryovial Elimine congélation lorsque les artefacts sont les tissus musculaires directement Immergé dans l&#39;azote liquide

Studies on skeletal muscle physiology face the technical challenge of appropriately processing the specimens to obtain sections with clearly visible ...