Cette procédure suit dans une large mesure de la technique de transplantation développés pour les poissons grotte par le laboratoire de Bill Jeffrey.

Partie 1: Préparation des embryons Dans ce protocole, nous allons utiliser le symbole JJ xy pour désigner un mutant de poisson zèbre hypothétique objectif. <ol><li> Zebrafish souches sont maintenus dans des conditions standard de facilité de poissons à 28,5 C sur un cycle de 14h light/10h sombre. </li><li> Dans la soirée, les mâles et les femelles lieu du poisson-zèbre souche JJ xy: AB / TU; tg (mGFP) dans un réservoir avec un diviseur de les séparer les uns des autres. La descendance de ce croisement exprimeront le transgène GFP dans tous les tissus, et seront utilisés comme donneurs. En parallèle, utilisez la même procédure pour mettre en place des croisements entre non transgéniques animaux de type sauvage. Selon le but de cette expérience, on peut traverser les animaux de type sauvage au locus JJ xy en tant que donneurs ou hôtes. </li><li> Dans une heure après la lumière s&#39;allume dans la matinée, retirer les séparateurs pour permettre aux poissons de se reproduire. </li><li> Après 15-30 minutes de recueillir des embryons dans 100 x 15 mm boîte de Pétri, les nettoyer et changer le milieu (eau oeuf). </li><li> Gardez les embryons dans un incubateur à 28,5 C jusqu&#39;au moment désiré. </li><li> Lorsque vous êtes prêt pour la transplantation, retirez le chorion manuellement avec deux paires de forceps pointu, et incuber les embryons dans 0,2% d&#39;EDTA en calcium sans ZFR pendant 30 minutes. </li></ol> Partie 2: Préparation des aiguilles de dissection <ol><li> La procédure de transplantation nécessite deux types d&#39;aiguilles: une aiguisée, qui est utilisé pour couper les tissus entourant la lentille, retirer le chorion, et les embryons libération de agarose et une aiguille émoussée utilisée pour déplacer la lentille donateurs et l&#39;insérer dans l&#39;hôte. Ici, nous allons vous montrer comment faire de ces aiguilles. </li><li> Tout d&#39;abord, la pointe d&#39;une pipette Pasteur doit être coupé avec un couteau de diamant. Ensuite, insérez un verre capillaire dans la pointe pipette Pasteur de sorte qu&#39;environ la moitié de sa longueur est à l&#39;intérieur. Ce sera plus facile pour immobiliser le fil de tungstène par fusion dans le verre à l&#39;étape suivante. </li><li> Ensuite, insérez un fil de tungstène minces dans l&#39;extrémité ouverte du capillaire. Faire fondre le verre sur le fil avec un bec Bunsen. En utilisant des pinces, rester stable à la fin de verre avec le fil métallique tout en tordant l&#39;autre extrémité de l&#39;aiguille à la main. Le verre ramolli devrait spirale autour du métal, il préhension en place. </li><li> Si vous effectuez une aiguille pointe arrondie, alors c&#39;est la fin de la procédure. Pour créer une aiguille aiguisée, tenir le fil de tungstène sur un bec Bunsen pendant 1-1,5 minutes, brûler le métal donc une pointe très fine est créé. Vérifier la qualité de pointe par microscropy.It est une bonne idée de stériliser les deux aiguilles dans la flamme pendant quelques secondes immédiatement avant la transplantation. </li></ol> Partie 3: Préparation des réactifs <ol><li> Sans calcium Zebrafish Ringer (ZFR) Solutions (116 mM NaCl, 2,9 mM de KCl, 5 mM d&#39;HEPES, pH 7,2) </li><li> 0,2% en calcium des solutions sans Zebrafish Ringer (pH 7,2) EDTA </li><li> 1,2% (point de fusion bas) agarose en calcium sans ZFR </li><li> 0,2% (point de fusion bas) agarose en calcium sans ZFR </li><li> Embryon moyenne (pH 7,0, contenant par litre: 10 ml Hanks Solution # 1, 1 ml de solution de Hanks n ° 2, 10ml Hanks Solution n ° 4, 10ml Hanks Solution # 5, le bicarbonate de sodium 0,35 g, 300 uL d&#39;HCl 2M, la pénicilline-streptomycine 500 000 U) comme dans le livre de poisson zèbre (Université de l&#39;Oregon Press, 2000). </li><li> Fils de tungstène, plaqué or, diamètre 0.1mm, Alfa Aesar </li><li> Capillaire, diamètre 1.0mm, Instruments de précision du monde </li></ol> Partie 4: procédure de transplantation <ol><li> Dans un tube à centrifuger en plastique, préparez 1,2% d&#39;agarose bas point de fusion en calcium sans ZFR préchauffé à 40 ° C dans un bain d&#39;eau. </li><li> Au stade désiré (30 HPF, par exemple), prennent les embryons dans une pipette Pasteur, lentement les expulser dans le tube de centrifugation, et lentement les expulser dans le tube de centrifugeuse et incuber pendant environ 5 secondes. Les donateurs et les hôtes doivent être incubés séparément. </li><li> Avec une pipette, prendre jusqu&#39;à les embryons dans agarose à 1,2% du tube de centrifugeuse et les organiser en deux rangées parallèles dans une boîte de Pétri en polystyrène de 100mm, en gardant les hôtes et les donateurs séparée. En les pipetage lentement et prudemment, la plupart des embryons seront couchés sur le flanc dans l&#39;agarose. Vous aurez besoin de réorienter ceux qui ne réside pas dans cette position en utilisant l&#39;aiguille émoussée de telle sorte que l&#39;œil est orienté vers le haut avant que l&#39;agarose se solidifie. </li><li> Attendez que l&#39;agarose à 1,2% à solidifier, puis superposer avec 0,2% de point de fusion solution à faible agarose. </li><li> Utilisation de l&#39;aiguille aiguisé, enlever tout d&#39;agarose autour des yeux, et très soigneusement coupés de la lentille à partir de l&#39;hôte et les embryons donneurs à l&#39;aide de petites touches. Assurez-vous de couper juste assez proche de l&#39;objectif afin de ne pas le déchirer, mais aussi afin que vous ne retirez pas trop de tissu par rapport au reste de l&#39;œil hôte. </li><li> Une fois libre, lentilles flottent dans le milieu. Utilisation de l&#39;aiguille émoussée, poussez délicatement le donateurlentilles juste au-dessus de l&#39;emplacement de l&#39;endroit où la lentille de l&#39;hôte serait normalement, puis enfoncez-la dans les yeux avec l&#39;aiguille émoussée. La lentille de l&#39;hôte peut être écarté. </li><li> Laisser embryons donateurs et d&#39;accueil en agarose à 1,2% pour les 30-60 minutes, puis les libérer de l&#39;agarose en utilisant l&#39;aiguille forte, et les transférer dans une plaque de 24 puits avec un milieu de l&#39;embryon. Chaque embryon doit occuper un autre puits. Assurez-vous de l&#39;étiquette du puits proprement qu&#39;il est clair, qui correspond à l&#39;hôte qui donateur. </li><li> Incuber à 28,5 C. L&#39;provenant de donneurs lentille peut être distinguée de la lentille de l&#39;hôte sur le côté non opéré du même animal repose sur fluorescence de la GFP. Les articles peuvent également être faites pour mesurer la taille d&#39;objectif et de déterminer si elle différencie correctement. </li></ol><img src="/files/ftp_upload/1258/1258fig1.jpg" alt="Figure 1" /> Fig. 1 image à faible grossissement d&#39;une aiguille aiguisée utilisée pour objectif de transplantation. Un capillaire en verre (B) est insérée dans la pipette Pasteur (A), et un mince fil de tungstène (C) est insérée dans l&#39;extrémité ouverte du capillaire. Le verre sur le fil est adoucie par un bec Bunsen. En utilisant des pinces, rester stable à la fin de verre avec le fil métallique tout en tordant l&#39;autre extrémité de la pipette Pasteur avec votre main. Le verre ramolli devrait spirale autour du métal, le saisissant à la place. <img src="/files/ftp_upload/1258/fig2 1258.png" alt="" width="600" height="204" /> Fig. 2 Les conseils des deux types d&#39;aiguilles utilisées pour objectif de transplantation. <img src="/files/ftp_upload/1258/1258fig3.jpg" alt="Figure 3" /> Fig. 3 embryons ont subi lentille de la transplantation au 30hpf. On voit ici un embryon donneur (A, B), un œil sur l&#39;hôte avec un objectif de transplanter (C, D), et le côté le contrôle de l&#39;embryon hôte (E, F). Chaque œil est montré à la fois dans la lumière transmise et éclairage UV tel qu&#39;indiqué. Les photographies ont été prises à 48 HPF. Le Q01 [9] transgéniques ligne a été utilisée dans cette expérience.

<ol>
	<li>Thut, C. J., Rountree, R. B., Hwa, M., Kingsley, D. 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+large-scale+in+situ+screen+provides+molecular+evidence+for+the+induction+of+eye+anterior+segment+structures+by+the+developing+lens.">A large-scale in situ screen provides molecular evidence for the induction of eye anterior segment structures by the developing lens.</a> Dev Biol. 231, 63-76 (2001).</li><li>Yamamoto, Y., Jeffery, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+role+for+the+lens+in+cave+fish+eye+degeneration.">Central role for the lens in cave fish eye degeneration.</a> Science. 289, 631-633 (2000).</li><li>Easter, S. S., Nicola, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+vision+in+the+zebrafish(Danio+rerio).">The development of vision in the zebrafish(Danio rerio).</a> Dev Biol. 180, 646-663 (1996).</li><li>Schmitt, E., Dowling, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+eye+morphogenesis+in+the+zebrafish.+Brachydanio+rerio.">Early eye morphogenesis in the zebrafish. Brachydanio rerio.</a> J.comp. Neuro. 344 (4), 532-542 (1994).</li><li>Malicki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+the+power+of+forward+genetics--analysis+of+neuronal+diversity+and+patterning+in+the+zebrafish+retina.">Harnessing the power of forward genetics--analysis of neuronal diversity and patterning in the zebrafish retina.</a> Trends Neurosci. 23 (11), 531-541 (2000).</li><li>Malicki, J., Pujic, Z., Thisse, C., Thisse, B., Wei, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forward+and+reverse+genetic+approaches+to+the+analysis+of+eye+development+in+zebrafish.">Forward and reverse genetic approaches to the analysis of eye development in zebrafish.</a> Vision Res. 42 (4), 527-533 (2002).</li><li>Avanesov, A., Malicki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approaches+to+study+neurogenesis+in+the+zebrafish+retina.">Approaches to study neurogenesis in the zebrafish retina.</a> Methods Cell Biol. 76, 333-384 (2004).</li><li>Vihtelic, T. S., Hyde, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+mutagenesis+yields+eye+morphological+mutants+with+retinal+and+lens+defects.">Zebrafish mutagenesis yields eye morphological mutants with retinal and lens defects.</a> Vision Res. 42, 535-540 (2002).</li><li>Godinho, L., Mumm, J. S., Williams, P. R., Schroeter, E. H., Koerber, A., Park, S. W., Leach, S. D., Wong, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+amacrine+cell+neurites+to+appropriate+synaptic+laminae+in+the+developing+zebrafish+retina.">Targeting of amacrine cell neurites to appropriate synaptic laminae in the developing zebrafish retina.</a> Development. Nov 132, 5069-5079 (2005).</li></ol>

Plusieurs étapes nécessitent une attention particulière lors de transplantations d&#39;objectif. <ol><li> Aiguisé aiguilles: Il est préférable de préparer plusieurs aiguilles avant d&#39;effectuer la transplantation lentilles, et la pointe de l&#39;aiguille doit être aussi mince que possible. Une aiguille plus épaisse va déchirer plus de tissu en raison de son plus grand diamètre, provoquant une panne dans les yeux pour guérir. </li><li> Organiser les embryons: Juste avant la transplantation, vous devez ...

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<td>0.1mm diameter tungsten wire</td>
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lens transplantation in zebrafish and its application in the analysis of eye mutants

L&#39;objectif joue un rôle important dans le développement de la cupule optique [1,2]. En utilisant le poisson zèbre comme organisme modèle, les questions concernant le développement lentille peut être adressée. Le poisson zèbre est utile pour des études génétiques en raison de plusieurs caractéristiques avantageuses, y compris de petite taille, taux de fécondité élevé, cycle de vie court, et la facilité d&#39;entretien. Objectif de développement se produit rapidement chez le poisson zèbre. Par 72 HPF, la lentille de poisson zèbre est fonctionnellement matures [3]. L&#39;abondance des ressources génétiques et moléculaires sont disponibles pour soutenir la recherche dans le poisson zèbre. En outre, la similitude de l&#39;œil de poisson zèbre à ceux des autres vertébrés fournit une base pour son utilisation comme un modèle animal d&#39;excellents défauts de l&#39;homme [4-7]. Plusieurs mutants poisson zèbre présentent des anomalies lentilles, y compris des niveaux élevés de mort cellulaire, ce qui dans certains cas, conduit à une dégénérescence complète des tissus lentille [8]. Afin de déterminer si des anomalies de lentilles sont dus à des causes intrinsèques ou à défaut les interactions avec les tissus environnants, la transplantation d&#39;une lentille mutant dans un oeil de type sauvage est effectuée. L&#39;utilisation d&#39;aiguilles métalliques polies au feu, les lentilles de mutant ou de type sauvage sont soigneusement disséquées de l&#39;animal donneur, et transférés dans l&#39;hôte. Afin de distinguer les tissus de type sauvage et mutant, une lignée transgénique est utilisé comme donneur. Cette ligne exprime membranaires GFP dans tous les tissus, y compris l&#39;objectif. Cette technique de transplantation est un outil essentiel dans les études de mutants lentilles poisson zèbre.

Watch this Scientific Journal Video about Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants at JoVE.com

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

Objectif de développement implique des interactions avec d&#39;autres tissus. Plusieurs mutants oeil de poisson zèbre sont caractérisés par une taille anormalement petite lentille. Ici nous démontrons une expérience de transplantation objectif pour déterminer si ce phénotype est dû à des causes intrinsèques ou des interactions défectueuses avec les tissus qui entourent la lentille.

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 ...

lens-transplantation-zebrafish-its-application-analysis-eye

Research

JoVE Journal

Biology

11.0K vues.  The Second Teaching Hospital of Jilin University. Objectif de développement implique des interactions avec d'autres tissus. Plusieurs mutants oeil de poisson zèbre sont caractérisés par une taille anormalement petite lentille. Ici nous démontrons une expérience de transplantation objectif pour déterminer si ce phénotype est dû à des causes intrinsèques ou des interactions défectueuses avec les tissus qui entourent la lentille.

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

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 ...

Recherche

Biologie

Preparation and Culture of Rat Lens Epithelial Explants for Studying Terminal Differentiation

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  Following division, these cells migrate posteriorly, where FGF diffusing from the retina induces them to differentiate into a posterior array of elongated lens fiber cells, which compose the bulk of the lens.  Differentiation of lens epithelial cells into lens fibers can be induced in vitro by culturing explants of the central region of the anterior epithelium in the presence of FGF-2.  Explants are prepared from lenses of neonatal rats by removing the lens from the eye and grasping the lens capsule on the posterior side with dissecting tweezers.  The posterior capsule is then gently torn open and pressed down into the plastic bottom of a tissue culture dish. The peripheral regions of the explant are removed with a scalpel and the central area is then cultured in the presence of 100ng/ml FGF-2 for as long as 2-3 weeks, depending on the parameters to be studied.  Since epithelial cells in cultured explants differentiate in approximate synchrony over a period of days to weeks, the time course of signaling and gene expression can be determined using molecular, biochemical, and pharmacological techniques.  Immunofluorescence microscopy is a powerful adjunct to these methods as it demonstrates the subcellular localization of proteins of interest and can reveal the physiological consequences of experimental manipulations of signaling pathways.

Explants of the central region of rat lens epithelia differentiate synchronously when cultured in the presence of FGF-2. Immunofluorescence microscopy of such cultures can provides novel information about gene expression and signaling events associated with terminal differentiation.

Préparation et de la culture d&#39;explants Objectif Rat épithéliales pour l&#39;étude de la différenciation terminale

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

Culture primaire et l&#39;électroporation plasmidique de l&#39;Organe de Corti murin.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

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 ...

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 ...

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here&#160;we describe the design and analysis of TPR in Drosophila.

Conception et analyse du comportement de préférence de température et de son rythme circadien chez la drosophile

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.

The zebrafish adult kidney is an excellent system for renal regeneration and disease studies. An essential aspect of such research is the assessment of nephron structure and function. This protocol describes several methodologies that can be implemented to assess nephron tubule composition and to evaluate renal reabsorption.

Analyse de néphron Composition et fonction du rein poisson zèbre adulte

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys ...

Sequential Application of Glass Coverslips to Assess the Compressive Stiffness of the Mouse Lens: Strain and Morphometric Analyses

The eye lens is a transparent organ that refracts and focuses light to form a clear image on the retina. In humans, ciliary muscles contract to deform the lens, leading to an increase in the lens&#39; optical power to focus on nearby objects, a process known as accommodation. Age-related changes in lens stiffness have been linked to presbyopia, a reduction in the lens&#39; ability to accommodate, and, by extension, the need for reading glasses. Even though mouse lenses do not accommodate or develop presbyopia, mouse models can provide an invaluable genetic tool for understanding lens pathologies, and the accelerated aging observed in mice enables the study of age-related changes in the lens. This protocol demonstrates a simple, precise, and cost-effective method for determining mouse lens stiffness, using glass coverslips to apply sequentially increasing compressive loads onto the lens. Representative data confirm that mouse lenses become stiffer with age, like human lenses. This method is highly reproducible and can potentially be scaled up to mechanically test lenses from larger animals.

Age-related increases in eye lens stiffness are linked to presbyopia. This protocol describes a simple, cost-effective method for measuring mouse lens stiffness. Mouse lenses, like human lenses, become stiffer with age. This method is precise and can be adapted for lenses from larger animals.

Application séquentielle de verre Lamelles pour évaluer la compression Rigidité de l&#39;objectif de la souris: Strain et morphométriques Analyses

The eye lens is a transparent organ that refracts and focuses light to form a clear image on the retina. In humans, ciliary muscles contract to deform the ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

Haute résolution Time-lapse d&#39;imagerie et d&#39;analyse automatisée des microtubules Dynamics Living ombilicale humaine Cellules endothéliales de veine

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

L&#39;Assemblée et l&#39;application des «Shear Anneaux»: A Novel Endothelial Modèle pour Orbital, unidirectionnel et périodique Fluid débit et contrainte de cisaillement

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...