Nous tenons à remercier les membres du Laboratoire Barresi pour leur soutien et leurs précieux commentaires sur ce manuscrit. Nous remercions Alexander Workman pour son aide technique constante ainsi que le personnel de soins aux animaux pour leur aide dans le maintien du Smith College Zebrafish colonie. Nous remercions également Hallacy Mike, Rudi Rottenfusser, et Carl Zeiss pour le prêt du matériel de microscopie pour l&#39;tournages comm de ce protocole. Ce travail a été soutenu par une subvention de recherche financée par la NSF, 0615594.

<ol>
	<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+guide+for+the+laboratory+use+of+zebrafish+(Danio.">A guide for the laboratory use of zebrafish (Danio.</a> The Zebrafish Book. , (2007).</li><li>N&#252;sslein-Volhard, C., Dahm, R. e. d. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+A+Practical+Approach.">Zebrafish A Practical Approach.</a> , (2002).</li><li>Graeden, E., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+the+zebrafish+embryonic+brain+by+confocal+microscopy.">Live imaging of the zebrafish embryonic brain by confocal microscopy.</a> J Vis Exp. (26), (2009).</li><li>Gilmour, D. T., Jessen, J. R., Lin, S., N&#252;sslein-Volhard, C., Dahm, R. e. d. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish,+a+practical+approach.">Zebrafish, a practical approach.</a> 1, 121-121 (2002).</li><li>Woo, K., Shih, J., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+maps+of+the+zebrafish+embryo.">Fate maps of the zebrafish embryo.</a> Curr Opin Genet Dev. 5 (4), 439-439 (1995).</li><li>Barresi, M. J., Hutson, L. D., Chien, C. B., Karlstrom, 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=Hedgehog+regulated+Slit+expression+determines+commissure+and+glial+cell+position+in+the+zebrafish+forebrain.">Hedgehog regulated Slit expression determines commissure and glial cell position in the zebrafish forebrain.</a> Development. 132 (16), 3643-3643 (2005).</li><li>Kozlowski, D. J., Murakami, T., Ho, R. K., Weinberg, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+cell+movement+and+tissue+patterning+in+the+zebrafish+embryo+revealed+by+fate+mapping+with+caged+fluorescein.">Regional cell movement and tissue patterning in the zebrafish embryo revealed by fate mapping with caged fluorescein.</a> Biochem Cell Biol. 75 (5), 551-551 (1997).</li><li>Halloran, M. C., Murakami, T., Ho, R. K., Weinberg, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-induced+gene+expression+in+specific+cells+of+transgenic+zebrafish.">Laser-induced gene expression in specific cells of transgenic zebrafish.</a> Development. 127 (9), 1953-1953 (2000).</li><li>Hardy, M. E., Ross, L. V., Chien, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+gene+misexpression+in+zebrafish+embryos+induced+by+local+heat+shock+using+a+modified+soldering+iron.">Focal gene misexpression in zebrafish embryos induced by local heat shock using a modified soldering iron.</a> Dev Dyn. 236 (11), 3071-3071 (2007).</li></ol>

The authors have nothing to disclose.

Génération embryons chimères est un outil puissant qui aborde les questions de recherche au sein de nombreux systèmes de plusieurs modèles, à savoir la mouche des fruits (D.melanogaster), un ver (C.elegans), le poisson-zèbre (D.rerio) et de la souris (M.musculus). Nous soutenons que le système modèle de poisson zèbre est particulièrement bien adapté pour générer des chimères dans un relativement facile, rapide et polyvalent. Le poisson zèbre est un animal vertébré qui se développe en dehors de la mère qui rend beaucoup plus accessible que par rapport au modèle de la souris. Le poisson zèbre sont aussi significativement plus grandes que des mouches ou des vers, ce qui simplifie les manipulations embryonnaires tels que la transplantation de cellules. En outre, la capacité de combiner les procédures de transplantation de cellules avec des techniques transgéniques permet un large éventail d&#39;expériences possibles dans lesquelles la fonction des gènes peuvent être manipulés dans un tissu-spécifique de manière localisée. Par exemple, les clones petite GFP + ou la rhodamine cellules marquées permettent la caractérisation de morphologies cellulaires complets qui sont souvent perdus dans un donneur homozygote transgéniques ou sont impossibles à visualiser en raison de l&#39;étiquetage différentiel subcellulaires avec des anticorps communs. Par ailleurs, en utilisant des cellules marquées ou transgénique fluorescent remplis, nous pouvons suivre les cellules du donneur de l&#39;embryon de poisson zèbre en direct sur le temps. Timelapse imagerie des cellules individuelles fournit une nouvelle analyse du comportement cellulaire dans le contexte de l&#39;organisme entier. Notre laboratoire associe également l&#39;utilisation de choc thermique inductible lignées transgéniques avec les greffes de cellules de telle sorte que certains signaux de guidage axonal peut être misexpressed dans les cellules du donneur après une élévation de la température d&#39;incubation (données montrent pas). Cette technique est une approche extrêmement puissante et directe localement misexpress une protéine d&#39;intérêt de manière spatiale et temporelle spécifique, ce qui nous permet de voir comment la protéine spécifique affecte le développement. Dans notre cas, cette technique peut étendre nos connaissances derrière la fonction de signaux de guidage à fente-Robo dans le positionnement des commissures et des cellules gliales lors de la 5 ligne médiane. D&#39;autres approches de gènes tels que localement misexpressing uncaging UV focale et outils heatshock localisée (laser ou fer à souder), offrent d&#39;autres façons de manipuler l&#39;expression des gènes et des marqueurs dans un petit amas de cellules 7-9; cependant, la transplantation de cellules établit un environnement final de l&#39;analyse qui est exempt de tout traumatisme induit par les UV, laser ou à la chaleur faisant une transplantation de cellules d&#39;une approche plus expérimentale contrôlée. En conclusion, nous avons trouvé des procédures transplantation de cellules pour être une partie essentielle de notre recherche en biologie du développement neurologique. Génération des clones de cellules ayant des propriétés différentes est un outil essentiel pour le biologiste du développement dans la lutte contre les comportements des questions fondamentales de la cellule, la fonction des gènes, des cellules et de l&#39;autonomie.

<table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Nom </td><td> Tapez </td><td> Société </td><td> Nombre de catalogue </td><td> Détails </td></tr><tr><td> Boîtes de Pétri </td><td> Outil </td><td> Fisherbrand </td><td> 08-757-13 </td><td> 100 x 15 mm </td></tr><tr><td> Les plaques de verre en bas de la culture </td><td> Outil </td><td> MatTek </td><td> P35G-1.5-2.0-C </td><td> 35 x 15mm, n ° 1.5, non couchés, irradiés par rayons gamma </td></tr><tr><td> Grand diamètre en verre pipettes Pasteur </td><td> Outil </td><td> Fisherbrand </td><td> 63A-53-WT </td><td></td></tr><tr><td> Verre à filament capillaires pour les moules creux (sans fil) et les aiguilles d&#39;injection (avec filament) </td><td> Outil </td><td> Instruments de précision du monde </td><td> 1B150F-4 </td><td> 4 po (100 mm), 1,5 / 0,84 OD / ID (mm) </td></tr><tr><td> Transplant capillaires (TransferTip) </td><td> Outil </td><td> Eppendorf </td><td> 83000122-8 </td><td> Type IV, stérile, Int. 15μm, 20 degrés courbure </td></tr><tr><td> Moisissures Transplant </td><td> Outil </td><td> Adaptive Science Tools </td><td> PT-1 </td><td> 150 triangulaires mottes de tenir embryons individuels </td></tr><tr><td> Agarose, Type I </td><td> Réactifs </td><td> Sigma-Aldrich </td><td> A6013-250G </td><td> 1,5% d&#39;agarose faite dans EM </td></tr><tr><td> Moyenne des embryons (antibiotique) </td><td> Réactifs </td><td></td><td></td><td> Voir Westerfield, 2007 </td></tr><tr><td> Tampon phosphate </td><td> Réactifs </td><td></td><td></td><td> Voir Westerfield, 2007 </td></tr><tr><td> Pince Watchman </td><td> Outil </td><td> Outils Fine Science </td><td></td><td> 100 + mm pointe (terne), pointe 50mm </td></tr><tr><td> Rhodamine B Dextran </td><td> Lineage colorant traceur </td><td> Molecular Probes, Invitrogen </td><td> D1824 </td><td> MW 10.000 Da, la lysine-fixable, rouge (590nm) colorant fluorescent excité avec le vert (570nm) de lumière </td></tr><tr><td> Microscope à dissection </td><td> Microscope </td><td> Olympus </td><td> SZX7 </td><td></td></tr><tr><td> Fluorescent Microscope stéréo </td><td> Microscope </td><td> Zeiss </td><td> Lumar </td><td> Joystick entièrement automatisé contrôlé (Sycop) </td></tr><tr><td> Appareil Transplant </td><td> Outil </td><td> Eppendorf Corp </td><td> Airtram </td><td></td></tr><tr><td> Micromanipulateur automatisé </td><td> Outil </td><td> Eppendorf Corp </td><td> TransferMan NK2 </td><td> Joystick contrôlée </td></tr><tr><td> Micromanipulateur </td><td> Outil </td><td> Applied Scientific Instruments, Inc </td><td> 00-42-101-0000 </td><td> MM33 droit </td></tr><tr><td> Microinjecteur avec l&#39;Unité de pression retour </td><td> Outil </td><td> Applied Scientific Instruments, Inc </td><td> MPPI-2 BPU </td><td></td></tr><tr><td> Flamming / Brown micropipette Puller </td><td> Outil </td><td> Sutter Instrument Company </td><td> Modèle P97 </td><td> Programme: Chaleur 550; Velocity 200; Heure / Retard 200; Tirez vigueur 120. </td></tr></tbody></table>

tissue targeted embryonic chimeras: zebrafish gastrula cell transplantation

Certaines questions fondamentales dans le domaine de la biologie du développement ne peut être répondu lorsque les cellules sont placées dans de nouveaux environnements ou lorsque de petits groupes de cellules dans un contexte plus large sont modifiées. Observer comment une cellule interagit et se comporte dans un environnement unique est essentiel pour caractériser les fonctions cellulaires. Déterminer comment le misexpression localisée d&#39;une protéine spécifique des influences qui entourent les cellules fournit des informations utiles sur les rôles que joue la protéine dans une variété de processus de développement. Notre laboratoire utilise le système de modèle de poisson zèbre pour combinent de façon unique avec des approches génétiques classiques techniques de transplantation de générer des chimères génotypiques ou phénotypiques. Nous étudions les interactions cellulaires des neurones-glie pendant la formation des commissures du cerveau antérieur chez le poisson zèbre. Cette vidéo décrit une méthode qui permet à notre laboratoire pour étudier le rôle des populations astrogliales dans le diencéphale et le rôle des signaux de guidage spécifiques qui influencent les axones projetant comme ils croisent la ligne médiane. En raison de leurs embryons de poisson zèbre transparence sont des modèles idéaux pour ce type de placement cellulaire ectopique ou misexpression gène localisé. Suivi des cellules transplantées peut être accompli en utilisant un colorant vital ou une ligne de poissons transgéniques exprimant une protéine fluorescente. Nous démontrons ici comment préparer embryons donneurs avec un traceur colorant vital pour la transplantation, ainsi que la façon d&#39;extraire et de greffe de cellules d&#39;un embryon de gastrula scène à l&#39;autre. Nous présentons des données démontrant la GFP + ectopique des cellules transgéniques dans le prosencéphale des embryons de poissons zèbres et de caractériser la localisation de ces cellules à l&#39;égard de prosencéphale commissures. En outre, nous montrons au laser microscopie à balayage confocal timelapse d&#39;Alexa 594 cellules marquées transplantées dans un embryon hôte GFP + transgéniques. Ces données fournissent la preuve que la gastrula scène greffe permet le positionnement ciblé des cellules ectopiques de répondre à une variété de questions en biologie du développement.

Watch this Scientific Journal Video about Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation at JoVE.com

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Zebrafish transplantation de cellules permet la combinaison de la génétique et l&#39;embryologie de générer des chimères tissulaires spécifiques. Cette vidéo montre gastrula scène transplantations de cellules qui ont permis à notre laboratoire pour étudier les rôles des populations astrogliales et les indices des directives précises lors de la formation commissure dans le prosencéphale.

Tissu ciblé chimères embryonnaires: Zebrafish cellulaire Gastrula transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

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Research

JoVE Journal

Biology

19.2K vues.  Smith College. Zebrafish transplantation de cellules permet la combinaison de la génétique et l'embryologie de générer des chimères tissulaires spécifiques. Cette vidéo montre gastrula scène transplantations de cellules qui ont permis à notre laboratoire pour étudier les rôles des populations astrogliales et les indices des directives précises lors de la formation commissure dans le prosencéphale.

Vidéo: Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

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

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

Co-actine sédimentation Assay; pour l&#39;analyse des Binding Protein à F-actine

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Generating Chimeric Zebrafish Embryos by Transplantation

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

Génération embryons de poissons zèbres chimériques par transplantation

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

Microdissection of Zebrafish Embryonic Eye Tissues

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post fertilization (dpf), the larvae will show the first visual response. Many genes have been identified to control a proper eye development, but we are far from a complete understanding of the underlying genetic architecture. Whole genome gene expression profiling is a useful tool to elucidate genetic regulatory network for eye development. However, the small size of the embryonic eye in zebrafish makes it challenging to obtain intact and pure eye tissues for expression analysis. For example, the anterior-posterior length of the eye between day 2 and 3 is only approximately 200-300 &mu;m, while the diameter of the lens is less 100 &mu;m. Also, the retinal pigment epithelium (RPE) underlying the retina is just a single-layer epithelium. While gene expression profiles can be obtained from the whole embryo, they do not accurately represent the expression of these tissues. Therefore pure tissue must be obtained for a successful gene expression profiling of eye development. To address this issue, we have developed an approach to microdissect intact retina and retina with RPE attached from 1-3 dpf, which cover major stages of eye morphogenesis. All procedures can be done with fine forceps and general laboratory supplies under standard stereomicroscopes. For retinal dissection, the single-layer RPE is removed and peeled off by brushing action and the preferential adherence of the RPE remnants to the surface of the culture plate for dissection. For RPE-attached retinal dissection, the adherence of RPE to the dissection plate is removed before the dissection so that the RPE can be completely preserved with the retina. A careful lifting action of this tissue can efficiently separate the presumptive choroid and sclera. The lens can be removed in both cases by a chemically etched tungsten needle. In short, our approach can obtain intact eye tissues and has been successfully utilized to study tissue-specific expression profiles of zebrafish retina1, 2 and retinal pigment epithelium3.

This article describes an approach to microdissect zebrafish retinas with and without retinal pigment epithelium attached, from one to three days postfertilization embryos.

Microdissection de tissus embryonnaires Zebrafish Eye

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post ...

Dechorionation of Medaka Embryos and Cell Transplantation for the Generation of Chimeras

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of the vertebrate genome functions. Manipulation of medaka embryos, such as dechorionation, mounting embryos for imaging and cell transplantation, are key procedures to work on both medaka and zebrafish in a laboratory. Cell transplantation examines cell autonomy of medaka mutations. Chimeras are generated by transplanting labeled cells from donor embryos into unlabeled recipient embryos. Donor cells can be transplanted to specific areas of the recipient embryos based on the fate maps 3 so that clones from transplanted cells can be integrated in the tissue of interest during development. Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. In this video, we show detailed procedures to manipulate medaka embryos.

Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. This video shows step-by-step procedures for how to manipulate medaka embryos, including dechorionation, mounting in agarose for imaging and cell transplantation for the production of chimeras. These procedures are essential to use medaka and zebrafish in a laboratory to take full advantage of their complementary features for the genetic dissection of vertebrate genome functions.

Dechorionation d&#39;embryons Medaka et la transplantation de cellules pour la production de chimères

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in ...

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound transgenic fish which express different fluorescent reporters in neighboring cell types provide a means of following cellular interactions 2 and/or tissue-level responses to experimental manipulations over time. In this video, we demonstrate methods that can be used for imaging multiple transgenically labeled cell types serially in individual fish over time courses that can span from minutes to several days. The techniques described are applicable to any study seeking to correlate the "behavior" of neighboring cells types over time, including: 1) serial 'catch and release' methods for imaging a large number of fish over successive days, 2) simplified approaches for separating fluorophores with overlapping excitation/emission profiles (e.g., GFP and YFP), 3) use of hypopigmented mutant lines to extend the time window available for high-resolution imaging into late larval stages of development, 4) use of membrane targeted fluorescent reporters to reveal fine morphological detail of individual cells as well as cellular details in larger populations of cells, and 5) a previously described method for chemically-induced ablation of transgenically targeted cell types; i.e., nitroreductase (NTR) mediated conversion of prodrug substrates, such as metronidazole (MTZ), to cytotoxic derivatives 3,5.
As an example of these approaches, we will visualize the ablation and regeneration of a subtype of retinal bipolar neuron within individual fish over several days. Simultaneously we will monitor several other retinal cell types, including neighboring non-targeted bipolar cells and potential degeneration-stimulated retinal stem cells (i.e., M&#971;ller glia). This strategy is being applied in our lab to characterize cell- and tissue-level (e.g., stem cell niche) responses to the selective loss and regeneration of targeted neuronal cell types.

In this video, techniques for multicolor confocal time-lapse imaging and targeted cell ablation are provided. Time-lapse imaging is used to monitor the behavior of multiple cell types of interest in vivo. Targeted cell ablation facilitates the study neural circuit function and cell-specific neuronal regeneration paradigms.

Multicolor imagerie time-lapse de poisson zèbre transgénique: Visualisation des cellules souches rétiniennes Activé par ablation cellulaire ciblée neuronale

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

Cultures de cellules primaires à partir Drosophile Embryons Gastrula

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

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 ...

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

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

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

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

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

Immunostaining of Dissected Zebrafish Embryonic Heart

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics 1,2. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects 3. The expression of any gene can be manipulated via morpholino technology or RNA injection 4. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis 5. 
Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue 6. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. 
 Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. 
Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals.

A rapid way to conduct immunostaining of zebrafish embryonic heart is described. Compared to the whole mount immunostaining approach, this method dramatically increases the penetration of the antibodies, which allows obtaining high resolution images that reveal cellular/subcellular structures in the heart within a much reduced processing time.

Immunocoloration des Disséqué coeur embryonnaire Zebrafish

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...