Vorremmo ringraziare i membri del Laboratorio Barresi per il sostegno e utili commenti su questo manoscritto. Ringraziamo Workman Alessandro per la sua costante assistenza tecnica e il personale la cura degli animali per il loro aiuto nel mantenere la Smith College colonia Zebrafish. Ringraziamo anche Hallacy Mike, Rudi Rottenfusser, e Carl Zeiss Microimaging per il prestito alcune attrezzature per la microscopia filmin di questo protocollo. Questo lavoro è stato supportato da un assegno di ricerca finanziato 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.

Generare embrioni chimera è un potente strumento che affronta problematiche di ricerca all&#39;interno di molti sistemi di modelli diversi, ovvero il moscerino della frutta (D.melanogaster), worm (C.elegans), pesce zebra (D.rerio) e mouse (M.musculus). Noi sosteniamo che il sistema modello Danio rerio è particolarmente adatto per la produzione di chimere in un relativamente facile, veloce e versatile. Il pesce zebra è un vertebrato che si sviluppa al di fuori della madre che lo rende molto più accessibile rispetto al modello murino. Zebrafish sono anche significativamente più grandi mosche o vermi, che semplifica la manipolazione embrionale come il trapianto di cellule. Inoltre, la capacità di combinare le procedure di trapianto di cellule con tecniche transgeniche consente una vasta gamma di esperienze possibili in cui la funzione del gene può essere manipolata in un localizzata tessuto-specifica. Per esempio, piccoli cloni di GFP + o rodamina cellule marcate consentono la caratterizzazione della morfologia delle cellule pieno che spesso si perdono in un donatore transgenici omozigoti o sono impossibili da visualizzare a causa di etichettatura differenziale subcellulare con anticorpi comune. Inoltre, utilizzando cellule transgenically tag o fluorescente pieno, siamo in grado di monitorare le cellule del donatore nell&#39;embrione zebrafish vivere nel tempo. Timelapse immagini delle singole cellule fornisce una nuova analisi del comportamento cellulare nel contesto di tutto l&#39;organismo. Il nostro laboratorio abbina anche l&#39;utilizzo di shock termico inducibile linee transgeniche con trapianti di cellule in modo che alcuni spunti di guida degli assoni possono essere misexpressed nelle cellule del donatore a seguito di un aumento della temperatura di incubazione (dati non dimostrano). Questa tecnica è un approccio estremamente potente e diretto a livello locale misexpress una proteina di interesse in modo spaziale e temporale-specifici, che ci permette di vedere come la proteina specifica influenza lo sviluppo. Nel nostro caso, questa tecnica può estendere le nostre conoscenze base del funzionamento di fessura-Robo spunti guida nel posizionamento delle commissure e cellule gliali sulla linea mediana 5. Altri approcci ai geni a livello locale misexpressing come uncaging UV focale e strumenti heatshock localizzate (laser o saldatore), offrono metodi alternativi per manipolare l&#39;espressione dei geni e marker in un piccolo gruppo di cellule 7-9, tuttavia, stabilisce un trapianto di cellule ambiente finale di analisi che è libero da qualsiasi trauma indotto da UV, laser o termica fare il trapianto di cellule di un approccio più sperimentale controllato. In conclusione, abbiamo trovato le procedure di trapianto di cellule da una parte cruciale della nostra ricerca della biologia dello sviluppo neurologico. Generazione di cloni di cellule con proprietà diverse è uno strumento critico per il biologo dello sviluppo per affrontare questioni fondamentali dei comportamenti delle cellule, la funzione del gene, e l&#39;autonomia delle cellule.

<table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Nome </td><td> Tipo </td><td> Azienda </td><td> Numero di catalogo </td><td> Specifiche </td></tr><tr><td> Petri </td><td> Strumento </td><td> Fisherbrand </td><td> 08-757-13 </td><td> 100 x 15 mm </td></tr><tr><td> Con fondo in vetro cultura piastre </td><td> Strumento </td><td> MatTek </td><td> P35G-1.5-2.0-C </td><td> 35 x 15mm, 1,5 No, non rivestito, gamma-irradiati </td></tr><tr><td> Ampio foro pipette Pasteur in vetro </td><td> Strumento </td><td> Fisherbrand </td><td> 63A-53-WT </td><td></td></tr><tr><td> Di vetro a filamento capillari per stampi attraverso (senza filamento) e aghi di iniezione (con filamento) </td><td> Strumento </td><td> Mondo Strumenti di precisione </td><td> 1B150F-4 </td><td> 4 pollici (100 mm), 1,5 / 0,84 OD / ID (mm) </td></tr><tr><td> Capillari trapianti (TransferTip) </td><td> Strumento </td><td> Eppendorf </td><td> 83000122-8 </td><td> Tipo IV, sterile, int. 15μm, 20 piegare grado </td></tr><tr><td> Trapianto di stampo </td><td> Strumento </td><td> Adaptive Scienza Tools </td><td> PT-1 </td><td> 150 divots triangolare di tenere gli embrioni singoli </td></tr><tr><td> Agarosio, tipo I </td><td> Reagente </td><td> Sigma Aldrich </td><td> A6013-250G </td><td> Agarosio 1,5% made in EM </td></tr><tr><td> (Antibiotico) Embrione Media </td><td> Reagente </td><td></td><td></td><td> Vedi Westerfield, 2007 </td></tr><tr><td> Tampone fosfato </td><td> Reagente </td><td></td><td></td><td> Vedi Westerfield, 2007 </td></tr><tr><td> Watchman Pinza </td><td> Strumento </td><td> Strumenti Scienza multa </td><td></td><td> 100 + mm punta (opaco), punta di 50 millimetri </td></tr><tr><td> Rodamina B Destrano </td><td> Lineage tracciante colorante </td><td> Sonde molecolari, Invitrogen </td><td> D1824 </td><td> Da 10.000 MW, lisina risolvibile, rosso (590nm) colorante fluorescente eccitato con verde (570nm) la luce </td></tr><tr><td> Microscopio da dissezione </td><td> Microscopio </td><td> Olimpo </td><td> SZX7 </td><td></td></tr><tr><td> Fluorescente Microscopio Stereo </td><td> Microscopio </td><td> Zeiss </td><td> Lumar </td><td> Fully Automated Joystick controllata (Sycop) </td></tr><tr><td> Apparecchi trapianto </td><td> Strumento </td><td> Eppendorf Corp. </td><td> Airtram </td><td></td></tr><tr><td> Micromanipolatore automatizzati </td><td> Strumento </td><td> Eppendorf Corp. </td><td> TransferMan NK2 </td><td> Joystick controllata </td></tr><tr><td> Micromanipolatore </td><td> Strumento </td><td> Applied Scientific Instruments, Inc. </td><td> 00-42-101-0000 </td><td> MM33 Destra </td></tr><tr><td> Microinjector con unità di pressione Indietro </td><td> Strumento </td><td> Applied Scientific Instruments, Inc. </td><td> MPPI-2 BPU </td><td></td></tr><tr><td> Flamming / Brown micropipetta Puller </td><td> Strumento </td><td> Sutter strumento aziendale </td><td> Modello P97 </td><td> Programma: Calore 550; Velocity 200; Tempo / ritardo 200; Forza di tiro 120. </td></tr></tbody></table>

tissue targeted embryonic chimeras: zebrafish gastrula cell transplantation

Alcune questioni fondamentali nel campo della biologia dello sviluppo può essere risolta solo quando le cellule si trovano in nuovi ambienti o ambienti in cui piccoli gruppi di cellule in un contesto più ampio sono alterati. Guardando come una cellula interagisce e si comporta in un ambiente unico è essenziale per caratterizzare le funzioni cellulari. Determinare come la misexpression localizzata di una influenza specifica proteina che circonda le cellule fornisce informazioni penetranti sui ruoli che le proteine ​​gioca in una varietà di processi di sviluppo. Il nostro laboratorio utilizza il sistema zebrafish modello di combinare in modo univoco approcci genetici con le tecniche di trapianto classica per generare chimere genotipica o fenotipica. Studiamo neuroni-cellule gliali interazioni durante la formazione delle commissure proencefalo in zebrafish. Questo video descrive un metodo che consente al nostro laboratorio per studiare il ruolo delle popolazioni astrogliali nel diencefalo ed i ruoli di spunti orientamenti specifici che influenzano la progettazione assoni che attraversano la linea mediana. Grazie alla loro trasparenza embrioni di zebrafish sono modelli ideali per questo tipo di posizionamento di cellule ectopiche o misexpression gene localizzato. Monitoraggio cellule trapiantate possono essere eseguite utilizzando un colorante vitale o una linea di pesce transgenici che esprimono una proteina fluorescente. Dimostriamo qui come preparare gli embrioni donatori con un tracciante colorante vitale per il trapianto, e come estrarre e trapianto di cellule da un embrione gastrula messo in scena ad un altro. Vi presentiamo i dati che mostrano ectopica GFP + cellule transgeniche nel proencefalo di embrioni di zebrafish e caratterizzare la posizione di queste cellule rispetto al proencefalo commessure. Inoltre, laser show microscopia confocale a scansione Timelapse di Alexa 594 cellule marcate trapiantate in un GFP + embrione ospitare transgenici. Questi dati forniscono evidenza che gastrula messo in scena il trapianto consente il posizionamento mirato di cellule ectopiche per affrontare una serie di domande di biologia dello sviluppo.

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 trapianto di cellule consente la combinazione di genetica e dell&#39;embriologia per generare chimere tessuto specifiche. Questo video dimostra gastrula scena trapianti di cellule che hanno permesso al nostro laboratorio per studiare il ruolo delle popolazioni astrogliali e spunti indicazioni specifiche durante la formazione commessura nel proencefalo.

Tessuto Chimere mirate embrionali: Zebrafish gastrula trapianto di cellule

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 Views.  Smith College. Zebrafish trapianto di cellule consente la combinazione di genetica e dell'embriologia per generare chimere tessuto specifiche. Questo video dimostra gastrula scena trapianti di cellule che hanno permesso al nostro laboratorio per studiare il ruolo delle popolazioni astrogliali e spunti indicazioni specifiche durante la formazione commessura nel proencefalo.

Video: 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.

Il trapianto di midollo renale intero in Zebrafish adulti

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

Ricerca

Biologia

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.

Actina Co-sedimentazione di analisi, per l&#39;Analisi del Binding Protein a F-Actina

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.

Generare embrioni di zebrafish Chimerico da trapianto

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.

Microdissezione dei tessuti oculari Zebrafish embrionali

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 di embrioni Medaka e trapianto di cellule per la generazione di chimere

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 time-lapse imaging di transgenici Zebrafish: Visualizzare cellule staminali della retina Attivato da mirata ablazione delle cellule neuronali

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.

Colture cellulari primarie da Drosophila Gastrula embrioni

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.

Il trapianto di cellule direttamente nel rene di Zebrafish adulti

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.

Quantificare la frequenza di tumori di moltiplicazione celle usando Limitare trapianto di cellule Diluizione in Singenico 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.

Immunocolorazione di Dissected Cuore Zebrafish embrionali

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