Nos gustaría agradecer a los miembros del Laboratorio de Barresi por su apoyo y sus valiosos comentarios sobre este manuscrito. Damos las gracias al Trabajador Alexander por su asistencia técnica permanente, así como el personal de cuidado de los animales por su ayuda en el mantenimiento de la colonia Smith College de pez cebra. También queremos agradecer a Mike Hallacy, Rottenfusser Rudi, y Microfilm Carl Zeiss para el préstamo algunos equipos de microscopía para la Filmin de este protocolo. Este trabajo fue apoyado por una beca de investigación financiada por 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.

La generación de embriones quiméricos es una poderosa herramienta que se ocupa de muchas cuestiones de investigación en sistemas de varios modelos, a saber, la mosca de la fruta (D.melanogaster), gusano (C. elegans), el pez cebra (D.rerio) y el ratón (M.musculus). Nosotros sostenemos que el sistema de modelo de pez cebra es especialmente adecuado para la generación de quimeras de una manera relativamente fácil, rápido y versátil. El pez cebra es un vertebrado que se desarrolla fuera de la madre por lo que es mucho más accesible en comparación con el modelo de ratón. Pez cebra también son significativamente más grandes que las moscas o los gusanos, lo que simplifica la manipulación embriológico, como el trasplante de células. Además, la capacidad de combinar los procedimientos de trasplante de células con las técnicas transgénicas permite una gran variedad de experiencias posibles en que puede ser manipulado en función de los genes de un tejido-específicos localizados manera. Por ejemplo, pequeños clones de GFP + o rodamina células marcadas permiten la caracterización de la morfología de la célula completa que a menudo se pierden en un donante de transgénicos homocigotos o son imposibles de visualizar debido a un etiquetado diferencial subcelular con los anticuerpos comunes. Además, mediante el uso de células transgénicamente etiquetados o llenado con fluorescencia, podemos rastrear las células del donante en el embrión del pez cebra en vivo a través del tiempo. Timelapse imágenes de células individuales ofrece un nuevo análisis del comportamiento celular en el contexto de todo el organismo. Nuestro laboratorio también se combina el uso de choque térmico inducible líneas transgénicas con trasplantes de células de tal manera que ciertas señales de guiado de los axones pueden ser misexpressed en las células del donante después de una elevación de la temperatura de incubación (datos no muestran). Esta técnica es un método extremadamente poderoso y directo a misexpress localmente una proteína de interés de manera espacial y temporal específico, lo que nos permite ver cómo la proteína específica afecta el desarrollo. En nuestro caso, esta técnica se puede ampliar nuestro conocimiento detrás de la función de las señales de orientación Robo de hendidura en el posicionamiento de las comisuras y las células gliales en la línea media 5. Otros enfoques de genes a nivel local como misexpressing uncaging UV focal y herramientas localizadas choque térmico (láser o soldadura de hierro), no ofrecen alternativas para manipular la expresión de genes y marcadores en un pequeño grupo de células 7-9, sin embargo, el trasplante de células establece un entorno final de análisis que está libre de cualquier trauma ocasionado por los rayos ultravioleta, láser o calor haciendo el trasplante de células de un enfoque experimental más controlada. En conclusión, hemos encontrado los procedimientos de trasplante de células a ser una parte crucial de nuestra investigación en biología del desarrollo neurológico. La generación de clones de células con propiedades diferentes es una herramienta fundamental para el biólogo del desarrollo para hacer frente a cuestiones fundamentales de comportamiento celular, la función del gen, y la autonomía de la célula.

<table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Nombre </td><td> Tipo </td><td> Empresa </td><td> Número de catálogo </td><td> Detalles específicos </td></tr><tr><td> Placas de Petri </td><td> Herramienta </td><td> Fisherbrand </td><td> 08-757-13 </td><td> 100 x 15 mm </td></tr><tr><td> Placas de vidrio fondo la cultura </td><td> Herramienta </td><td> MatTek </td><td> P35G-1.5-2.0-C </td><td> 35 x 15 mm, no 1.5, sin estucar ni recubrir, irradiados con rayos gamma </td></tr><tr><td> De ancho Diámetro pipetas Pasteur de vidrio </td><td> Herramienta </td><td> Fisherbrand </td><td> 63A-53-WT </td><td></td></tr><tr><td> Capilares de vidrio de filamento a través de moldes (sin filamento) y agujas de inyección (con filamento) </td><td> Herramienta </td><td> Mundial de Instrumentos de Precisión </td><td> 1B150F-4 </td><td> 4 pulgadas (100 mm), 1,5 / 0,84 OD / ID (mm) </td></tr><tr><td> Trasplante capilares (TransferTip) </td><td> Herramienta </td><td> Eppendorf </td><td> 83000122-8 </td><td> Tipo IV, Int. estéril. 15μm, 20 doble grado </td></tr><tr><td> Trasplante de molde </td><td> Herramienta </td><td> Herramientas de adaptación Ciencia </td><td> PT-1 </td><td> 150 chuletas triangular para mantener los embriones individuales </td></tr><tr><td> Agarosa, Tipo I </td><td> Reactivo </td><td> Sigma Aldrich </td><td> A6013-250G </td><td> Agarosa al 1,5% hecha en EM </td></tr><tr><td> (Antibióticos) de embriones medio </td><td> Reactivo </td><td></td><td></td><td> Ver Westerfield, 2007 </td></tr><tr><td> Tampón fosfato </td><td> Reactivo </td><td></td><td></td><td> Ver Westerfield, 2007 </td></tr><tr><td> Pinzas vigilante </td><td> Herramienta </td><td> Herramientas de Bellas Ciencia </td><td></td><td> 100 + mm punta (sordo), punta de 50 mm </td></tr><tr><td> Rodamina B Dextran </td><td> Lineage trazador de tinta </td><td> Molecular Probes, Invitrogen </td><td> D1824 </td><td> Da 10.000 MW, la lisina puede arreglar, rojo (590nm) tinte fluorescente excitado con el verde (570 nm) la luz </td></tr><tr><td> Microscopio de disección </td><td> Microscopio </td><td> Olimpo </td><td> SZX7 </td><td></td></tr><tr><td> Fluorescente Microscopio Estéreo </td><td> Microscopio </td><td> Zeiss </td><td> Lumar </td><td> Completamente automatizado Joystick Controlados (Sycop) </td></tr><tr><td> Trasplante de aparato </td><td> Herramienta </td><td> Eppendorf Corp. </td><td> Airtram </td><td></td></tr><tr><td> Micromanipulador automatizado </td><td> Herramienta </td><td> Eppendorf Corp. </td><td> TransferMan NK2 </td><td> Joystick controlada </td></tr><tr><td> Micromanipulador </td><td> Herramienta </td><td> Aplicada Scientific Instruments, Inc. </td><td> 00-42-101-0000 </td><td> MM33 Derecho </td></tr><tr><td> Microinyector con la Unidad de contrapresión </td><td> Herramienta </td><td> Aplicada Scientific Instruments, Inc. </td><td> MPPI-2 BPU </td><td></td></tr><tr><td> Flamming / Brown micropipeta Extractor </td><td> Herramienta </td><td> Sutter Instrument Company </td><td> Modelo P97 </td><td> Programa: Heat 550; Velocidad de 200; Tiempo / Delay 200; Tire de la fuerza 120. </td></tr></tbody></table>

tissue targeted embryonic chimeras: zebrafish gastrula cell transplantation

Algunas cuestiones fundamentales en el campo de la biología del desarrollo sólo pueden ser respondidas cuando las células se colocan en entornos nuevos o cuando pequeños grupos de células en un contexto más amplio se han modificado. Ver cómo una célula interactúa y se comporta en un entorno único es esencial para la caracterización de las funciones celulares. Determinar cómo el misexpression localizada de una proteína específica que influye en torno a las células proporciona información detallada sobre el papel que juega la proteína en una variedad de procesos de desarrollo. Nuestro laboratorio utiliza el sistema de modelo de pez cebra para combinar exclusivamente con métodos genéticos clásicos técnicas de trasplante para generar quimeras genotípicos o fenotípicos. Se estudian las neuronas gliales interacciones celulares durante la formación de las comisuras del cerebro anterior en el pez cebra. Este video describe un método que permite a nuestro laboratorio para investigar el papel de las poblaciones astroglial en el diencéfalo y el papel de las señales de orientación específicos que influyen en los axones proyectan al cruzar la línea media. Debido a su transparencia embriones de pez cebra son un modelo ideal para este tipo de colocación de células ectópico o misexpression gen localizado. Seguimiento de las células trasplantadas se puede lograr usando un colorante vital o una línea de peces transgénicos que expresan una proteína fluorescente. Estamos aquí demostrar cómo preparar embriones de donantes con un trazador de tinta vitales para el trasplante, así como la extracción y trasplante de células de un embrión en etapas gástrula a otro. Se presentan datos que muestran ectópico GFP + células transgénicas en el cerebro anterior de embriones de pez cebra y caracterizar la ubicación de estas células con respecto al cerebro anterior comisuras. Además, show de rayos láser microscopía confocal timelapse de Alexa 594 células marcadas trasplantadas a un GFP + embriones transgénicos de acogida. Estos datos proporcionan evidencia de que la gástrula etapas de trasplante permite el posicionamiento específico de las células ectópico para hacer frente a una serie de preguntas de Biología del Desarrollo.

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

Pez cebra trasplante de células permite la combinación de la genética y la embriología para generar quimeras tejido específico. Este vídeo demuestra gástrula etapas trasplantes de células que han permitido a nuestro laboratorio para investigar el papel de las poblaciones de astro y señales específicas de orientación durante la formación de la comisura del cerebro anterior.

Tejido diana quimeras embrionarias: pez cebra Gástrula Trasplante de células

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

tissue-targeted-embryonic-chimeras-zebrafish-gastrula-cell

Research

JoVE Journal

Biology

19.2K vistas.  Smith College. Pez cebra trasplante de células permite la combinación de la genética y la embriología para generar quimeras tejido específico. Este vídeo demuestra gástrula etapas trasplantes de células que han permitido a nuestro laboratorio para investigar el papel de las poblaciones de astro y señales específicas de orientación durante la formación de la comisura del cerebro anterior.

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.

El trasplante de médula del riñón entero en adultos de pez cebra

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

Investigación

Biología

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-sedimentación de ensayo, pues el análisis de proteína de unión a la 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.

La generación de embriones quiméricos de pez cebra mediante un trasplante

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.

Microdisección de los tejidos del ojo de pez cebra embrionarias

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 de embriones de medaka y trasplante de células para la generación de quimeras

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 de imágenes de pez cebra transgénico: Visualización de células madre retinianas activado por la ablación selectiva de células neuronales

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.

Cultivos de células primarias de Drosophila Gástrula embriones

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.

El trasplante de células directamente en el riñón de adultos de pez cebra

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.

Cuantificar la frecuencia de tumores se propagan las células usando trasplante de células limitación de dilución en el pez cebra singénico

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.

Inmunotinción de Disección del corazón embrionario de pez cebra

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