Los métodos para la fertilización in vitro y diploides gynogenetic fueron desarrollados para el pez cebra por Charline Walker en el laboratorio de George Streisinger en la Universidad de Oregon en los años 1970 y 1980. El método descrito aquí no ha cambiado desde los protocolos de Charline que están disponibles en su totalidad en el Libro de pez cebra 3. Los métodos para la fertilización in vitro y diploides gynogenetic fueron desarrollados para el pez cebra por Charline Walker en el laboratorio de George Streisinger en la Universidad de Oregon en los años 1970 y 1980. El método descrito aquí no ha cambiado desde los protocolos de Charline que están disponibles en su totalidad en el Libro de pez cebra 3.

Resumen de la presión a principios Nota: las recetas de muchos de los reactivos utilizados en este protocolo, como tricaína, peces de agua y solución salina Hanks están disponibles en línea en el capítulo 10 del Libro de pez cebra 3 ( <a href="http://zfin.org/zf_info/zfbook/zfbk.html">http://zfin.org/zf_info/zfbook/zfbk.html</a> ) <ol><li> Producir embriones por fecundación in vitro con esperma inactivado UV. </li><li> Inmediatamente la transferencia de los óvulos fertilizados en un frasco de vidrio (con un tapón de presión de plástico) lleno hasta el borde con agua del huevo. Tapar el extremo abierto de la ampolla con una fina capa de látex de caucho y encaje la parte superior (en la que un agujero se ha reducido) en el vial sobre el caucho. </li><li> Coloque el vial en una prensa hidráulica y en el 1,4 min después de la fertilización aumentar la presión a 8000 psi (Tenga en cuenta que muchas máquinas están calibradas para leer la presión ejercida sobre un pistón de 2 pulgadas de diámetro, y las lecturas deben ser convertidos a psi) </li><li> A partir de los 6 minutos después de la fecundación, liberar la presión gradualmente durante un período de 1 min. </li></ol> Procedimiento detallado presión inicial <ol><li> Día antes del experimento <ol style="list-style-type:lower-alpha;"><li> Por la tarde, los hombres y mujeres por separado para ser exprimidos. Cualquier hombre de edad se puede utilizar ya que no estará contribuyendo material genético. Las mujeres deben parecer grande y la grasa en el vientre. Los machos deben tener un color amarillento y ágil. </li><li> Mantenga mujeres y hombres por separado durante la noche en los tanques donde se puede acceder fácilmente por la mañana. </li></ol></li><li> Mañana del experimento <ol style="list-style-type:lower-alpha;"><li> Prepare una solución de Hanks, añadir bicarbonato de sodio. </li><li> Llevar a la instalación de los peces: cubo de hielo, Hanks, tricaína, contador de tiempo, los tubos de recogida de esperma. </li><li> Diluir tricaína en peces de agua </li></ol></li><li> Recoger el esperma <ol style="list-style-type:lower-alpha;"><li> Prepare un frasco transparente de la solución de Hanks en el hielo. Medida ~ 50 l por hombres que se apretó. </li><li> Anestesiar a 3 ó 4 hombres a la vez por la inmersión en tricaína. Que se pueden levantar de la anestesia con una cuchara de plástico. </li><li> Enjuague con agua y secar pescado &quot;húmedo-seco&quot; en una toalla de papel (muy importante: el agua de esperma activa). </li><li> Monte de pescado en un soporte de espuma bajo un microscopio estereoscópico a bajo aumento con epi-iluminación. Separar las aletas pélvicas con una pinza y una posición microcapilar en la apertura de la cloaca. </li><li> Accidente cerebrovascular de los lados del pescado con cuidado pero con firmeza, con suave (Millipore) con fórceps. </li><li> Mientras que la esperma lechosa salir del poro genital, que se acumulan en el capilar se utiliza una succión suave. </li><li> La piscina del esperma de varios machos en una solución salina helada de Hank. Esperma de los machos 50-10 es la adecuada para la fecundación de varios cientos de huevos. Esperma en frío Hanks seguirá para fertilizar los óvulos de manera eficiente por varias horas o incluso días. &quot;Ojo&quot; de la concentración de espermatozoides, recogiendo lo suficiente como para hacer una suspensión turbia. </li></ol></li><li> UV inactivar los espermatozoides <ol style="list-style-type:lower-alpha;"><li> Llene el fondo de un plato de Petri de vidrio con hielo. </li><li> Poner un vidrio de reloj en la parte superior del hielo en el plato. </li><li> Con una pipeta Pasteur, transferir el esperma de los tubos de ensayo en el vidrio de reloj. Asegúrese de obtener la mayor cantidad de esperma en la pipeta como sea posible sin asumir las burbujas de aire con ella. Además, no se la burbuja de los espermatozoides en el vidrio de reloj a medida que se les expulsa de la pipeta. Expulsar a los espermatozoides en una capa fina y uniforme sobre vidrio de reloj. DESCARTE esta pipeta. </li><li> Coloque la placa de Petri en una UV reticulante </li><li> Encienda el reticulante durante 2 minutos </li><li> Usando una pipeta limpia, eliminar los espermatozoides desde el vidrio de reloj y transferirlo a un tubo eppendorf marcados esperma UV y el lugar en el hielo. </li></ol></li><li> Recolección de huevos y la fecundación in vitro <ol style="list-style-type:lower-alpha;"><li> Anestesiar a tres o cuatro mujeres en un momento en tricaína. </li><li> Enjuague con agua y secar pescado &quot;húmedo-seco&quot; en una toalla de papel. El exceso de agua se hinchan los huevos y evitar la fecundación. </li><li> Colocar la hembra en un plato de plástico 35 mm de Petri y con el húmedo (no mojado) los dedos, presione suavemente pero con firmeza en el vientre. Si ella está dispuesta a poner sus huevos, van a salir con bastante facilidad. </li><li> Recolectan los huevos con una espátula y volver a la hembra en el agua. Buenos huevos son de color amarillento, transparente, mientras que los huevos que han permanecido en la mujer demasiado tiempo son blancas y acuosas (ver más abajo). Para asegurarse de obtener buenos huevos, que se acumulan durante las primeras 2 horas después de que las luces se encienden en las instalaciones de los peces. Mantenga los huevos cubiertos para prevenir la desecación. </li><li> Añadir 30-50 l de la suspensión de esperma UV a los huevos, mezclar suavemente con la punta de la pipeta. </li><li> Inmediatomente añadir aproximadamente 1 ml de agua y los peces iniciar el temporizador. Los huevos de izquierda a desarrollar en este punto se haploides. </li></ol></li><li> . Presión temprano Nota: los pasos ag debe llevarse a cabo dentro de los 90 segundos de la fertilización! <ol style="list-style-type:lower-alpha;"><li> Iniciar el temporizador en un momento que los huevos son activados por la adición de agua </li><li> Espere unos minutos y agregar más agua, agitar los huevos en el centro del plato. </li><li> Utilizando una línea de corte y pulido pipeta Pasteur, la transferencia de los óvulos fertilizados a los viales de presión. </li><li> Si es necesario, añada más agua de huevos a los viales para que sean casi desbordante, dejando una gota de agua en el borde de los viales. </li><li> Coloque una tapa de goma sobre la gota de agua y asegurar la tapa de plástico sobre la goma. </li><li> Coloque el vial (s) boca abajo en el cilindro de presión, agregando más agua de huevos para volver a llenar el cilindro. Ponga la tapa en el cilindro de forma segura. </li><li> Coloque el cilindro en la prensa con el émbolo y aplicar 8.000 libras / pie cuadrado. in </li><li> Deja la botella en la prensa hasta 6 minutos después de que el momento de la activación (por un tiempo total de 4,5 minutos de presión). </li><li> A los 6 minutos después de la activación, retire el cilindro de la prensa, y retirar los viales del cilindro. Secar cuidadosamente los viales para evitar el enfriamiento de los embriones. </li><li> Restablecer el cilindro y volverlo a llenar con agua los huevos. </li><li> Preparar los viales próximos por parte llenándolos con agua de huevos, según sea necesario. </li><li> Repita este procedimiento para todos los lotes de huevos. </li></ol></li></ol> NOTA: El cilindro con capacidad para 3 viales a la vez, por lo que puede retrasar la fertilización de los huevos unos minutos para ver si otras mujeres que ya están en el tricaína dan huevos. Esto ayudará a acelerar las cosas mediante el tratamiento de varios lotes de una sola vez. Además, recuerda una vez que la prensa se está utilizando no poner todos los peces más en el tricaína hasta que se complete el tratamiento. Permitiendo que los peces para mantenerse en la anestesia mucho tiempo puede provocar la muerte. En segundo lugar, si usted aprieta los peces, mientras que la prensa está siendo utilizado, y obtener los huevos, pueden ser demasiado seca para ser viable por el momento se puede volver a utilizar la prensa.

<ol>
	<li>Streisinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segregation+analyses+and+gene-centromere+distances+in+zebrafish.">Segregation analyses and gene-centromere distances in zebrafish.</a> Genetics. 112 (2), 311-311 (1986).</li><li>Streisinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+clones+of+homozygous+diploid+zebra+fish+(Brachydanio+rerio).">Production of clones of homozygous diploid zebra fish (Brachydanio rerio).</a> Nature. 291 (5813), 293-293 (1981).</li><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> The Zebrafish Book: A guide for the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>Johnson, S. L., Africa, D., Horne, S., Postlethwait, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Half-tetrad+analysis+in+zebrafish:+mapping+the+ros+mutation+and+the+centromere+of+linkage+group+I.">Half-tetrad analysis in zebrafish: mapping the ros mutation and the centromere of linkage group I.</a> Genetics. 139 (4), 1727-1727 (1995).</li><li>Beattie, C. E., Raible, D. W., Henion, P. D., Eisen, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+pressure+screens.">Early pressure screens.</a> Methods Cell Biol. 237 (71), 2575-25 (1999).</li><li>Johnson, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centromere-linkage+analysis+and+consolidation+of+the+zebrafish+genetic+map.">Centromere-linkage analysis and consolidation of the zebrafish genetic map.</a> Genetics. 142 (4), 1277-1277 (1996).</li><li>Trede, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+mutants+with+disrupted+early+T-cell+and+thymus+development+identified+in+early+pressure+screen.">Zebrafish mutants with disrupted early T-cell and thymus development identified in early pressure screen.</a> Dev Dyn. 237 (9), 2575-2575 (1999).</li></ol>

Es importante asegurarse de que los embriones producidos por este método son diploides cierto gynogenetic. Como controles, generar una nidada de huevos diploides normales (al mantener a un lado algunos de los espermatozoides sin UV-inactivación) y un grupo de embriones haploides (por un lado mantener una nidada de huevos fecundados con el esperma de UV, sin pasar por el procedimiento de EP). En un mensaje embriones haploides días de fertilización tienen un eje cuerpo corto, el cerebro y la morfología irregular somi...

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making gynogenetic diploid zebrafish by early pressure

Heterocigosidad en eucariotas diploides a menudo hace que los estudios genéticos engorroso. Métodos que producen descendencia viable diploides homocigóticos directamente de las mujeres heterocigotas permite F1 hembras mutadas que se proyectarán directamente de mutaciones deletéreas en una pantalla acelerado avance genético. Streisinger et al.<sup&gt; 1,2</sup&gt; Describe los métodos para la toma de gynogenetic (homocigotos) pez cebra diploide mediante la activación de los huevos de pez cebra con la luz ultravioleta inactiva los espermatozoides y la prevención sea la segunda meiosis o la primera división celular cigóticos el uso de tratamientos físicos (calor o presión) que los microtúbulos deploymerize. La &quot;presión inicial&quot; (EP) método se bloquea la meiosis II, que se produce poco después de la fecundación. El método EP produce un alto porcentaje de embriones viables que se pueden desarrollar a adultos fértiles de ambos sexos. El método genera embriones que son homocigotos en todos los lugares excepto aquellos que fueron separados de sus centrómero por la recombinación durante la meiosis I. mutaciones homocigóticas se detectan en las garras del Parlamento Europeo entre el 50% de loci centromérica y menos del 1% para los loci telomérica. Este método se reproduce textualmente del Libro de pez cebra<sup&gt; 3</sup&gt;.

Watch this Scientific Journal Video about Making Gynogenetic Diploid Zebrafish by Early Pressure at JoVE.com

Making Gynogenetic Diploid Zebrafish by Early Pressure

Este es un método para la generación de embriones de pez cebra gynogenetic diploide (embriones, cuya única contribución genética proviene de la madre) por el bloqueo de la segunda división meiótica inmediatamente después de la fertilización con luz ultravioleta inactiva los espermatozoides. EP embriones no son totalmente homocigotos debido a la recombinación en la primera división meiótica, sin embargo, son homocigotos en todos los lugares que no han sido separados de sus centrómero por recombinación.

Hacer pez cebra Gynogenetic diploides por presión inicial

Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. Methods that produce viable homozygous diploid offspring directly from ...

making-gynogenetic-diploid-zebrafish-by-early-pressure

Research

JoVE Journal

Biology

13.4K vistas.  University of Oregon. Este es un método para la generación de embriones de pez cebra gynogenetic diploide (embriones, cuya única contribución genética proviene de la madre) por el bloqueo de la segunda división meiótica inmediatamente después de la fertilización con luz ultravioleta inactiva los espermatozoides. EP embriones no son totalmente homocigotos debido a la recombinación en la primera división meiótica, sin embargo, son homocigotos en todos los lugares que no han sido sepa...

Video: Making Gynogenetic Diploid Zebrafish by Early Pressure

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

Imágenes en vivo del cerebro del embrión de pez cebra mediante microscopía confocal

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Investigación

Biología

Brain Banking: Making the Most of your Research Specimens

Unbiased stereology is a method for accurately and efficiently estimating the total neuron number (or other cell type) in a given area of interest1. To achieve this goal 6-10 systematic sections should be probed covering the entire structure. Typically this involves processing 1/5 sections which leaves a significant amount of material unprocessed. In order to maximize the material, we propose an inexpensive method for preserving fixed tissue as part of a long-term storage research plan. As tissue is sliced and processed for the desired stain or antibody, alternate sections should be systematically placed in antigen preserve at -20&deg;C for future processing. Using 24-well plates, sections can be placed in order for future retrieval. Using this method, tissue can be stored and processed for immunohistochemistry over the course of years.

Brain banking and systematic sampling of biological material provides the basis for unbiased stereology and maximizes the potential data obtained from each specimen.

Banca cerebro: Aprovechar al máximo las muestras de su investigación

Unbiased stereology is a method for accurately and efficiently estimating the total neuron number (or other cell type) in a given area of interest1.  To ...

Making MR Imaging Child's Play - Pediatric Neuroimaging Protocol, Guidelines and Procedure

Within the last decade there has been an increase in the use of structural and functional magnetic resonance imaging (fMRI) to investigate the neural basis of human perception, cognition and behavior 1, 2. Moreover, this non-invasive imaging method has grown into a tool for clinicians and researchers to explore typical and atypical brain development. Although advances in neuroimaging tools and techniques are apparent, (f)MRI in young pediatric populations remains relatively infrequent 2. Practical as well as technical challenges when imaging children present clinicians and research teams with a unique set of problems 3, 2. To name just a few, the child participants are challenged by a need for motivation, alertness and cooperation. Anxiety may be an additional factor to be addressed. Researchers or clinicians need to consider time constraints, movement restriction, scanner background noise and unfamiliarity with the MR scanner environment2,4-10. A progressive use of functional and structural neuroimaging in younger age groups, however, could further add to our understanding of brain development. As an example, several research groups are currently working towards early detection of developmental disorders, potentially even before children present associated behavioral characteristics e.g.11. Various strategies and techniques have been reported as a means to ensure comfort and cooperation of young children during neuroimaging sessions. Play therapy 12, behavioral approaches 13, 14,15, 16-18 and simulation 19, the use of mock scanner areas 20,21, basic relaxation 22 and a combination of these techniques 23 have all been shown to improve the participant's compliance and thus MRI data quality. Even more importantly, these strategies have proven to increase the comfort of families and children involved 12. One of the main advances of such techniques for the clinical practice is the possibility of avoiding sedation or general anesthesia (GA) as a way to manage children's compliance during MR imaging sessions 19,20. In the current video report, we present a pediatric neuroimaging protocol with guidelines and procedures that have proven to be successful to date in young children.

Making MR Imaging Child&#039;s Play - Pediatric Neuroimaging Protocol, Guidelines and Procedure

Despite an increase in the use of structural and functional magnetic resonance imaging (fMRI) in humans, the study of young pediatric populations remains a challenge. We present a hands-on, step-by-step video protocol including guidelines for clinicians and researchers intending to perform (f)MRI in young children.

Hacer jugar MR Imaging Niño - Protocolo neuroimagen pediátrica, Directrices y Procedimientos

Within the last decade there has been an increase in the use of structural and functional magnetic resonance imaging (fMRI) to investigate the neural ...

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 ...

Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like experimental conditions. Laser ablation electrospray ionization (LAESI) is one such development and is particularly well-suited for the investigation of water-containing specimens. LAESI utilizes a mid-infrared laser beam (2.94 &mu;m wavelength) to excite the water molecules of the sample. When the ablation fluence threshold is exceeded, the sample material is expelled in the form of particulate matter and these projectiles travel to tens of millimeters above the sample surface. In LAESI, this ablation plume is intercepted by highly charged droplets to capture a fraction of the ejected sample material and convert its chemical constituents into gas-phase ions. A mass spectrometer equipped with an atmospheric-pressure ion source interface is employed to analyze and record the composition of the released ions originating from the probed area (pixel) of the sample. A systematic interrogation over an array of pixels opens a way for molecular imaging in the microprobe analysis mode. A unique aspect of LAESI mass spectrometric imaging is depth profiling that, in combination with lateral imaging, enables three-dimensional (3D) molecular imaging. With current lateral and depth resolutions of ~100 &mu;m and ~40 &mu;m, respectively, LAESI mass spectrometric imaging helps to explore the molecular structure of biological tissues. Herein, we review the major elements of a LAESI system and provide guidelines for a successful imaging experiment.

Laser ablation electrospray ionization (LAESI) is an atmospheric-pressure ion source for mass spectrometry. In the imaging mode, a mid-infrared laser probes the distributions of molecules across a tissue section or a biofilm. This technique presents a new approach for diverse bioanalytical studies carried out under native experimental conditions.

Presión atmosférica de Imagen Molecular de los tejidos biológicos y biopelículas por LAESI Espectrometría de Masas

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like ...

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell types exhibit heterogeneous biochemical makeup depending on their physiological conditions and interactions with the environment. Conventional methods of mass spectrometry (MS) used for the analysis of biomolecules in single cells rely on extensive sample preparation. Removing the cells from their natural environment and extensive sample processing could lead to changes in the cellular composition. Ambient ionization methods enable the analysis of samples in their native environment and without extensive sample preparation.1 The techniques based on the mid infrared (mid-IR) laser ablation of biological materials at 2.94 &mu;m wavelength utilize the sudden excitation of water that results in phase explosion.2 Ambient ionization techniques based on mid-IR laser radiation, such as laser ablation electrospray ionization (LAESI) and atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), have successfully demonstrated the ability to directly analyze water-rich tissues and biofluids at atmospheric pressure.3-11 In LAESI the mid-IR laser ablation plume that mostly consists of neutral particulate matter from the sample coalesces with highly charged electrospray droplets to produce ions. Recently, mid-IR ablation of single cells was performed by delivering the mid-IR radiation through an etched fiber. The plume generated from this ablation was postionized by an electrospray enabling the analysis of diverse metabolites in single cells by LAESI-MS.12 This article describes the detailed protocol for single cell analysis using LAESI-MS. The presented video demonstrates the analysis of a single epidermal cell from the skin of an Allium cepa bulb. The schematic of the system is shown in Figure 1. A representative example of single cell ablation and a LAESI mass spectrum from the cell are provided in Figure 2.

Single cell analysis is performed by mass spectrometry on plant and animal cells at atmospheric pressure by using a sharpened optical fiber to sample the cells for laser ablation electrospray ionization (LAESI) mass spectrometry.

El análisis directo de las células individuales mediante espectrometría de masas a presión atmosférica

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell ...

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharides1, 2. The modified monosaccharides, processed by the glycan biosynthetic machinery, are incorporated into cell surface glycoconjugates. The bioorthogonal azide or alkyne tags then allow covalent conjugation with fluorescent probes for visualization, or with affinity probes for enrichment and glycoproteomic analysis. This protocol describes the procedures typically used for noninvasive imaging of fucosylated glycans in zebrafish embryos, including: 1) microinjection of one-cell stage embryos with GDP-5-alkynylfucose (GDP-FucAl), 2) labeling fucosylated glycans in the enveloping layer of zebrafish embryos with azide-conjugated fluorophores via biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and 3) imaging by confocal microscopy3. The method described here can be readily extended to visualize other classes of glycans, e.g. glycans containing sialic acid4 and N-acetylgalactosamine5, 6, in developing zebrafish and in other living organisms.

A click-chemistry based method that allows for the rapid, noninvasive, and robust labeling of alkyne-tagged glycans in zebrafish embryos is described. Fucosylated glycans in the enveloping layer of zebrafish embryos in the late gastrulation stage were imaged in this study.

Glicanos imágenes en embriones de pez cebra mediante marcado metabólico y Química Haga clic Bioorthogonal

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

En electroporación in vivo de Morpholinos en la retina de pez cebra adultos

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

En Vivo Tracking 4-Dimensional de madre hematopoyéticas y células progenitoras en ratones adultos de calota de Médula Ósea

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity &#8211; a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish is a powerful model system for developmental biology and human disease research due to their genetic similarity with higher vertebrates. This protocol describes a methodology to create haploid zebrafish embryos that can be utilized for forward screen strategies to identify recessive mutations in genes essential for early embryogenesis.

La producción de embriones de pez cebra mediante haploide<em&gt; In Vitro</em&gt; Fertilización

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for ...

Methods Article

Hacer pez cebra Gynogenetic diploides por presión inicial