Wir möchten die Mitglieder des Barresi Lab für ihre Unterstützung und hilfreichen Kommentare zu diesem Manuskript danken. Wir danken Alexander Workman für seine ständige technische Unterstützung sowie die Tierpfleger für ihre Hilfe bei der Aufrechterhaltung der Smith College Zebrafisch Kolonie. Wir danken auch Mike Hallacy, Rudi Rottenfusser und Carl Zeiss MicroImaging für die Kreditvergabe einige Mikroskopie Ausrüstung für die filmin dieses Protokolls. Diese Arbeit wurde durch einen NSF finanzierten Forschung zu gewähren, 0615594 unterstützt.

<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.

Generierung von chimären Embryonen ist ein leistungsstarkes Tool, dass viele Fragestellungen Adressen in mehreren Modellsystemen, nämlich der Fruchtfliege (D.melanogaster), Wurm (C. elegans), Zebrafisch (D.rerio) und Maus (M.musculus). Wir argumentieren, dass der Zebrafisch-Modell-System einzigartig ist zur Erzeugung von Chimären in einer relativ einfach, schnell und vielseitig geeignet. Der Zebrafisch ist ein Wirbeltier, die außerhalb der Mutter macht es deutlich leichter zugänglich als die Maus-Modell im Vergleich entwickelt. Zebrafische sind auch deutlich größer als Fliegen oder Würmern, die embryologische Manipulationen wie Zelltransplantation vereinfacht. Darüber hinaus ermöglicht die Fähigkeit, Zelltransplantation Verfahren mit transgenen Techniken kombinieren eine große Bandbreite von möglichen Experimenten, in denen Gen-Funktion in einer lokalisierten Gewebe-spezifische Art und Weise manipuliert werden kann. Zum Beispiel können kleine Klone von GFP + oder Rhodamin markierten Zellen der Charakterisierung von vollständigen Zellmorphologien, die oft in einer homozygoten transgenen Spender verloren oder sind nicht zu visualisieren durch differenzierte subzelluläre Kennzeichnung mit Common-Antikörper. Darüber hinaus durch transgen markiert oder fluoreszierend gefüllte Zellen, können wir Spenderzellen in die Live-Zebrafischembryo im Laufe der Zeit zu verfolgen. Timelapse Imaging von einzelnen Zellen stellt eine neue Analyse von zellulären Verhalten im Kontext des gesamten Organismus. Unser Labor verbindet auch die Verwendung von Hitzeschock induzierbare transgene Linien mit Zell-Transplantationen, so dass bestimmte Axon Führung Cues in Spenderzellen kann nach einer Erhebung in Inkubationstemperatur (Daten nicht gezeigt) misexpressed werden. Diese Technik ist eine extrem leistungsfähige und direkten Zugang zu lokal misexpress ein Protein von Interesse in einem räumlichen und zeitlichen spezifische Art und Weise, die uns zu sehen, wie das spezifische Protein Entwicklung beeinflusst werden kann. In unserem Fall, kann diese Technik unser Wissen hinter der Funktion der Slit-Robo Führung Cues in der Positionierung der Kommissuren und Gliazellen an der Mittellinie 5 erstrecken. Andere Ansätze zur lokal misexpressing Gene wie fokale UV Uncaging und lokalisierten Hitzeschock-Tools (Laser-oder Lötkolben), nicht bieten alternative Möglichkeiten, um die Expression von Genen und Markern in einer kleinen Gruppe von Zellen 7-9 manipulieren, allerdings stellt Zelle Transplantation ein Endumgebung der Analyse, die frei von Traumata durch UV-, Laser-oder Wärme machen Zelltransplantation einer kontrollierten experimentellen Ansatz induziert wird. Zusammenfassend haben wir Zelltransplantation Verfahren festgestellt, dass ein entscheidender Teil unserer neurologischen Erforschung der Biologie werden. Generieren Klone von Zellen mit unterschiedlichen Eigenschaften ist ein wichtiges Werkzeug für die Entwicklungsbiologe bei der Bewältigung grundlegender Fragen der Zell Verhaltensweisen, Genfunktion und Zelle Autonomie.

<table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Name </td><td> Typ </td><td> Firma </td><td> Catalog Number </td><td> Besonderheiten </td></tr><tr><td> Petrischalen </td><td> Werkzeug </td><td> Fisherbrand </td><td> 08-757-13 </td><td> 100 x 15 mm </td></tr><tr><td> Glasboden Kulturplatten </td><td> Werkzeug </td><td> MatTek </td><td> P35G-1,5-2,0-C </td><td> 35 x 15mm, No 1,5, Uncoated, Gamma-bestrahlt </td></tr><tr><td> Weite Bohrung Glas Pasteur Pipetten </td><td> Werkzeug </td><td> Fisherbrand </td><td> 63A-53-WT </td><td></td></tr><tr><td> Glasfilamentgewebe Kapillaren für Mulde formen (ohne Faden) und Injektionsnadeln (mit Faden) </td><td> Werkzeug </td><td> World Precision Instruments </td><td> 1B150F-4 </td><td> 4 Zoll (100 mm), 1.5 / 0.84 OD / ID (mm) </td></tr><tr><td> Transplant Kapillaren (TransferTip) </td><td> Werkzeug </td><td> Eppendorf </td><td> 83000122-8 </td><td> Typ IV, steril, Int. 15 um, 20-Grad-Kurve </td></tr><tr><td> Transplant Form </td><td> Werkzeug </td><td> Adaptive Science Tools </td><td> PT-1 </td><td> 150 dreieckige Einschläge auf einzelne Embryonen halten </td></tr><tr><td> Agarose, Type I </td><td> Reagens </td><td> Sigma Aldrich </td><td> A6013-250G </td><td> 1,5% Agarose made in EM </td></tr><tr><td> (Antibiotikum) Embryo Medium </td><td> Reagens </td><td></td><td></td><td> Siehe Westerfield, 2007 </td></tr><tr><td> Phosphatpuffer </td><td> Reagens </td><td></td><td></td><td> Siehe Westerfield, 2007 </td></tr><tr><td> Watchman Zange </td><td> Werkzeug </td><td> Feine Science Tools </td><td></td><td> 100 + mm Spitze (matt), 50mm Spitze </td></tr><tr><td> Rhodamin B Dextran </td><td> Lineage Markierungsfarbstoff </td><td> Molecular Probes, Invitrogen </td><td> D1824 </td><td> MW 10.000 Da, Lysin-fixierbare, rot (590nm) Fluoreszenzfarbstoff mit grünem (570 nm) Licht angeregt </td></tr><tr><td> Präpariermikroskop </td><td> Mikroskop </td><td> Olymp </td><td> SZX7 </td><td></td></tr><tr><td> Fluorescent Stereo-Mikroskop </td><td> Mikroskop </td><td> Zeiss </td><td> Lumar </td><td> Vollautomatische Joystick gesteuert (SyCoP) </td></tr><tr><td> Transplant Apparatus </td><td> Werkzeug </td><td> Eppendorf Corp </td><td> Airtram </td><td></td></tr><tr><td> Automatisierte Mikromanipulator </td><td> Werkzeug </td><td> Eppendorf Corp </td><td> TransferMan NK2 </td><td> Joystick gesteuert </td></tr><tr><td> Mikromanipulator </td><td> Werkzeug </td><td> Applied Scientific Instruments, Inc. </td><td> 00-42-101-0000 </td><td> MM33 mit der rechten </td></tr><tr><td> Mikroinjektor mit Back-Pressure-Einheit </td><td> Werkzeug </td><td> Applied Scientific Instruments, Inc. </td><td> MPPI-2 BPU </td><td></td></tr><tr><td> Flamming / Brown Mikropipette Puller </td><td> Werkzeug </td><td> Sutter Instrument Company </td><td> Modell P97 </td><td> Programm: Heat 550; Velocity 200; Zeit / Delay 200; Zugkraft 120. </td></tr></tbody></table>

tissue targeted embryonic chimeras: zebrafish gastrula cell transplantation

Bestimmte grundlegende Fragen auf dem Gebiet der Entwicklungsbiologie kann nur beantwortet werden, wenn die Zellen in neue Umgebungen platziert werden oder wenn kleine Gruppen von Zellen in einem größeren Kontext verändert werden. Beobachten, wie eine Zelle mit interagiert und verhält sich in einer einzigartigen Umgebung ist wesentlich für die Charakterisierung Zellfunktionen. Die Festlegung, wie die lokalisierte misexpression eines spezifischen Proteins beeinflusst umgebenden Zellen aufschlussreiche Auskunft über die Rollen, die Protein spielt in einer Vielzahl von Entwicklungsprozessen. Unser Labor verwendet die Zebrafisch-Modell-System zur eindeutigen kombinieren genetische Ansätze mit den klassischen Techniken der Transplantation genotypische oder phänotypische Chimären zu erzeugen. Wir studieren Neuron-Glia-Zell-Interaktionen während der Bildung des Vorderhirns Kommissuren im Zebrafisch. Dieses Video beschreibt eine Methode, unser Labor, um die Rolle von astroglialen Populationen im Zwischenhirn und die Rolle von spezifischen Leitlinien Hinweise, dass projizieren Axone Einfluss, da sie die Mittellinie überqueren untersucht werden können. Aufgrund ihrer Transparenz Zebrafischembryonen sind ideale Modelle für diese Art von ektopischen Zellen Platzierung oder lokalisierte Gen misexpression. Tracking-transplantierten Zellen kann mit Hilfe einer vitalen Farbstoff oder ein transgener Fische Linie Ausdruck eines fluoreszierenden Proteins werden. Wir zeigen hier, wie Sie Spender Embryonen mit einem vitalen Farbtracer für die Transplantation vorbereitet, und wie zu extrahieren und zu verpflanzen Zellen aus einer Gastrula inszeniert Embryo zu einem anderen. Wir präsentieren Daten, die zeigen ektopische GFP + transgenen Zellen im Vorderhirn von Zebrafisch-Embryonen und charakterisieren die Lage dieser Zellen im Hinblick auf Kommissuren Vorderhirn. Darüber hinaus zeigen wir Laser-Scanning-konfokale Mikroskopie Zeitraffer von Alexa 594 markierte Zellen in einem GFP + transgenen Wirt Embryo verpflanzt. Diese Daten belegen, dass Gastrula inszeniert Transplantation die gezielte Positionierung der ektopischen Zellen ermöglicht, eine Vielzahl von Fragen in der Entwicklungsbiologie Adresse.

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

Zebrafisch-Transplantation ermöglicht die Kombination von Genetik und Embryologie an gewebespezifischen Chimären zu erzeugen. Dieses Video zeigt Gastrula inszeniert zelltransplantationen, dass es unserem Labor auf den Rollen von astroglialen Bevölkerung und spezifische Anleitungen Cues während Kommissur Bildung im Vorderhirn zu untersuchen.

Tissue Gezielte Embryonale Chimären: Zebrafisch Gastrula Cell Transplantation

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

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

Research

JoVE Journal

Biology

19.3K Aufrufe.  Smith College. Zebrafisch-Transplantation ermöglicht die Kombination von Genetik und Embryologie an gewebespezifischen Chimären zu erzeugen. Dieses Video zeigt Gastrula inszeniert zelltransplantationen, dass es unserem Labor auf den Rollen von astroglialen Bevölkerung und spezifische Anleitungen Cues während Kommissur Bildung im Vorderhirn zu untersuchen.

Serie: 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.

Die Transplantation von Whole Kidney Marrow in Adult Zebrafisch

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

Forschung

Biologie

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

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

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

Aktin Co-Sedimentation Assay; für die Analyse von Protein-Bindung an F-Aktin

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

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

Generation of Human CD40-aktivierten B-Zellen

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

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.

Generieren Chimäre Zebrafischembryonen durch Transplantation

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

Microdissection of Zebrafish Embryonic Eye Tissues

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

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

Mikrodissektion von Zebrafisch Embryonale Augentücher

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 von Medaka Embryonen und Zell-Transplantation für die Erzeugung von Chimären

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 Zeitraffer-Imaging transgener Zebrafische: Visualisierung von Retinal Stammzellen durch gezielte Nervenzellen Ablation Activated

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.

Primären Zellkulturen aus Drosophila Gastrula Embryonen

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

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.

Die Quantifizierung der Häufigkeit von Tumor-Ausbreitung Cells Mit Limiting Dilution Cell Transplantation in Syngene Zebrafisch

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.

Immunfärbung von Dissected Zebrafisch embryonale Herz

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