Diese Arbeit wurde von der Deutschen Forschungsgemeinschaft (DFG) unterstützt wird, wollen wir Ayele Tsedeke Taddese für technische Unterstützung danken.

1. Herstellung der Glas-Nadeln (Abbildung 1) <ol><li> Verwenden Glaskapillaren mit einem Durchmesser von 0,75 mm bis Injektionsnadeln (Abbildung 1A) vorzubereiten. </li><li> Legen Sie die Glaskapillare in eine Nadel Zieher und ziehen Sie die Nadel mit dem folgenden Parameter: Heizung Zykluswert: 463; Ziehen Zykluswert: 230; Geschwindigkeit: 150 ms; Zeit: 150 ms (Fig. 1B). </li><li> Brechen Sie das Glas gezogen Kapillare mit Uhrmacher-Pinzette, eine Nadel Durchmesser 20 um unter einem Stereoskop mit einem Mikrometer-Okular zu produzieren. </li><li> Verwenden Sie eine Drehbank mit einem angefeuchteten Gummi Spinnrad, um die Nadel zu schärfen und produzieren ein 20 um Fase (1C und 1D). Hinweis: Sharp, abgeschrägten Nadeln Verbesserung der Leichtigkeit des Einsetzens in die Herzkammer und damit Minimierung der Schädigung des Gewebes und damit die Loch Muskel nach dem Entfernen der Nadel (Fig. 1E-G) wieder zu verschließen. </li></ol> 2. Preparation der Morpholino-Lösung <ol><li> Bereiten Sie die Morpholino Lager durch Auflösen des lyophilisierten Morpholino in 1x Phosphat-gepufferte Kochsalzlösung (PBS) in einer Endkonzentration von 7,5 mM. [Siehe Anweisungen des Herstellers für Details (Material Tabelle)]. </li><li> Bereiten Sie die Morpholino Injektionslösung durch Mischen von 2,5 ul Morpholin-Stammlösung (7,5 mM) mit 2,8 ul Endo-Porter-Stammlösung (1 mM) (Siehe Tabelle Materialien) für eine endgültige Konzentration von 3,5 mM und 0,5 mM Morpholino Endo-Porter. </li></ol> 3. Herstellung der Morpholino-Injektion (Fig. 2) <ol><li> Laden Sie die abgeschrägte Glas Nadel mit 5 ul dieser Lösung mit einer Mikropipette mit einem 10 ul Microloader Pipettenspitze. </li><li> Einfügen der Glas Nadel in den Nadelhalter des Mikromanipulators mit dem pneumatischen pico Pumpe (Fig. 2A-C) verbunden ist. </li><li> Platzieren des Nadelhalters neben dem Mikroskop, so daß die Nadel nur Bedürfnissenin einer planaren Richtung bewegt werden, um in die Herzkammer der Larve (2A) einzufügen. </li><li> Einstellen des Winkels für die Injektionen bei 45 °. </li><li> Stellen Sie die Mikroinjektor Werte wie folgt: Haltedruck: 20 Pfund pro Quadratzoll (psi); Ausstoßdruck: 15 psi; 100 ms Bereich von Gating, Periodenwert von 1,9 (entspricht 10,9 ms). </li><li> Schmelz Agarose in 1x PBS unter Verwendung eines Standard-Mikrowellen zu 20 ml einer 1,5% (w / v) Gel zu erzeugen. Gießen geschmolzenen Gel in eine 10 cm-Petrischale. Legen Sie eine gerillte Form der Injektion in die warme Agarose, so dass einmal ausgehärtet, wird die Agarose-Furchen, in denen die Larve sediert platziert werden 11 (Figuren 2D und 2E) haben. </li></ol> 4. Injektionen (Fig. 3) <ol><li> Anesthetize Larve in 100 ml Aquarienwasser mit 20 mg / L Tricaine (L-Ethyl-m-Amino-benzoat-Methan-Sulfonat), bis sie reagiert auf Berührung. </li><li> Mit einem Kunststoff Pasteur pipette, um die Larve sediert transferieren in eine Nut des nassen Agarose-Form, so dass die Bauchseite zugewandt ist gegen die vertikale Agarose Wand der Nut (3A). </li><li> Platzieren der Agarose Platte unter dem Stereomikroskop, so daß die Herzkammer abgewandt ist die Injektionsnadel (Fig. 3A). </li><li> Senken Sie die Nadel, um sie in der Herzkammer einfügen. Führen Sie die Nadel nur 1-2 um in die Herzkammer kümmert sich nicht um die Nadel zu tief (3B und 3C) einfügen. </li><li> Sobald die Nadel eingeführt ist, spritzen die Morpholino-Lösung in die Kammer mit 4-6 Pulse, jeder, die 3 nl der Lösung mit Warteintervalle Lichtung des Herzens (Fig. 3D und 3E) zu ermöglichen. </li><li> Nach der Injektion die Nadel entfernen (parallel zur Ebene der Insertion) und dann sorgfältig übertragen die Larven mit einem Kunststoff-Pasteur-Pipette mit E3 Medium gefüllt back in eine Petrischale mit frischen E3-Medium. </li><li> Platzieren der Nadel in eine Petrischale mit 1x PBS, um das Trocknen der Nadel zu verhindern, wenn die Übertragung der Larven injiziert. </li><li> Wiederholen Sie die Schritte 4.1-4.6 jede 12-24 h für die Dauer des Experiments. Hinweis: Wiederholen der Injektionen gewährleistet die Aufnahme und Aufrechterhaltung der Morpholino in Zellen. </li></ol> 5. Analysen Injektionen (Fig. 4) <ol><li> Anesthetize die Fische im Aquarium 100 ml Wasser mit 20 mg / L Tricaine, bis sie reagiert auf Berührung. </li><li> Legen Sie die Larve seitlich auf einer flachen nassen 1,5% (w / v) Agarose-Platte mit E3 Medium bedeckt. </li><li> Bild Larven mit einem Stereomikroskop mit Hellfeld-und Fluoreszenzbildgebung. Hinweis: Da die Larven durchsichtig sind, sollte der Fluorescein-markierten Morpholino als fluoreszierende grün in den Blutgefäßen im gesamten Tier direkt nach der Injektion sichtbar sein. Diese Fluoreszenz wird weiter in die vaskularisierten Geweben zerstreuen durch15 min (in den 4A-4C). </li></ol> 6. Beurteilung der Regenerative Outgrowth <ol><li> Bewerten Sie die aufgenommenen Bilder mit Fiji Bild J freie Software (http://fiji.sc/Fiji). Zur Untersuchung der Regeneration, verwenden Sie die Line-Tracing-Tool, um die Menge der regenerativen Wachstum, das auf Kontrolle und Morpholino-injizierten Larve tretenen bestimmen. </li><li> Um Bilder zu kalibrieren, öffnen Sie eine der Original-Bilddateien in Fidschi durch Drag &amp; Drop die Datei in das Hauptfenster Fidschi. </li><li> Um den Maßstab für alle folgenden Bilder einzustellen, gehen Sie auf &quot;Analysieren&quot; im Hauptmenü. </li><li> In der Drop-Down-Menü klicken Sie auf &quot;Set scale&quot;. </li><li> In diesem Fenster aktivieren Sie die Option &quot;Global&quot;, indem Sie das Kontrollkästchen. </li><li> Nun öffnen Sie das Bild, um die Menge der regenerativen Wachstum wieder per Drag &amp; Drop (siehe Schritt 6.1) zu messen. </li><li> Wählen Sie die &quot;Freihand-Auswahl&quot;-Werkzeug in der Werkzeugleiste. </li><li> Umkreisen Sie die zu messenden Bereich (Figures 4J-4L). </li><li> Drücken Sie Strg + M. Damit wird ein neues Fenster &quot;Ergebnisse&quot;, die den Wert des eingekreisten Bereichs in quadratischen Pixeln zu öffnen. </li></ol>

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Die Autoren haben nichts zu offenbaren.

Die intraventrikuläre Injektion bietet eine schnelle und zuverlässige Beurteilung Verfahren zur Prüfung der Genfunktion in späteren Phasen der Entwicklung oder der Homöostase des Körpers, ohne die Gen-Funktion während der Embryogenese. Um den Erfolg dieser Technik zu gewährleisten, sollte man sich bewusst sein, vier kritische Punkte: 1) Nadel, 2) Trocknen aus der Nadel, 3) zu minimieren Volumen und 4) minimale Belichtungszeit in der Sedierung Lösung. Nadeln, die zu klein sind, werden häufig verstopfen, währen...

Regeneration von Organen und Anhängsel ist grundlegend wichtig für das Überleben und Fitness; jedoch haben mehrere Wirbeltieren einschließlich des Menschen regenerative Fähigkeiten beschränkt. Während mehreren Tiermodellen existieren, die umfangreichen Regenerationsfähigkeit haben, Reverse genetische Techniken, um Gen-Funktion während der Organregeneration und Anhängsel zu beurteilen sind sehr begrenzt oder gar nicht vorhanden. Daher sind neue Methoden erforderlich, um die Molekularbiologie der Regeneration in diesen Modellorganismen zu sezieren. Der Zebrafisch ist ein gut etabliertes Modell für das Verständnis der Zell-und Molekularbiologie der Organregeneration und ein Anhängsel, nicht nur wegen ihrer großen Fähigkeit, mehrere Organe, Gewebe und Anhängsel zu regenerieren, sondern auch, weil mehrere transgene Fische Linien existieren, um Zellen zu verfolgen und Gen-Konstrukte überexprimieren 2, 3. Allerdings ist Gen-Hemmung in Zebrafisch-Larven hauptsächlich auf die overexpre begrenztssion der dominant-negative Konstrukte, die nicht für alle Gene von Interesse sind oder deren Transgen Produkt kann gain-of-function-Effekte, die die endogene Aktivität des Gens spiegeln nicht erwerben. Somit wird eine alternative Methode, um speziell zu entfernen Genexpression durch KO oder Zuschlags notwendig, um diese Probleme zu überwinden. Gen-spezifischen Targeting mit TALENS als Reverse genetische Mittel, um Gen-Knockout-Funktion besteht; Dies ist jedoch Knockout Strategie während der frühen Embryogenese sehr häufig zu funktionellen Beurteilungen, da ursprünglichen Anforderungen des Gens zu verhindern weiteren Verlauf der Embryonalentwicklung. So, das Studium später Phänomene wie Regeneration oder Organ Homöostase nach der Entwicklung mit TALENS ausgeschlossen 4, 5. Daher ist eine alternative Strategie zum Entfernen Gen benötigt, die Gen-Funktion richtet sich nach der frühen Entwicklung bis zur Gen-Anforderungen voll ausgebildete Organe und Strukturen zu beurteilen. </p&gt; Morpholino-Injektion wurde als wirksam bei der Targeting-Gene in einigen adulten Organen und der Erwachsenen Regenerieren Rippen 6-8, aber diese Methoden erfordern Elektroporation und viele innere Organe schwer zu elektroporieren entweder aufgrund ihrer Lage oder wegen ihrer Empfindlichkeit gegenüber elektrischen Störung. Darüber hinaus sind einige Gewebe in der Larve schwer direkt zu injizieren, da eine direkte Injektion kann ihre strukturelle Integrität stören oder weil ihre Größe ist die Begrenzung. Die Schwanzflosse der Larve ist eine solche Struktur, da eine direkte Injektion in die finfold ist nicht möglich. Somit eine Alternative zur Elektroporation und direkte Injektion erforderlich war, um Gene in Geweben, die entweder zu klein oder zu injizieren kann elektroporiert werden Ziel. Um während der Regeneration des Larvenschwanzflosse Ziel und hemmen die Funktion von spezifischen Genen haben wir Morpholino bestehenden Technologien erlauben die eine modifizierteEURTEILUNG der Gen-Funktion während der Schwanzflosse Regeneration in der späten inszeniert Larve. Dieses Verfahren verwendet intraventrikuläre Liefer 9 von Fluorescein-markierten morpholinos zusammen mit Endo-Porter Transfektionsreagenz 10. Sobald im Ventrikel, schnell der Morpholino-Endo-Porter Mischung während der Larve über das Gefäßsystem verteilt und tritt Gewebe, die bisher nicht zum Ziel gewesen. Diese Injektionsverfahren kann modifiziert werden, um Zielgene in spezifischen Geweben und möglicherweise auch in anderen Tiermodellen, die gegenwärtig nicht reversen genetischen Methoden Genfunktion hemmen angewendet werden. So bietet es eine schnelle und einfache Methode mit dem Potenzial für eine breite Palette verwenden, um Gen-Funktion sofort studieren während der allgemeinen Organ-Homöostase und Regeneration bei Larvenstadien.

<table><tbody><tr><td>Morpholino with 3&amp;acute;fluorescein tag</td><td>Gene Tools Inc.</td><td>Customized</td><td>More information at www.gene-tools.com</td></tr><tr><td>Endo-Porter</td><td>Gene Tools Inc.</td><td></td><td>More information at www.gene-tools.com</td></tr><tr><td>Agarose</td><td>Serva</td><td>120623</td><td>For pouring plates to place the fish during injections and imaging</td></tr><tr><td>Tricaine (L-Ethyl-m-amino-benzate-methane sulfonate)</td><td>Applichem</td><td></td><td>For anesthesia</td></tr><tr><td>E3 medium (5 mM NaCl; 0.17 mM KCl; 0.33 mM CaCl&lt;sub&gt;2&lt;/sub&gt;; 0.33 mM MgSO&lt;sub&gt;4&lt;/sub&gt;)</td><td></td><td></td><td>For larval husbandry</td></tr><tr><td>Petri Dishes</td><td>Sarstedt</td><td>821.472</td><td>For placing the fish during injections and imaging</td></tr><tr><td>Thin Wall Glass Capillaries</td><td>World Precision Instruments, Inc.</td><td>TW100F-3</td><td>For preparing injection needles</td></tr><tr><td>Microloader pipette tips</td><td>Eppendorf</td><td>5,242,956,003</td><td>For loading the needle</td></tr><tr><td>Pasteur Pipettes (3 ml/1 ml)</td><td>Brand</td><td>747760/ 74775</td><td>For transfering larva</td></tr><tr><td>Flaming/Brown needle puller</td><td>Sutter</td><td></td><td>More information at www.sutter.com</td></tr><tr><td>Watchmaker (Dumont) Tweezers (size 5)</td><td>World Precision Instruments, Inc.</td><td>501985</td><td>To brake the needle before sharpening</td></tr><tr><td>Dissecting microscope</td><td>Olympus, Leica, Zeiss</td><td>Varies with the manufacturer</td><td>Part of the whole injection setup</td></tr><tr><td>Fluorescence camera</td><td>Olympus, Leica, Zeiss</td><td>Varies with the manufacturer</td><td>To image the fluorescence after injeciton</td></tr><tr><td>PicoNozzle kit (microinjector holder)</td><td>World Precision Instruments</td><td>5430-12</td><td>For microinjections</td></tr><tr><td>Pneumatic PicoPump (microinjector)</td><td>World Precision Instruments</td><td>SYS-PV820</td><td>For microinjections</td></tr></tbody></table>

reverse genetic morpholino approach using cardiac ventricular injection to transfect multiple difficult-to-target tissues in the zebrafish larva

Der Zebrafisch ist ein wichtiges Modell, um die Zell-und Molekularbiologie der Organ-und Anhängsel Regeneration zu verstehen. Allerdings molekularen Strategien zur reversen Genetik beschäftigen haben noch nicht ausreichend entwickelt, um Gen-Funktion bei der Regeneration oder Gewebe-Homöostase während der Larvenstadien nach Zebrafischembryogenese zu bewerten und mehrere Gewebe innerhalb des Zebrafisch-Larve sind schwer zu zielen. Intraventrikuläre Injektion von genspezifischen Morpholinos bieten ein alternatives Verfahren für die aktuelle Unfähigkeit genomisch Zielzebrafisch-Gene in einer zeitlich gesteuerten Weise in diesen Phasen. Dieses Verfahren ermöglicht eine vollständige Dispersion und anschließende Einarbeitung des Morpholino in verschiedenen Geweben im ganzen Körper, einschließlich Strukturen, die bisher unmöglich zu erreichen, wie sie im Larvenschwanzflosse, eine Struktur oft verwendet, um nicht-invasiv zu erforschen Geweberegeneration waren. Mehrere Gene während der Larven finfold Regeneration aktiviert werden auch Präsentationt bei der Regeneration von Geweben erwachsener Wirbeltiere, so dass die Larve ist ein nützliches Modell, um die Regeneration bei Erwachsenen zu verstehen. Diese Morpholino Dispersionsmethode erlaubt die schnelle und einfache Identifizierung von Genen, die für die Regeneration von Gewebe Larven sowie anderen physiologischen Erscheinungen regulieren Gewebshomöostase nach Embryogenese erforderlich. Daher stellt diese Methode ein Liefer aktuell benötigten Strategie zur zeitlichen Steuerung der Auswertung von Genfunktionen nach Embryogenese.

Die scharfe, abgeschrägte Injektionsnadel wird einfach in die Herzkammer des Zebrafisch-Larve platziert, wenn dorsal angefahren (3A). Das Herz pumpt weiter und trotz der Anwesenheit der Nadel (3B und 3C) Blutstrom aufrechterhalten wird. Vorsichtig Injektionen nicht stören die Morphologie der Herzkammer oder der Herzkontraktionen (Fig. 3D), trotz der Injektion des Morpholino in das Herz (Fig. 3E). Innerhalb...

Watch this Scientific Journal Video about Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva at JoVE.com

Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva

Ein anpassungsfähiges reversen genetischen Methode für Zebrafisch-Gen-Funktion während der späteren Phasen der Entwicklung und physiologischen Homöostase wie Geweberegeneration unter Verwendung intraventrikuläre Injektion von Gen-spezifischen morpholinos beurteilen.

Rückwärts Genetische Morpholino Ansatz mit Herz ventrikuläre Injektion auf mehrere Schwer zu Zielgeweben in der Zebrafisch-Larve Transfektion

The zebrafish is an important model to understand the cell and molecular biology of organ and appendage regeneration. However, molecular strategies to ...

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Biology

10.5K Aufrufe.  Technische Universität Dresden.  Ein anpassungsfähiges reversen genetischen Methode für Zebrafisch-Gen-Funktion während der späteren Phasen der Entwicklung und physiologischen Homöostase wie Geweberegeneration unter Verwendung intraventrikuläre Injektion von Gen-spezifischen morpholinos beurteilen. 

Serie: Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

Analyse von Zebrabärbling Nierenentwicklung mit Zeitraffer-Imaging einem Binokular Mit Ausgerüstet für Optische Schnitte

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

Forschung

Entwicklungsbiologie

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments.
In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

In - vivo - Bildgebung von Expression Transgene Gene in einzelnen Retinal Vorläufern in Chimäre Zebrabärblingembryonen Studie Zellnichtautonome Einflüsse

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.

Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.

Mikroinjektion von mRNA und Morpholino Antisense Oligonukleotide in Zebrafisch-Embryonen.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression ...

Biologie

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

Visualisierung von zellulären elektrische Aktivität im frühen Zebrafish Embryos und Tumoren

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

Manipulation of gene expression in tissues is required to perform functional studies. In this paper, we demonstrate the cerebroventricular microinjection (CVMI) technique as a means to modulate gene expression in the adult zebrafish brain. By using CVMI, substances can be administered into the cerebroventricular fluid and be thoroughly distributed along the rostrocaudal axis of the brain. We particularly focus on the use of antisense morpholino oligonucleotides, which are potent tools for knocking down gene expression in vivo. In our method, when applied, morpholino molecules are taken up by the cells lining the ventricular surface. These cells include the radial glial cells, which act as neurogenic progenitors. Therefore, knocking down gene expression in the radial glial cells is of utmost importance to analyze the widespread neurogenesis response in zebrafish, and also would provide insight into how vertebrates could sustain adult neurogenesis response. Such an understanding would also help the efforts for clinical applications in human neurodegenerative disorders and central nervous system regeneration. Thus, we present the cerebroventricular microinjection method as a quick and efficient way to alter gene expression and neurogenesis response in the adult zebrafish forebrain. We also provide troubleshooting tips and other useful information on how to carry out the CVMI procedure.

In this article, we demonstrate a method for manipulation of gene expression in the ventricular cells of the adult zebrafish telencephalon using antisense morpholino oligonucleotides. We present this method as an efficient and quick protocol that can be used for functional studies in the adult vertebrate brain.

Mikromanipulation der Genexpression in der Erotik-Zebrafischgehirn Mit cerebroventricular Mikroinjektion von Morpholino Oligonukleotiden

Manipulation of gene expression in tissues is  required to perform functional studies. In this paper, we demonstrate the  cerebroventricular ...

Neurowissenschaften

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In the zebrafish model, effective reverse genetic techniques have been developed using microinjection of gene-specific antisense morpholinos. Morpholinos target an mRNA through specific base-pairing and block gene function transiently by inhibiting translation or splicing for several days during embryogenesis (knockdown). However, in vertebrates such as mouse or zebrafish, some gene functions can be obscured by these approaches due to the presence of another gene that compensates for the loss. This is especially true for gene families containing sister genes that are co-expressed in the same developing tissues. In zebrafish, functional compensation can be tested in a relatively high-throughput manner, by co-injection of morpholinos that target knockdown of both genes simultaneously. Likewise, using morpholinos, a genetic interaction between any two genes can be demonstrated by knockdown of both genes together at sub-threshold levels. For example, morpholinos can be titrated such that neither individual knockdown generates a phenotype. If, under these conditions, co-injection of both morpholinos causes a phenotype, a genetic interaction is shown. Here we demonstrate how to show functional redundancy in the context of two related GATA transcription factors. GATA factors are essential for specification of cardiac progenitors, but this is revealed only by the loss of both Gata5 and Gata6. We show how to carry out microinjection experiments, validate the morpholinos, and evaluate the compensated phenotype for cardiogenesis.

Gene function can be obscured in loss-of-function experiments if there is compensation by another gene. The zebrafish model provides a relatively high-throughput means to reveal such functional redundancy in living embryos.

Ein Reverse Genetic Ansatz zur funktionellen Redundanz während der Embryogenese-Test

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In ...

Design and Microinjection of Morpholino Antisense Oligonucleotides and mRNA into Zebrafish Embryos to Elucidate Specific Gene Function in Heart Development

The morpholino oligomer-based knockdown system has been used to identify the function of various gene products through loss or reduced expression. Morpholinos (MOs) have the advantage in biological stability over DNA oligos because they are not susceptible to enzymatic degradation. For optimal effectiveness, MOs are injected into 1-4 cell stage embryos. The temporal efficacy of knockdown is variable, but MOs are believed to lose their effects due to dilution eventually. Morpholino dilution and injection amount should be closely controlled to minimize the occurrence of off-target effects while maintaining on-target efficacy. Additional complementary tools, such as CRISPR/Cas9 should be performed against the target gene of interest to generate mutant lines and to confirm the morphant phenotype with these lines. This article will demonstrate how to design, prepare, and microinject a translation-blocking morpholino against hand2 into the yolk of 1-4 cell stage zebrafish embryos to knockdown hand2 function and rescue these "morphants" by co-injection of mRNA encoding the corresponding cDNA. Subsequently, the efficacy of the morpholino microinjections is assessed by first verifying the presence of morpholino in the yolk (co-injected with phenol red) and then by phenotypic analysis. Moreover, cardiac functional analysis to test for knockdown efficacy will be discussed. Finally, assessing the effect of morpholino-induced blockage of gene translation via western blotting will be explained.

The present protocol describes designing, preparing, and microinjecting a translational-blocking morpholino against a representative cardiac gene; Heart And Neural Crest Derivatives Expressed2 (hand2) into the yolk of newly fertilized zebrafish embryos to knock down gene function. It also shows a transient rescue of these "morphants" by co-injection of mRNA encoding this gene product.

The morpholino oligomer-based knockdown system has been used to identify the function of various gene products through loss or reduced expression.  ...

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.

In-vivo-Elektroporation von Morpholinos in die Adult Zebrafisch Retina

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

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

In-vivo-Elektroporation von Morpholinos in das Regenerations Adult Zebrafisch Leitwerk

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Gene Silencing with Morpholinos

Morpholino-mediated gene silencing is a common technique used to study roles of specific genes during development. Morpholinos inhibit gene expression by hybridizing to complementary mRNAs. Due to their unique chemistry, morpholinos are easy to produce and store, which makes them remarkably cost effective compared to other gene silencing methods.
This video reviews proper experimental design when using these oligonucleotides. Following that, an explanation of morpholino microinjection techniques in zebrafish and the analysis of resulting phenotypes will be discussed. Finally, we showcase examples of specific applications where morpholino technology is used to model developmental disorders or to study tissue regeneration.

Morpholino Technology: Gene Silencing in Zebrafish

Gen-Silencing mit Morpholinos

Morpholino-mediated gene silencing is a common technique used to study roles of specific genes during development. Morpholinos inhibit gene expression by ...