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.

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		<col />
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:171px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Reagent/Material</td>
			<td style="width:171px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Morpholino with 3&#180;fluorescein tag</td>
			<td style="width:171px;">Gene Tools Inc.&#160;</td>
			<td style="width:119px;">Customized</td>
			<td style="width:167px;">More information on www.genetools.com</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Endo-Porter</td>
			<td style="width:171px;">Gene Tools Inc.&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">More information on www.genetools.com</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Agarose</td>
			<td style="width:171px;">Serva</td>
			<td style="width:119px;">120623</td>
			<td style="width:167px;">For pouring plates to place the fish during injections and imaging</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tricane (L-Ethyl-m-amino-benzate-methane sulfonate)</td>
			<td style="width:171px;">Applichem</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">For anesthesia</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">E3 medium (5 mM NaCl; 0.17 mM KCl; 0.33 mM CaCl2; 0.33 mM MgSo4)</td>
			<td style="width:171px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">For larval husbandry</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Petri Dishes</td>
			<td style="width:171px;">Sarstedt</td>
			<td style="width:119px;">821.472</td>
			<td style="width:167px;">For placing the fish during injections and imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#160;Equipment</td>
			<td style="width:171px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Thin Wall Glass Capillaries</td>
			<td style="width:171px;">World Precision Instruments, Inc.&#160;</td>
			<td style="width:119px;">TW100F-3</td>
			<td style="width:167px;">For preparing injection needles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microloader pipette tips</td>
			<td style="width:171px;">Eppendorf</td>
			<td style="width:119px;">5,242,956,003</td>
			<td style="width:167px;">For loading the needle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pasteur Pipettes (3 ml/1 ml)</td>
			<td style="width:171px;">Brand</td>
			<td style="width:119px;">747760/ 74775</td>
			<td style="width:167px;">For transfering larva</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Flaming / Brown&#160; needle puller&#160;</td>
			<td style="width:171px;">Sutter</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">More information on www.sutter.com</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Watchmaker (Dumont) Tweezers (size 5)</td>
			<td style="width:171px;">World Precision Instruments, Inc.&#160;</td>
			<td style="width:119px;">501985</td>
			<td style="width:167px;">To brake the needle before sharpening</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dissecting microscope</td>
			<td style="width:171px;">Olympus, Leica, Zeiss</td>
			<td style="width:119px;">Varies with the manufacturer</td>
			<td style="width:167px;">Part of the whole injection setup</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Fluorescence camera</td>
			<td style="width:171px;">Olympus, Leica, Zeiss</td>
			<td style="width:119px;">Varies with the manufacturer</td>
			<td style="width:167px;">To image the fluorescence after injeciton</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PicoNozzle kit (microinjector holder)</td>
			<td style="width:171px;">World Precision Instruments</td>
			<td style="width:119px;">5430-12</td>
			<td style="width:167px;">For microinjections</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pneumatic PicoPump (microinjector)</td>
			<td style="width:171px;">World Precision Instruments</td>
			<td style="width:119px;">SYS-PV820</td>
			<td style="width:167px;">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 ...

reverse-genetic-morpholino-approach-using-cardiac-ventricular

Research

JoVE Journal

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

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 ...

Forschung

Biologie

Zebrafish Brain Ventricle Injection

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved cavities within the brain that are filled with cerebrospinal fluid.  In zebrafish, after neural tube formation, the neuroepithelium undergoes a series of constrictions and folds while it fills with fluid resulting in brain ventricle formation.  In order to understand the process of ventricle formation, and the neuroepithelial shape changes that occur at the same time, we needed a way to visualize the ventricle space in comparison to the brain tissue.  However, the nature of transparent zebrafish embryos makes it difficult to differentiate the tissue from the ventricle space.  Therefore, we developed a brain ventricle injection technique where the ventricle space is filled with a fluorescent dye and imaged by brightfield and fluorescent microscopy.  The brightfield and the fluorescent images are then processed and superimposed in Photoshop.  This technique allows for visualization of the ventricle space with the fluorescent dye, in comparison to the shape of the neuroepithelium in the brightfield image.  Brain ventricle injection in zebrafish can be employed from 18 hours post fertilization through early larval stages.  We have used this technique extensively in our studies of brain ventricle formation and morphogenesis as well as in characterizing brain morphogenesis mutants (1-3).

After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.

Zebrafisch Gehirn Ventrikel Injection

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved ...

Retro-orbital Injection in Adult Zebrafish

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is often the most effective and noninvasive form of delivery of an agent of interest. In the zebrafish (Danio rerio), IV injection of drugs has long been a challenge because of the small vessel diameter. This has also proved a significant hurdle for the injection of cells during hematopoeitic stem cell transplantation. Historically, injections into the bloodstream were done directly through the heart. However, this intra-cardiac procedure has a very high mortality rate as the heart is often punctured during injection leaving the fish prone to infection, massive blood loss or fatal organ damage. Drawing on our experience with the mouse, we have developed a new injection procedure in the zebrafish in which the injection site is behind the eye and into the retro-orbital venous sinus. This retro-orbital (RO) injection technique has been successfully employed in both the injection of drugs in the adult fish as well as transplantation of whole kidney marrow cells. RO injection has a much lower mortality rate than traditional intra-cardiac injection. Fish that are injected retro-orbitally tend to bleed less following injection and are at a much lower risk of injury to a major organ like the heart. Further, when performed properly, injected cells and/or drugs quickly enter the bloodstream allowing compounds to exert their effect on the whole fish and kidney cells to easily home to their niche. Thus, this new injection technique minimizes mortality while allowing efficient delivery of material into the bloodstream of adult fish. Here we exemplify this technique by retro-orbital injection of Tg(globin:GFP) cells into adult casper fish as well as injection of a red fluorescent dye (dextran, Texas Red ) into adult casper fish. We then visualize successful injections by whole animal fluorescence microscopy.

Here we show how to do retro-orbital injection in adult zebrafish.

Retro-Orbital-Injection in Adult Zebrafisch

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Mit dem Gene Pulser MXcell Elektroporation System zur Primary Cells mit hohem Wirkungsgrad zu transfizieren

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage.
GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreERT). CreERT contains a modified estrogen receptor ligand binding domain which renders CreERT sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreERT, which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP.
Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreERT release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreERT.
We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders.
This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreERT;mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark und Track Cells In Vivo</em

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing ...

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 ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

Intraperitoneale Injektion in Adult Zebrafisch

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

Gavaging Adult Zebrafisch

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Nephrotoxin Mikroinjektion in Zebrabärbling akuten Nierenschädigung zu Modell

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A Multi-hole Cryovial Eliminates Freezing Artifacts when Muscle Tissues are Directly Immersed in Liquid Nitrogen

Studies on skeletal muscle physiology face the technical challenge of appropriately processing the specimens to obtain sections with clearly visible cytoplasmic compartments. Another hurdle is the tight apposition of myofibers to the surrounding tissues. Because the process of tissue fixation and paraffin embedding leads to the shrinkage of muscle fibers, freezing is an optimal means of hardening muscle tissue for sectioning. However, a commonly encountered issue, the formation of ice crystals, occurs during the preparation of frozen sections because of the high water content of muscle. The protocol presented here first describes a simple and efficient method for properly freezing muscle tissues by immersing them in liquid nitrogen. The problem with using liquid nitrogen alone is that it causes the formation of a nitrogen gas barrier next to the tissue, which acts as an insulator and inhibits the cooling of the tissues. To avoid this "vapor blanket" effect, a new cryovial was designed to increase the speed of liquid flow around the tissue surface. This was achieved by punching a total of 14 inlet holes in the wall of the vial. According to bubble dynamics, a higher rate of liquid flow results in smaller bubbles and fewer chances to form a gas barrier. When liquid nitrogen flows into the cryovial through the inlet holes, the flow velocity around the tissue is fast enough to eliminate the gas barrier. Compared to the method of freezing muscle tissues using pre-chilled isopentane, this protocol is simpler and more efficient and can be used to freeze muscle in a throughput manner. Furthermore, this method is optimal for institutions that do not have access to isopentane, which is extremely flammable at room temperature.

This protocol describes a procedure to freeze muscle tissues by plunging them directly into liquid nitrogen. This protocol also highlights a new cryovial that can avoid the &#34;blanket effect&#34; of nitrogen gas when liquid nitrogen contacts the tissue surface of a specimen.

Ein Multi-Loch Cryovial Eliminiert Einfrieren Artefakte, wenn Muskelgewebe wird in flüssigem Stickstoff direkt tauchend

Studies on skeletal muscle physiology face the technical challenge of appropriately processing the specimens to obtain sections with clearly visible ...