Questo lavoro è stato sostenuto dalla Deutsche Forschungsgemeinschaft (DFG), desideriamo ringraziare Ayele Tsedeke Taddese per il supporto tecnico.

1. Preparazione degli aghi di vetro (Figura 1) <ol><li> Utilizzare capillari di vetro con diametro 0,75 millimetri per preparare aghi per iniezione (Figura 1A). </li><li> Posizionare il capillare di vetro in un estrattore ago e tirare l&#39;ago con il seguente parametro: valore del ciclo di riscaldamento: 463; tirando valore del ciclo: 230; Velocità: 150 msec; Tempo: 150 msec (Figura 1B). </li><li> Rompere il capillare di vetro tirato con le pinzette da orologiaio per produrre un diametro ago 20 micron sotto uno stereoscopio con un micrometro. </li><li> Utilizzare un tornio con una ruota che gira gomma bagnata per affinare l&#39;ago e produrre un 20 micron smusso (figure 1C e 1D). Nota: Sharp, aghi smussati migliorare la facilità di inserimento nel ventricolo e quindi ridurre al minimo il danno al tessuto e consentono il muscolo forata per richiudere dopo la rimozione dell&#39;ago (Figure 1E-G). </li></ol> 2. Preparzione della soluzione Morpholino <ol><li> Preparare il morpholino magazzino sciogliendo la morpholino liofilizzato in 1x Phosphate-Buffered Saline (PBS) ad una concentrazione finale di 7,5 mm. [Riferimento alle istruzioni del produttore per i dettagli (tabella materiali)]. </li><li> Preparare la soluzione iniettabile morfolino mescolando soluzione 2,5 microlitri morfolino magazzino (7,5 mm) con 2,8 ml soluzione Endo-Porter magazzino (1 mm) (vedere la tabella dei materiali) per una concentrazione finale di morfolino 3,5 mm e 0,5 mM Endo-Porter. </li></ol> 3. Preparazione dell&#39;iniezione Morpholino (Figura 2) <ol><li> Caricare l&#39;ago di vetro smussato con 5 ml di questa soluzione utilizzando una micropipetta con 10 microlitri punta microloader pipetta. </li><li> Inserire l&#39;ago di vetro nel supporto dell&#39;ago del micromanipolatore collegato alla pompa pico pneumatico (figure 2A-C). </li><li> Posizionare il supporto dell&#39;ago accanto al microscopio in modo che solo le esigenze agodi essere spostati in una direzione planare per inserirla nel ventricolo cardiaco della larva (Figura 2A). </li><li> Regolare l&#39;angolo per le iniezioni a circa 45 °. </li><li> Impostare i valori microinjector come segue: pressione di mantenimento: 20 libbre per pollice quadrato (psi); Pressione di espulsione: 15 psi; 100 msec gamma di gating, valore di periodo di 1,9 (corrisponde a 10,9 msec). </li><li> Sciogliere agarosio in 1x PBS utilizzando un forno standard per produrre 20 ml di 1,5% (w / v) di gel. Versare gel fuso in una capsula Petri 10 centimetri. Posizionare una forma di iniezione scanalato in agarosio caldo in modo che, una volta indurito, il agarosio avrà solchi in cui la larva sedato sarà posto 11 (figure 2D e 2E). </li></ol> 4. Iniezioni (Figura 3) <ol><li> Anestetizzare larva in 100 ml di acqua dell&#39;acquario con 20 mg / L tricaine (L-etil-m-amino-benzoato-metano solfonato) finché smettono di rispondere al tatto. </li><li> Utilizzare un Pasteur pipett plasticae di trasferire accuratamente la larva sedato in una scanalatura dello stampo agarosio bagnato in modo che il lato ventrale è rivolto contro la parete agarosio verticale della scanalatura (Figura 3A). </li><li> Posizionare la piastra di agarosio allo stereomicroscopio in modo che il ventricolo è rivolto verso l&#39;ago di iniezione (figura 3A). </li><li> Abbassare l&#39;ago per inserirlo nel ventricolo del cuore. Inserire l&#39;ago solo 1-2 micron nel ventricolo avendo cura di non inserire l&#39;ago troppo in profondità (Figure 3B e 3C). </li><li> Una volta inserito l&#39;ago, iniettare la soluzione morfolino nel ventricolo con 4-6 impulsi, ciascuno consegna 3 nl della soluzione, con intervalli di attesa per consentire compensazione del cuore (figure 3D e 3E). </li><li> Dopo l&#39;iniezione, rimuovere l&#39;ago (parallelo al piano di inserimento) e poi trasferire accuratamente la larva con una pipetta Pasteur plastica riempito di E3 medio BACk in una capsula di Petri contenente mezzo E3 fresco. </li><li> Posizionare l&#39;ago in una capsula di Petri contenente PBS 1x per impedire l&#39;essiccamento dell&#39;ago quando si trasferisce la larva iniettata. </li><li> Ripetere i passaggi 4,1-4,6 ogni 12-24 ore per la durata dell&#39;esperimento. Nota: Ripetendo le iniezioni assicura l&#39;assorbimento e la manutenzione del morfolino nelle cellule. </li></ol> 5. Analisi di iniezioni (figura 4) <ol><li> Anestetizzare il pesce in 100 ml di acqua dell&#39;acquario con 20 mg / L tricaine fino a che non smettono di rispondere al tatto. </li><li> Posizionare la larva lateralmente su un piatto bagnato 1,5% (w / v) piastra di agarosio coperto con E3 medie. </li><li> Larve di immagine utilizzando uno stereomicroscopio con campo chiaro e imaging di fluorescenza. Nota: Poiché le larve sono trasparenti, il morfolino fluoresceina-tag dovrebbe essere visibile come verde fluorescente nei vasi sanguigni in tutto l&#39;animale immediatamente dopo l&#39;iniezione. Questa fluorescenza ulteriormente disperdere nei tessuti vascolarizzati da15 min (Figure 4A-4C). </li></ol> 6. Valutazione della Regenerative escrescenza <ol><li> Valutare le immagini catturate utilizzando Fiji Immagine software libero J (http://fiji.sc/Fiji). Per indagare la rigenerazione, utilizzare lo strumento di line-analisi per determinare la quantità di crescita rigenerativa che si è verificato sul controllo e la larva morfolino-iniettato. </li><li> Per calibrare le immagini, aprire uno dei file di immagine originali in Fiji trascinando e rilasciando il file nella finestra principale di Fiji. </li><li> Per impostare la scala per tutte le immagini che seguono, andare su &quot;Analizza&quot; nel menu principale. </li><li> Nel menu a discesa fare clic su &quot;Imposta scala&quot;. </li><li> In questa finestra, attivare l&#39;opzione &quot;Global&quot; facendo clic sulla casella di controllo. </li><li> Ora aprite l&#39;immagine per misurare la quantità di crescita rigenerativa di nuovo con il drag and drop (vedi punto 6.1). </li><li> Scegliere lo strumento &quot;selezione a mano libera&quot; nella barra degli strumenti. </li><li> Circondare la zona da misurare (Figure 4J-4L). </li><li> Premere Ctrl + M. Si aprirà una nuova finestra &quot;Risultati&quot; che mostra il valore dell&#39;area circondata in pixel quadrati. </li></ol>

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L&#39;iniezione intraventricolare fornisce un metodo di valutazione rapida e affidabile per analizzare la funzione genica nelle fasi successive di sviluppo o di omeostasi del corpo senza alterare la funzione del gene durante l&#39;embriogenesi. Per assicurare il successo di questa tecnica, si deve essere consapevoli di quattro punti critici: 1) dimensioni dell&#39;ago, 2) essiccazione dell&#39;ago, 3) minimizzare il volume, e 4) tempo di esposizione minima nella soluzione sedazione. Aghi che sono troppo piccoli saranno ...

Rigenerazione di organi e appendici è di fondamentale importanza per la sopravvivenza e fitness; Tuttavia, alcuni vertebrati compreso l&#39;uomo hanno limitato la capacità rigenerative. Mentre diversi modelli animali esistere che hanno ampia capacità rigenerativa, invertire tecniche genetiche per valutare la funzione del gene durante organo e la rigenerazione appendice rimangono molto limitato o inesistente. Pertanto, sono necessari nuovi approcci per sezionare la biologia molecolare di rigenerazione in questi organismi modello. Il zebrafish è un modello ben consolidato per comprendere la biologia cellulare e molecolare di organo e rigenerazione appendice 1, non solo per la sua notevole capacità di rigenerare più organi, tessuti e appendici, ma anche perché diverse linee transgeniche di pesce esistono per monitorare le cellule e di iperespressione del gene costruisce 2, 3. Tuttavia, l&#39;inibizione del gene in zebrafish larvale è limitata principalmente al overexpreSsion di costrutti dominanti negativi, che non sono a disposizione di tutti i geni di interesse o il cui prodotto del transgene in grado di acquisire il guadagno-di-funzione degli effetti che non riflettono l&#39;attività endogena del gene. Pertanto, è necessario un metodo alternativo per rimuovere specificamente espressione genica per KO o smontabile per superare questi problemi. Gene-specifica il targeting utilizzando TALENS esiste come un mezzo genetica inversa per knockout funzione del gene; Tuttavia, questa strategia knockout è molto spesso limitata a valutazioni funzionali durante embriogenesi precoce, perché i requisiti iniziali del gene impedire ulteriore progressione dello sviluppo embrionale. Così, studiando i fenomeni successivi come la rigenerazione o organo omeostasi dopo di sviluppo utilizzando TALENS è preclusa 4, 5. Pertanto, è necessaria una strategia di rimozione gene supplente che gli obiettivi di funzione del gene dopo lo sviluppo presto per valutare i requisiti del gene in organi e strutture completamente formate. </p&gt; Morfolino iniezione ha dimostrato di essere efficace nel rivolti geni in alcuni organi adulti e l&#39;adulto rigenerante pinna 6-8, ma questi metodi richiedono elettroporazione e molti organi interni sono difficili da elettroporazione sia a causa della loro posizione o per la loro sensibilità elettrica interruzioni. Inoltre, alcuni tessuti del larva sono difficili da iniettare direttamente, perché l&#39;iniezione diretta può compromettere la loro integrità strutturale o perché la loro dimensione è limitante. La pinna caudale della larva è una tale struttura, in quanto l&#39;iniezione diretta nel finfold non è possibile. Così, alternativa alla elettroporazione e iniezione diretta era necessaria per indirizzare geni nei tessuti che sono o troppo piccolo per iniettare o non possono essere elettroporate. Al fine di indirizzare e inibire la funzione dei geni specifici durante la rigenerazione della pinna caudale larvale, abbiamo modificato morpholino tecnologie esistenti consentendo unVALUTAZIONE della funzione del gene durante la rigenerazione pinna caudale in larva tardo-messa in scena. Questo metodo impiega consegna intraventricolare 9 del Morpholinos fluoresceina-tagged con Endo-Porter trasfezione reagente 10. Una volta nel ventricolo, la miscela morfolino-Endo-Porter diffonde rapidamente in tutto il larva attraverso il sistema vascolare ed entra tessuti che sono stati precedentemente impossibile bersaglio. Questo metodo di iniezione può essere modificato per geni bersaglio in tessuti specifici e molto probabilmente può essere applicato in altri modelli animali che attualmente mancano metodi genetici inverso per inibire la funzione del gene. Così, offre un metodo rapido e facile con la possibilità di ampio utilizzo gamma di studiare immediatamente la funzione del gene durante l&#39;omeostasi organo generale e rigenerazione nelle fasi larvali.

<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

Lo zebrafish è un modello importante per comprendere la biologia cellulare e molecolare di organo e rigenerazione appendice. Tuttavia, le strategie molecolari di impiegare genetica inversa non sono ancora stati adeguatamente sviluppati per valutare la funzione del gene nella rigenerazione o omeostasi dei tessuti durante le fasi larvali dopo embriogenesi zebrafish, e diversi tessuti all&#39;interno della larva zebrafish sono difficili da colpire. Iniezioni intraventricolare di Morpholinos gene-specifici offrono un metodo alternativo per l&#39;attuale incapacità di indirizzare genomically geni di zebrafish in modo controllato temporalmente in queste fasi. Questo metodo consente la completa dispersione e successiva incorporazione del morfolino in vari tessuti dell&#39;organismo, comprese le strutture che prima erano impossibili da raggiungere come quelli nella pinna caudale larvale, una struttura spesso usato per ricercare in modo non invasivo rigenerazione tissutale. Diversi geni attivati ​​durante la rigenerazione finfold larvale sono anche Present nella rigenerazione dei tessuti vertebrati adulti, in modo che la larva è un modello utile per capire la rigenerazione negli adulti. Questo metodo dispersione morfolino consente l&#39;identificazione rapida e facile di geni necessari per la rigenerazione di tessuti larvali nonché altri fenomeni fisiologici che regolano l&#39;omeostasi del tessuto dopo l&#39;embriogenesi. Pertanto, questo metodo di consegna fornisce una strategia attualmente necessari per il controllo temporale per la valutazione della funzione genica dopo embriogenesi.

Il forte, smussato ago per l&#39;iniezione è facilmente inserito nel ventricolo cardiaco della larva zebrafish quando si avvicinò dorsale (Figura 3A). Il cuore continua a pompare e il flusso di sangue viene mantenuta nonostante la presenza dell&#39;ago (Figure 3B e 3C). Iniezioni attente non perturbare la morfologia del ventricolo o contrazioni cardiache (Figura 3D) nonostante iniezione del morfolino nel cuore (Figura 3E). <p clas...

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

Un metodo genetica inversa adattabile per zebrafish per valutare la funzione del gene durante le fasi successive di sviluppo e omeostasi fisiologica, come la rigenerazione dei tessuti utilizzando iniezioni intraventricolare di Morpholinos gene-specifici.

Reverse genetica Morpholino approccio Uso cardiaca ventricolare iniezione per transfettare diversi tessuti difficili da bersaglio in Zebrafish Larva

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 Views.  Technische Universität Dresden.  Un metodo genetica inversa adattabile per zebrafish per valutare la funzione del gene durante le fasi successive di sviluppo e omeostasi fisiologica, come la rigenerazione dei tessuti utilizzando iniezioni intraventricolare di Morpholinos gene-specifici. 

Video: 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.

Analisi dello sviluppo del rene Zebrafish con time-lapse imaging Utilizzando una dissezione microscopio dotato di sezionamento ottico

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

Ricerca

Biologia dello sviluppo

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.

Imaging in vivo del transgenico espressione genica nei singoli retinica Progenitori in embrioni di zebrafish Chimerico da Cell studio Influenze non autonomo

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.

Microiniezione di mRNA e oligonucleotidi antisenso Morpholino in embrioni di zebrafish.

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

Biologia

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.

Visualizzazione dell'attività elettrica cellulare in Zebrafish embrioni in anticipo e tumori

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.

Micromanipolazione di espressione genica in Zebrafish adulti cervello con microiniezione Cerebroventricular di Morpholino oligonucleotidi

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

Neuroscienze

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.

Un approccio inverso di test genetici per ridondanza funzionale durante l&#39;embriogenesi

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 elettroporazione in vivo dei Morpholinos nella retina Zebrafish per adulti

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 elettroporazione in vivo di Morpholinos nella Adult Regenerating 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 ...

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

Silenziamento genico con Morpholinos

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