Questo lavoro è stato supportato dal NIH e PKD fondazione a ZS. Tutti gli esperimenti sugli animali sono state condotte in base a Yale Animal Resources Center (Yarc) e cura degli animali istituzionali e Usa Committee (IACUC) le linee guida.

Parte 1: Preparazione di micropipette, e piatti da camera microiniezione <ol><li> Fabbricare micropipette da riscaldamento e tirando tubi in vetro borosilicato capillari (World Precision Instruments, Inc., 1B100-4) in un dispositivo estrattore micropipetta (Sutter Instruments Inc., Flaming / Brown P-97). Conservare in una capsula di Petri in cima alla piccola quantità di argilla o di nastro adesivo. </li><li> Versare 1,5% agarosio (American Bioanlytical, Inc., AB00972-00500) a 1x piatti medio E3 stampato con depressioni a forma di cuneo che servirà come un metodo semplice per tenere gli embrioni durante l&#39;iniezione (come descritto in &#39;Il libro Zebrafish &quot;) 1. Lasciate piastre di agar camera di solidificare prima dell&#39;uso e conservare a 4 ° C. </li></ol> Parte 2: Preparazione di RNA Un approccio potente per gli studi la funzione del gene è quello di microinject in vitro l&#39;RNA trascritto ricoperto in embrioni di zebrafish. Capped RNA si comporta in modo simile a mRNA eucariotici presenti in vivo per la presenza della analogico PAC. Zebrafish ricercatori utilizzano abitualmente questo metodo per iperespressione o misexpress loro gene di interesse. In questa dimostrazione, ci sarà uno microinject EGFP-tagged trascrizione e l&#39;uso dal vivo tutto-embrione GFP-espressione come una lettura visibile per una iniezione di successo. <ol><li> Eseguire un reazione in vitro la trascrizione dell&#39;RNA PAC sul tuo trascrizione di interesse. Nel nostro laboratorio, abbiamo routine inserire un cDNA di interesse in un vettore pCS2 + ed eseguire un reazione di sintesi in vitro di grandi quantità di RNA ricoperto utilizzando la mMessage mMachine SP6 kit (Ambion, Inc., AM1340). </li><li> Purificare il campione di RNA mediante l&#39;esecuzione attraverso il kit RNeasy Mini (Qiagen, Inc., 74104) o fenolo: estrazione con cloroformio e precipitazione isopropanolo. </li><li> Accuratamente determinare la concentrazione del preparato RNA e conservare a -80 ° C fino al momento dell&#39;utilizzo. </li><li> Il giorno della iniezione, scongelare il campione di RNA, vortice leggermente e spin down brevemente. </li><li> Preparare una soluzione di lavoro con acqua sterile e una concentrazione finale di 0,025-0,050% rosso fenolo (Sigma-Aldrich, P0290). Rosso fenolo serve come marcatore visibile per l&#39;iniezione della soluzione in embrione. In questo articolo, si prepara una soluzione di lavoro di 100 ng / uL mRNA EGFP con il 0,050% rosso fenolo. </li><li> Tenere campione lavorando su ghiaccio e rendimenti azionari RNA a -80 ° C. </li><li> Procedere alla parte 4 quando è pronto per iniettare questo esempio. </li></ol> Parte 3: Preparazione del morfolino Morfolino oligonucleotidi antisenso sono ampiamente utilizzati per modificare l&#39;espressione genica, bloccando la traduzione di una proteina bersaglio o modificando pre-mRNA splicing 2,3. Morpholinos nel zebrafish servire come un potente strumento di genetica inversa abbattendo la funzione del gene. In questo articolo, ci sarà uno microinject oligo morfolino mirato al sito di inizio traslazionale di PKD2 (5&#39;-AGGACGAACGCGACTGGAGCTCATC-3 &#39;). Sulla base di lavoro stabiliti in precedenza, ci aspettiamo che gli embrioni iniettati per imitare fenotipicamente PKD2 4 mutanti di pesce. <ol><li> Noi di routine per 300 nmol di un oligo morfolino specificamente progettato per un gene di interesse da Strumenti Gene, LLC (Philomath, OR). </li><li> Aggiungere 100 uL di acqua sterile per ottenere un 3 soluzione madre mM. Aliquota della soluzione e conservare a -20 ° C fino al momento dell&#39;utilizzo. </li><li> Il giorno della iniezione, riscaldare la soluzione morfolino a 65 ° C per 5 minuti. Scatto raffreddare su ghiaccio immediatamente e spin brevemente. Questo passo denatura le strutture secondarie nella oligo e assicura che la soluzione è completamente solubilizzato. </li><li> Preparare una soluzione di lavoro diluendo la soluzione morfolino in acqua sterile e una concentrazione finale di 0,025-0,050% rosso fenolo. In questa dimostrazione, ci prepareremo una soluzione di lavoro di 0,50 mm PKD2 morfolino con il 0,050% rosso fenolo. </li><li> Continuare a lavorare soluzione a temperatura ambiente. </li><li> Procedere alla parte 4 quando è pronto per iniettare il campione .---- </li></ol> Parte 4: Riempimento della micropipetta con la soluzione di lavoro <ol><li> Tagliare la punta distale di una micropipetta con una pinza o una lametta chirurgico per creare un&#39;apertura che è appena visibile sotto un microscopio da dissezione (Nikon Instruments, Inc., SM2645) con ingrandimento 50X. </li><li> Posto 2 uL della soluzione di lavoro come una goccia su un vetrino. </li><li> Fissare il retro di una micropipetta di una siringa da 5 ml dotato di tubi su un ago ipodermico e leggermente immergere la punta distale del micropipetta nella tua goccia di soluzione di lavoro. Fare attenzione a non rompere la punta della micropipetta. </li><li> Sifone la soluzione di lavoro attraverso l&#39;estremità distale del micropipetta tirando lo stantuffo della siringa. </li></ol> Parte 5: Calibrazione del volume di iniezione micropipetta <ol><li> Mettere una goccia di olio minerale (American Bioanalytical, Inc., AB00921-00025) su un vetrino micrometrico (FiSher, 12-561-SM1). </li><li> Accendere e alimentazione di aria alla pressione pulsata micro-apparato iniettore (World Precision Instruments Inc., PV830). </li><li> Fissare la micropipetta al titolare micropipetta dell&#39;apparato micro iniettore. L&#39;iniettore è micro pressione regolata e scarichi vengono attivate premendo il pedale. </li><li> Sotto il microscopio dissezione, verificare il volume di iniezione utilizzando l&#39;iniettore micro posto una goccia di soluzione di lavoro sul petrolio micrometro. Misurare il diametro della goccia in quanto galleggia come una sfera sulla parte superiore del petrolio. </li><li> Regolare la durata e la pressione di iniezione di calibrare con attenzione il volume di iniezione. Questa fase aiuta la riproducibilità dell&#39;esperimento microiniezione. In questa dimostrazione, tutte le microiniezioni sarà fatto con un diametro calo di 0,15 mm (circa 1,76 nL in volume). </li><li> Una volta che il volume di iniezione è stata calibrata, utilizzare la manopola &quot;Hold&quot; sulla micro iniettore per prevenire il riempimento e la fuoriuscita della micropipetta. </li></ol> Parte 6: Preparazione di embrioni di zebrafish fecondate per microiniezione <ol><li> Zebrafish in modo casuale compagno nelle prime ore di ogni mattina. Raccogliere embrioni a 1-cellula stadio e metterli in 1x medio E3. </li><li> Usando una pipetta di trasferimento 3 ml (Becton Dickinson mediche di laboratorio, 357.524), organizzare gli embrioni lungo le depressioni a forma di cuneo in lastre camera di microiniezione. </li><li> Rimuovere il supporto in modo che gli embrioni sono superficialmente sommerse e non allagata. Questa fase aiuta a risolvere gli embrioni al fondo del depressioni così come nella penetrazione del corion dalla micropipetta. </li></ol> Parte 7: Microiniezione attraverso il corion <ol><li> Manipolare gli embrioni con la micropipetta in modo che il citoplasma della cellula embrionale 1-fase è visibile sotto il microscopio da dissezione. Fare attenzione a non rompere la punta della micropipetta. </li><li> Penetrare il corion e poi il tuorlo con la micropipetta per iniettare l&#39;embrione. In questa dimostrazione, ci sarà la prima microinjecting nel tuorlo direttamente sotto la cella, e permettono il flusso citoplasmatico e diffusione di portare la soluzione di lavoro all&#39;interno della cellula. Questo flusso è visibile a causa del rosso fenolo aggiunto alla soluzione di lavoro. </li><li> Ci sarà anche dimostrare l&#39;iniezione diretta nel citoplasma cellulare. Microiniezione diretta nella cella è più robusto, ma in termini di tempo l&#39;orientamento corretto embrione e la tecnica microiniezione è richiesto. Come la membrana cellulare è più rigoroso rispetto della membrana tuorlo, è spesso più veloce per entrare nel micropipetta attraverso il tuorlo per raggiungere il citoplasma della cellula. Orientando il polo animale verso il fondo della vasca e di lavoro con mano ferma può aiutare in questo metodo di iniezione. </li><li> Dopo la microiniezione, luogo gli embrioni in una capsula di Petri con media E3 e incubare loro a 28,5 ° C per il normale sviluppo. </li><li> Nelle prossime ore e giorni diversi dello sviluppo embrionale, osservare gli embrioni per la fenotipi di interesse. </li></ol> Parte 8: I risultati rappresentativi EGFP mRNA iperespressione: Per verificare il successo della microiniezione, che monitorerà l&#39;espressione della GFP in embrioni di sviluppo a partire fase scudo (~ 6 HPF) dal vivo in tutto-embrione microscopia a fluorescenza (Leica Microsystems Gmbh, MZFLIII). Espressione del costrutto è più forte durante gli eventi precoci dello sviluppo embrionale e spesso diminuiscono nel corso dello sviluppo come l&#39;iniezione di RNA è ricoperto a poco a poco degradato e dipende dalla stabilità della proteina espressa. PKD2 traslazionale-blocking morfolino: Sulla base dei risultati precedentemente pubblicato, ci aspettiamo che il PKD2 morphants per imitare la curvatura dorsale asse del corpo come si trova nel PKD2 zebrafish mutanti e cisti renali presenti a circa 2-3 dpf 4. <img src="/files/ftp_upload/1113/1113fig1.png" alt="Figura 1" /> Figura 1: i risultati Rappresentante microiniezione di mRNA EGFP e PKD2 agosto morfolino. (A) gli embrioni sono stati TAB microiniettati a 1-cellula palco con 0,17 ng di mRNA EGFP e visualizzati a 1 dpf per l&#39;espressione in vivo GFP. (B, c) gli embrioni sono stati TAB microiniettati a 1-cellula palco con ~ 1.7 nl di un PKD2 traslazionale morfolino blocco a 0,50 mm e ha segnato per la curvatura dorsale del corpo a 4 dpf.

<ol>
	<li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>Nasevicius, A., Ekker, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+targeted+gene+'knockdown'+in+zebrafish.">Effective targeted gene 'knockdown' in zebrafish.</a> Nat Genet. 26, 216-220 (2000).</li><li>Draper, B., Morcos, P., Kimmel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+zebrafish+fgf8+pre-mRNA+splicing+with+morpholino+oligos:+a+quantifiable+method+for+gene+knockdown.">Inhibition of zebrafish fgf8 pre-mRNA splicing with morpholino oligos: a quantifiable method for gene knockdown.</a> Genesis. 30, 154-156 (2001).</li><li>Sun, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetic+screen+in+zebrafish+identifies+cilia+genes+as+a+principal+cause+of+cystic+kidney.">A genetic screen in zebrafish identifies cilia genes as a principal cause of cystic kidney.</a> Development. 131, 4085-4093 (2004).</li><li>Stuart, G., McMurray, J., Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication,+integration+and+stable+germ-line+transmission+of+foreign+sequences+injected+into+early+zebrafish+embryos.">Replication, integration and stable germ-line transmission of foreign sequences injected into early zebrafish embryos.</a> Development. 103, 403-412 (1988).</li><li>Stuart, G., Vielkind, J., McMurray, J., Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+lines+of+transgenic+zebrafish+exhibit+reproducible+patterns+of+transgene+expression.">Stable lines of transgenic zebrafish exhibit reproducible patterns of transgene expression.</a> Development. 109, 577-584 (1990).</li><li>Culp, P., N&#252;sslein-Volhard, C., Hopkins, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-frequency+germ-line+transmission+of+plasmid+DNA+sequences+injected+into+fertilized+zebrafish+eggs.">High-frequency germ-line transmission of plasmid DNA sequences injected into fertilized zebrafish eggs.</a> Proc Natl Acad Sci U S A. 88, 7953-7957 (1991).</li><li>Strehlow, D., Heinrich, G., Gilbert, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fates+of+the+blastomeres+of+the+16-cell+zebrafish+embryo.">The fates of the blastomeres of the 16-cell zebrafish embryo.</a> Development. 120, 1791-1798 (1994).</li><li>Helde, K., Wilson, E., Cretekos, C., Grunwald, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+early+cells+to+the+fate+map+of+the+zebrafish+gastrula.">Contribution of early cells to the fate map of the zebrafish gastrula.</a> Science. 265, 517-520 (1994).</li><li>Lin, S., Long, W., Chen, J., Hopkins, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+germ-line+chimeras+in+zebrafish+by+cell+transplants+from+genetically+pigmented+to+albino+embryos.">Production of germ-line chimeras in zebrafish by cell transplants from genetically pigmented to albino embryos.</a> Proc Natl Acad Sci U S A. 89, 4519-4523 (1992).</li><li>Long, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GATA-1+expression+pattern+can+be+recapitulated+in+living+transgenic+zebrafish+using+GFP+reporter+gene.">GATA-1 expression pattern can be recapitulated in living transgenic zebrafish using GFP reporter gene.</a> Development. 124, 4105-4111 (1997).</li><li>Murphey, R., Zon, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule+screening+in+the+zebrafish.">Small molecule screening in the zebrafish.</a> Methods. 39, 255-261 (2006).</li></ol>

Microiniezione in embrioni di zebrafish è una tecnica consolidata e robusta per esplorare il ruolo di un particolare gene nello sviluppo. Le applicazioni includono l&#39;iperespressione, misexpression e saggi knockdown del gene di interesse così come l&#39;analisi epistatico fra più geni. Microiniezione in zebrafish è stato ampiamente utilizzato per la generazione di animali transgenici, e mappatura destino delle cellule ai primi embrioni blastula 5,6,7,8,9. Inoltre, l&#39;applicazione di questa tecnica s...

Tutti gli altri materiali sono elencati nel protocollo.

microinjection of mrna and morpholino antisense oligonucleotides in zebrafish embryos.

Uno strumento essenziale per indagare il ruolo di un gene durante lo sviluppo è la capacità di eseguire knockdown gene, l&#39;iperespressione, e gli studi misexpression. In zebrafish (<em&gt; Danio rerio</em&gt;), La microiniezione di RNA, DNA, proteine, oligonucleotidi antisenso e di altre piccole molecole in sviluppo dell&#39;embrione fornisce ai ricercatori un test rapido e affidabile per esplorare la funzione del gene<em&gt; In vivo</em&gt;. In questo video-articolo, mostreremo come preparare e microinject<em&gt; In vitro</em&gt; Sintetizzato EGFP mRNA e una traslazione di blocco morfolino oligo contro<em&gt; PKD2</em&gt;, Un gene associato a malattia autosomica dominante del rene policistico (ADPKD), in 1 a cellule di embrioni fase zebrafish. Ci sarà quindi analizzare il successo del mRNA e microiniezioni morfolino verificando espressione GFP e analisi fenotipo. Vaste applicazioni di questa tecnica sono la generazione degli animali transgenici e la linea germinale chimere, cellule destino mappatura e lo screening del gene. Qui descriviamo un protocollo per la sovraespressione di EGFP e knockdown di<em&gt; PKD2</em&gt; Di mRNA e l&#39;iniezione oligonucleotide morfolino.

Watch this Scientific Journal Video about Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos. at JoVE.com

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

Microiniezione è un metodo consolidato ed efficace per l&#39;introduzione di sostanze estranee in embrioni di zebrafish fecondato. Qui, dimostriamo una tecnica robusta microiniezione per l&#39;esecuzione di sovraespressione di mRNA, e morfolino oligonucleotide studi gene knockdown 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 ...

microinjection-mrna-morpholino-antisense-oligonucleotides-zebrafish

Research

JoVE Journal

Biology

36.6K Views.  Yale University School of Medicine. Microiniezione è un metodo consolidato ed efficace per l'introduzione di sostanze estranee in embrioni di zebrafish fecondato. Qui, dimostriamo una tecnica robusta microiniezione per l'esecuzione di sovraespressione di mRNA, e morfolino oligonucleotide studi gene knockdown in zebrafish.

Video: Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

L&#39;infezione di embrioni di zebrafish con batteri patogeni intracellulari

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

Ricerca

Immunologia e infezioni

Microinjection of Zebrafish Embryos to Analyze Gene Function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

Microiniezione di embrioni di zebrafish per analizzare la funzione del gene

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

Biologia

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

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.  ...

Biologia dello sviluppo

Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the eye, brain, and somites of zebrafish. The advantage of electroporation over other methods of genetic manipulation is that specific tissues can be targeted, both spatially and temporally, for the introduction of macromolecules by the application of electrical current. Here we describe the use of electroporation for transfecting mif and mif-like morpholinos into the tissues of the developing inner ear of the zebrafish. In past studies, mif morpholino injected into embryos at the 1- to 8-cell stage resulted in widespread morphological changes in the nervous system and eye, as well as the ear. By targeting the tissues of the inner ear at later stages in development, we can determine the primary effects of MIF in the developing inner ear, as opposed to secondary effects that may result from the influence of other tissues. By using phalloidin and acetylated tubulin staining to study the morphology of neurons, neuronal processes, and hair cells associated with the posterior macula, we were able to assess the efficacy of electroporation as a method for targeted transfection in the zebrafish inner ear. The otic vesicles of 24hpf embryos were injected with morpholinos and electroporated and were then compared to embryos that had received no treatment or had been only injected or electroporated. Embryos that were injected and electroporated showed a decrease in hair cell numbers, decreased innervation by the statoacoustic ganglion (SAG) and fewer SAG neurons compared with control groups. Our results showed that direct delivery of morpholinos into otocysts at later stages avoids the non-specific nervous system and neural crest effects of morpholinos delivered at the 1-8 cell stage. It also allows examination of effects that are directed to the inner ear and not secondary effects on the ear from primary effects on the brain, neural crest or periotic mesenchyme.

A method to deliver morpholinos directly into the zebrafish otocyst at 24hpf has been developed. Using microinjection of morpholinos into the lumen of otic vesicle and electroporation to effect penetration, we were able to bypass the effect of morpholinos on the brain and obtain effects specific to the inner ear.

Consegna diretta di Morpholinos MIF Into the Otocyst Zebrafish di iniezione e di elettroporazione influenza lo sviluppo dell&#39;orecchio interno

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the ...

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 ...

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

The zebrafish is an important model to understand the cell and molecular biology of organ and appendage regeneration. However, molecular strategies to employ reverse genetics have not yet been adequately developed to assess gene function in regeneration or tissue homeostasis during larval stages after zebrafish embryogenesis, and several tissues within the zebrafish larva are difficult to target. Intraventricular injections of gene-specific morpholinos offer an alternative method for the current inability to genomically target zebrafish genes in a temporally controlled manner at these stages. This method allows for complete dispersion and subsequent incorporation of the morpholino into various tissues throughout the body, including structures that were formerly impossible to reach such as those in the larval caudal fin, a structure often used to noninvasively research tissue regeneration. Several genes activated during larval finfold regeneration are also present in regenerating adult vertebrate tissues, so the larva is a useful model to understand regeneration in adults. This morpholino dispersion method allows for the quick and easy identification of genes required for the regeneration of larval tissues as well as other physiological phenomena regulating tissue homeostasis after embryogenesis. Therefore, this delivery method provides a currently needed strategy for temporal control to the evaluation of gene function after embryogenesis.&#160;

An adaptable reverse genetic method for zebrafish to assess gene function during later stages of development and physiological homeostasis such as tissue regeneration using intraventricular injections of gene-specific morpholinos.

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 ...

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 ...

Didattica

JoVE Science Education

Biologia avanzata

Zebrafish Microinjection Techniques

One of the major advantages to working with zebrafish (Danio rerio) is that their genetics can be easily manipulated by microinjection of early stage embryos. Using this technique, solutions containing genetic material or knockdown constructs are delivered into the blastomeres: the embryonic cells sitting atop the yolk of the newly fertilized egg. Delivery into the cytoplasm is achieved either through direct injection into the blastomere, or via natural cytoplasmic movements that occur after a solution is injected into the yolk. Successful genetic manipulations are usually followed by quantification of embryonic phenotypes in order to elucidate the genetic mechanisms of development.
This video will provide an introduction to carrying out microinjections in zebrafish embryos. The discussion begins with a review of the essential tools for the technique, including the injection apparatus and the microinjector, which controls fluid movement with pressure pulses of air. Next, important preparatory steps are demonstrated, such as the pouring of agar plates to stabilize embryos during injection and calibration of the microinjection apparatus. The injection procedure is then presented along with tips on when and where injections should be performed. Finally, applications of the microinjection technique are discussed, including gene overexpression via mRNA injection, gene silencing by delivery of antisense morpholino oligonucleotides, and the generation of transgenic zebrafish using specially engineered plasmid DNA.

Introduction to Microinjection of Early Zebrafish Embryos

Tecniche di microiniezione di Zebrafish

One of the major advantages to working with zebrafish (Danio rerio) is that their genetics can be easily manipulated by microinjection of early stage ...

Biologia di base

Biologia II: topo, zebrafish e pollo

Morpholino Injection: A Method to Knockdown Target Gene Expression to Study Function in Zebrafish

Source: Hyde D.R., et al. <a href="https://www.jove.com/v/3632/" target="_blank">In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin</a>. J. Vis. Exp. (2012)
The protocol describes targeted knockdown of gene expression in zebrafish by morpholino injection in the tail.

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

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Capitoli in questo video

L'infezione di embrioni di zebrafish con batteri patogeni intracellulari