Este trabajo fue apoyado por el NIH y la fundación PKD de ZS. Todos los experimentos con animales se realizaron de acuerdo con Yale Centro de Recursos Animales (YARC) y Atención Institucional para las directrices del Comité (IACUC).

Parte 1: Preparación de pipetas y placas de microinyección de cámara <ol><li> Fabricar micropipetas de calefacción y tirar los tubos capilares de vidrio de borosilicato (World Precision Instruments, Inc., 1B100-4) en un dispositivo extractor de micropipeta (Sutter Instruments Inc., Flaming / Brown P-97). Almacenar en una placa de Petri en la parte superior de la pequeña cantidad de arcilla o cinta adhesiva. </li><li> Vierta el 1,5% de agarosa (American Bioanlytical, Inc., AB00972-00500) en placas de medio 1x E3 moldeado con forma de cuña canales que sirven como un método fácil para la celebración de los embriones durante la inyección (como se describe en &#39;El libro de pez cebra &quot;) 1. Deje que la cámara de placas de agar se solidifique antes de usar y almacenar a 4 º C. </li></ol> Parte 2: Preparación de ARN Un método poderoso para los estudios de la función del gen es microinject in vitro transcritos de ARN tope en embriones de pez cebra. Cubiertas de ARN se comporta de manera similar a los ARNm eucariotas en vivo debido a la presencia del análogo de la PAC. Investigadores del pez cebra rutinariamente utilizan este método para sobreexpresar o misexpress su gen de interés. En esta demostración, vamos a microinject una transcripción EGFP de etiquetado y el uso directo de todo el embrión GFP-expresión como una lectura visible de una inyección de éxito. <ol><li> Realizar una reacción de transcripción in vitro de la PAC en su transcripción de ARN de interés. En nuestro laboratorio, de manera rutinaria insertar un cDNA de interés en un vector pCS2 + y llevar a cabo una reacción de síntesis in vitro de grandes cantidades de ARN tope con el mMessage mMachine SP6 kit (Ambion, Inc., AM1340). </li><li> Purificar la muestra de ARN mediante la ejecución a través de la RNeasy Mini Kit (Qiagen, Inc., 74104) o con fenol: cloroformo extracción y precipitación con isopropanol. </li><li> Con cuidado, determinar la concentración de la preparación de ARN y almacenar a -80 º C hasta que esté listo para su uso. </li><li> En el día de la inyección, descongelar la muestra de ARN, vortex ligeramente y girar hacia abajo brevemente. </li><li> Prepare una solución de trabajo con agua estéril y una concentración final de 0,025 a 0,050% de rojo fenol (Sigma-Aldrich Co., P0290). Rojo fenol sirve como un marcador visible para la inyección de la solución en el embrión. En este artículo, vamos a preparar una solución de trabajo de 100 ARNm EGFP ng / uL con 0,050% de rojo fenol. </li><li> Mantenga la muestra de trabajo en el hielo y acciones retorno de ARN a -80 ° C. </li><li> Proceder a la parte 4 cuando esté listo para inyectar la muestra. </li></ol> Parte 3: Preparación de morfolino Morfolino oligonucleótidos antisentido son ampliamente utilizados para modificar la expresión de genes mediante el bloqueo de la traducción de una proteína específica o mediante la modificación de pre-mRNA de empalme 2,3. Morfolinos en el pez cebra servir como una poderosa herramienta genética inversa por derribar la función del gen. En este artículo vamos a microinject un oligo morfolino dirigidos al sitio de iniciación de la traducción de PKD2 (5&#39;-AGGACGAACGCGACTGGAGCTCATC-3 &#39;). Basado en el trabajo previamente establecido, se espera que los embriones inyectados para imitar fenotípicamente PKD2 4 peces mutantes. <ol><li> En forma rutinaria para 300 nmol de un oligo morfolinos diseñado específicamente para un gen de interés por parte de Gene Herramientas, LLC (Philomath, OR). </li><li> Añadir 100 uL de agua estéril para obtener una solución 3 mM de valores. Alícuota de la solución y almacenar a -20 ° C hasta que esté listo para su uso. </li><li> En el día de la inyección, calentar la solución morfolino a 65 ° C durante 5 minutos. Ajustar enfriar en hielo inmediatamente y girar brevemente. Este paso desnaturaliza las estructuras secundarias en el oligo y asegura que la solución esté completamente solubilizados. </li><li> Prepare una solución de trabajo mediante la dilución de la solución morfolino en agua estéril y una concentración final de 0,025 a 0,050% de rojo fenol. En esta demostración, que se prepara una solución de trabajo de 0,50 mm PKD2 morfolino con 0,050% de rojo fenol. </li><li> Seguir trabajando solución a temperatura ambiente. </li><li> Proceder a la parte 4 cuando esté listo para inyectar la muestra .---- </li></ol> Parte 4: Llenado de la micropipeta con su solución de trabajo <ol><li> Cortar el extremo distal de una micropipeta con fórceps o una hoja de afeitar quirúrgico para crear una abertura que es apenas visible bajo un microscopio de disección (Nikon Instruments, Inc., SM2645) con un aumento de 50X. </li><li> Coloque 2 l de la solución de trabajo como una gota en un portaobjetos. </li><li> Coloque la parte de atrás de una micropipeta a una jeringa de 5 ml equipado con un tubo de una aguja hipodérmica y un poco sumergir la punta distal de la micropipeta en su gota de solución de trabajo. Tenga cuidado de no romper la punta de la micropipeta. </li><li> Sifón de la solución de trabajo a través del extremo distal de la micropipeta, tirando del émbolo de la jeringa. </li></ol> Parte 5: Calibración del volumen de inyección micropipeta <ol><li> Coloque una gota de aceite mineral (American Inc. Bioanalytical, AB00921-00025) en una diapositiva micrómetro (Fisher, 12-561-SM1). </li><li> A su vez en el poder y el suministro de aire a la presión de pulso micro inyectores aparato (World Precision Instruments Inc., PV830). </li><li> Fije la micropipeta al titular de la micropipeta de los aparatos de micro inyectores. El inyector de micro es la presión regulada y las descargas se activan pulsando el pedal. </li><li> Bajo el microscopio de disección, prueba el volumen de inyección con inyector micro para colocar una gota de la solución de trabajo sobre el aceite de micrómetro. Mida el diámetro de la gota que flota como una esfera en la parte superior del aceite. </li><li> Ajustar la duración y la presión de inyección para calibrar su cuidado volumen de inyección. Este paso ayuda a la reproducibilidad de los experimentos de microinyección. En esta demostración, todas las microinyecciones se llevará a cabo con un diámetro de la gota de 0,15 mm (aproximadamente 1,76 nL en volumen). </li><li> Una vez que el volumen de la inyección ha sido calibrado, utilice el &quot;Hold&quot; botón en el inyector de micro para evitar que el relleno y las fugas de la micropipeta. </li></ol> Parte 6: Preparación fecundado embriones de pez cebra para la microinyección <ol><li> Pez cebra al azar mate en las primeras horas de cada mañana. Recoger los embriones en la etapa 1 de las células y colocarlas en 1x medio E3. </li><li> Con una pipeta de transferencia de 3 ml (Becton Dickinson Labware, 357524), organizar los embriones a lo largo de los valles en forma de cuña en las placas de microinyección de cámara. </li><li> Eliminar el medio para que los embriones son superficialmente sumergida y no inundadas. Este paso ayuda a la solución de los embriones en el fondo de los valles, así como en la penetración del corion de la micropipeta. </li></ol> Parte 7: Microinyección a través del corion <ol><li> Manipular los embriones con la micropipeta de modo que el citoplasma de las embrión en su fase 1-célula ya es visible bajo el microscopio de disección. Tenga cuidado de no romper la punta de la micropipeta. </li><li> Penetrar en el corion y la yema con la micropipeta con el fin de inyectar en el embrión. En esta demostración, que será la primera microinyección en la yema directamente debajo de la celda, y permitir el flujo citoplásmico y difusión para que la solución de trabajo en la célula. Este flujo es visible debido a la rojo fenol a la solución de trabajo. </li><li> También vamos a demostrar de inyección directa en el citoplasma celular. Microinyección directa en la célula es más robusto, pero mucho tiempo como la orientación adecuada del embrión y la técnica de microinyección es necesario. Como la membrana celular es más riguroso que la membrana de la yema, a menudo es más rápido para entrar en la micropipeta a través de la yema de huevo para alcanzar el citoplasma de la célula. Orientar el polo animal hacia la parte inferior de la cubeta y el trabajo con mano firme puede ayudar en este método de la inyección. </li><li> Después de la microinyección, colocar los embriones en una placa de Petri con medio E3 y se incuba en los 28,5 ° C para el desarrollo normal. </li><li> Durante las próximas horas y días diferentes del desarrollo del embrión, observar los embriones para su fenotipos de interés. </li></ol> Parte 8: Los resultados representativos EGFP sobreexpresión de ARNm: Para verificar el éxito de la microinyección, vamos a observar la expresión de GFP en los embriones en desarrollo a partir de la etapa escudo (~ 6 HPF) de la microscopia de fluorescencia in vivo de todo el embrión (Leica Microsystems GmbH, MZFLIII). Expresión de la construcción es el más fuerte durante los primeros eventos del desarrollo embrionario y, a menudo disminuirá durante el desarrollo como la inyección de ARN tope es degradado gradualmente y depende de la estabilidad de la proteína expresada. PKD2 traslación de bloqueo morfolino: En base a los resultados publicados anteriormente, esperamos que el PKD2 morphants para imitar la curvatura del cuerpo del eje dorsal que se encuentra en PKD2 pez cebra mutante y quistes del riñón en la actualidad aproximadamente 2 - 3 fdd 4. <img src="/files/ftp_upload/1113/1113fig1.png" alt="Figura 1" /> Figura 1: Los resultados representativos de la microinyección de ARNm EGFP y PKD2 agosto morfolino. (A) los embriones fueron TAB microinyección en la etapa de una célula con 0,17 ng de ARNm EGFP y se visualizan en un DPF en vivo la expresión de GFP. (B, c) los embriones fueron TAB microinyección en la fase 1 de las células con ~ 1,7 nl de una PKD2 traslacional bloqueo morfolino a 0,50 mm y anotó para la curvatura dorsal del cuerpo a las 4 de fdd.

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	<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>

Microinyección en embriones de pez cebra es una técnica bien establecida y robusta para explorar el papel de un gen en particular en el desarrollo. Las aplicaciones incluyen la sobre-expresión, misexpression, y los ensayos de caída de su gen de interés, así como el análisis de epistasis entre múltiples genes. Microinyección en el pez cebra ha sido ampliamente utilizado para la generación de animales transgénicos y la asignación de destino de las células de los embriones tempranos de blástula 5,6,7,8,9...

Todos los otros materiales se enumeran en el protocolo.

microinjection of mrna and morpholino antisense oligonucleotides in zebrafish embryos.

Una herramienta esencial para investigar el papel de un gen durante el desarrollo es la capacidad de realizar caída del gen, la sobreexpresión y estudios misexpression. En el pez cebra (<em&gt; Danio rerio</em&gt;), La microinyección de ARN, el ADN, las proteínas, los oligonucleótidos antisentido y otras moléculas pequeñas en el embrión en desarrollo ofrece a los investigadores un ensayo rápido y robusto para explorar la función de genes<em&gt; In vivo</em&gt;. En este video-artículo, vamos a demostrar cómo preparar y microinject<em&gt; In vitro</em&gt; Sintetizado EGFP ARNm y un oligo-traslacional bloqueo contra morfolino<em&gt; PKD2</em&gt;, Un gen asociado con la enfermedad de herencia autosómica dominante del riñón poliquístico (PQRAD), en una de las células de embriones de pez cebra etapa. A continuación, se analizará el éxito de los ARNm y microinyecciones morfolino mediante la verificación de buenas prácticas agrarias expresión y análisis de fenotipo. Amplia gama de aplicaciones de esta técnica incluyen la generación de animales transgénicos y las quimeras de línea germinal, la cartografía de la célula-destino y la detección de genes. En este documento se describe un protocolo para la sobreexpresión de EGFP y caída de<em&gt; PKD2</em&gt; Por ARNm y la inyección de morfolino oligonucleótidos.

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.

Microinyección es un método bien establecido y eficaz para la introducción de sustancias extrañas en embriones de pez cebra fecundado. Este sentido, demuestran una sólida técnica de microinyección para la realización de la sobreexpresión de ARNm, y morpholino oligonucleótidos estudios caída de genes en el pez cebra.

Microinyección de ARNm y oligonucleótidos antisentido Morpholino en embriones de pez cebra.

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 vistas.  Yale University School of Medicine. Microinyección es un método bien establecido y eficaz para la introducción de sustancias extrañas en embriones de pez cebra fecundado. Este sentido, demuestran una sólida técnica de microinyección para la realización de la sobreexpresión de ARNm, y morpholino oligonucleótidos estudios caída de genes en el pez cebra.

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.

La infección de embriones de pez cebra con patógenos bacterianos intracelulares

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

Investigación

Inmunología e Infección

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.

Microinyección de embriones de pez cebra para analizar la función génica

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

Biología

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.

Micromanipulación de la expresión génica en el cerebro de pez cebra adulto Uso Microinyección cerebroventricular de oligonucleótidos Morpholino

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

Neurociencias

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.  ...

Biología del Desarrollo

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.

Entrega directa de Morpholinos FOMIN Hacia el otocyst pez cebra por inyección y electroporación afecta el desarrollo del oído 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.

En electroporación in vivo de Morpholinos en la retina de pez cebra adultos

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.

Invierta genética Enfoque Morpholino Usando ventricular cardíaca inyección para transfectar múltiples tejidos difíciles de conectar por objetivo en el pez cebra 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

Silenciamiento de genes con morfolinos

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

Educación

JoVE Science Education

Biología avanzada

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

Técnicas de microinyección en el pez cebra

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

Biología básica

Biología II: Ratón, Pez cebra y Polluelo

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.