Este trabajo fue apoyado por la Deutsche Forschungsgemeinschaft (DFG), queremos agradecer Ayele Tsedeke Taddese para soporte técnico.

1. Preparación de las agujas de vidrio (Figura 1) <ol><li> Utilice capilares de vidrio con un diámetro de 0,75 mm para preparar agujas de inyección (Figura 1A). </li><li> Coloque el capilar de vidrio en un extractor de aguja y tire de la aguja con el siguiente parámetro: valor del ciclo de calentamiento: 463; tirando de valor del ciclo: 230; velocidad: 150 mseg; tiempo: 150 ms (Figura 1B). </li><li> Romper el capilar de vidrio tirado con pinzas de relojero para producir una aguja de 20 m de diámetro bajo un estereoscopio con un ocular micrométrico. </li><li> Utilice un torno con una rueca de goma mojada para afilar la aguja y producir unas 20 micras de bisel (figuras 1C y 1D). Nota: agudo, agujas biseladas mejorar la facilidad de inserción en el ventrículo y por lo tanto minimizar el daño al tejido y permitir que el músculo perforada para volver a sellar después de la retirada de la aguja (figuras 1E-G). </li></ol> 2. Preparación de la Solución Morpholino <ol><li> Preparar la población de morfolino disolviendo el morfolino liofilizado en 1x solución salina tamponada con fosfato (PBS) a una concentración final de 7,5 mM. [Vea las instrucciones del fabricante para obtener más información (tabla Materiales)]. </li><li> Preparar la solución de inyección morfolino mezclando disolución 2,5 l morfolino acciones (7,5 mM) con 2,8 l solución Endo-Porter de valores (1 mM) (Ver tabla Materiales) para obtener una concentración final de 3,5 mM morfolino y 0,5 mM Endo-Porter. </li></ol> 3. Preparación de la inyección de morfolino (Figura 2) <ol><li> Cargue la aguja de vidrio biselado con 5 l de esta solución utilizando una micropipeta con una punta de pipeta microloader 10 l. </li><li> Insertar la aguja de vidrio en el soporte de la aguja de la micromanipulador conectada a la bomba neumática Pico (Figuras 2A-C). </li><li> Coloque el soporte de la aguja al lado del microscopio, de manera que sólo las necesidades de la agujapara moverse en una dirección plana para insertarla en el ventrículo cardíaco de la larva (Figura 2A). </li><li> Ajuste el ángulo de la inyección en los alrededores de 45 °. </li><li> Establezca los valores microinyector como sigue: presión de mantenimiento: 20 libras por pulgada cuadrada (psi); presión de expulsión: 15 psi; Alcance de 100 ms de gating, valor del periodo de 1,9 (corresponde a 10,9 ms). </li><li> Derretir de agarosa en 1x PBS utilizando un horno de microondas estándar para producir 20 ml de un 1,5% (w / v) en gel. Verter en gel fundida en un plato de Petri de 10 cm. Coloque una forma de inyección ranurado en la agarosa caliente para que una vez endurecido, la agarosa tendrá surcos en los que la larva sedado se colocará 11 (Figuras 2D y 2E). </li></ol> 4. Inyecciones (Figura 3) <ol><li> Anestesie larva en 100 ml de agua del acuario con 20 mg / L tricaína (sulfonato L-etil-m-amino-benzoato-metano) hasta que dejan de responder al tacto. </li><li> Utilice una Pasteur pipetee plásticoe para transferir cuidadosamente la larva sedado en una ranura del molde de agarosa húmeda de modo que el lado ventral se enfrenta contra la pared de agarosa vertical de la ranura (Figura 3A). </li><li> Coloque la placa de agarosa bajo el microscopio estereoscópico para que el ventrículo está de espaldas a la aguja de inyección (Figura 3A). </li><li> Baje la aguja para insertarla en el ventrículo del corazón. Inserte la aguja sólo 1-2 micras en el ventrículo teniendo cuidado de no insertar la aguja profundamente (Figuras 3B y 3C). </li><li> Una vez que se inserta la aguja, se inyecta la solución morfolino al ventrículo con 4-6 pulsos, cada entrega de 3 nl de la solución, con intervalos de espera para permitir la limpieza del corazón (Figuras 3D y 3E). </li><li> Después de la inyección, retire la aguja (paralelo al plano de la inserción) y, a continuación transferir cuidadosamente la larva usando una pipeta Pasteur de plástico llena de E3 medio BACk en una placa de Petri que contiene medio de E3 fresco. </li><li> Coloque la aguja en una placa de Petri que contiene PBS 1x para evitar el secado de la aguja cuando la transferencia de la larva inyectado. </li><li> Repita los pasos 4.1 a 4.6 de cada 12 a 24 horas durante la duración del experimento. Nota: La repetición de las inyecciones asegura la absorción y el mantenimiento de la morfolino en las células. </li></ol> 5. Los análisis de las inyecciones (Figura 4) <ol><li> Anestesiar los peces en 100 ml de agua del acuario con 20 mg / L tricaína hasta que dejan de responder al tacto. </li><li> Coloque la larva lateralmente en un piso mojado 1,5% (w / v) de agarosa placa de cubierta con medio E3. </li><li> Larvas de imagen usando un microscopio estereoscópico con campo claro y fluorescencia de imagen. Nota: Como las larvas son transparentes, la fluoresceína morfolino-etiquetado debe ser visible como verde fluorescente en los vasos sanguíneos en todo el animal directamente después de la inyección. Esta fluorescencia se dispersará más en los tejidos vascularizados por15 min (Figuras 4A-4C). </li></ol> 6. Evaluación de Regenerativa excrecencia <ol><li> Evaluar las imágenes capturadas utilizando Fiji Image J software libre (http://fiji.sc/Fiji). Para la investigación de la regeneración, utilice la herramienta de línea de trazado para determinar la cantidad de crecimiento regenerativo que se ha producido en el control y la larva morpholino inyectado. </li><li> Para calibrar las imágenes, abrir uno de los archivos de imagen originales en Fiji por arrastrar y soltar el archivo en la ventana principal de Fiji. </li><li> Para definir la escala para todas las siguientes imágenes, vaya a &quot;Analizar&quot; en el menú principal. </li><li> En el menú desplegable, haga clic en &quot;escala Ajuste&quot;. </li><li> En esta ventana, active la opción &quot;Global&quot; haciendo clic en la casilla de verificación. </li><li> Ahora abra la imagen para medir la cantidad de crecimiento regenerativo de nuevo por arrastrar y soltar (véase el paso 6.1). </li><li> Elija la herramienta &quot;selecciones a mano alzada&quot; en la barra de herramientas. </li><li> Rodear el área a ser medido (Figuras 4J-4L). </li><li> Presione Ctrl + M. Esto abrirá una nueva ventana &quot;Resultados&quot; muestra el valor del área cercada en píxeles cuadrados. </li></ol>

<ol>
	<li>Gemberlin, M., Bailey, T. J., Hyde, D. R., Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+as+a+model+for+complex+tissue+regeneration.">The zebrafish as a model for complex tissue regeneration.</a> Trends in Genetics. 29, 611-620 (2013).</li><li>Suster, M. L., Abe, G., Schouw, A., Kawakami, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposon-mediated+BAC+transgenesis+in+zebrafish.">Transposon-mediated BAC transgenesis in zebrafish.</a> Nature Protocols. 6, 2011 (1998).</li><li>Skromne, I., Prince, V. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+perspectives+in+zebrafish+reverse+genetics:+moving+forward.">Current perspectives in zebrafish reverse genetics: moving forward.</a> Developmental Dynamics. 237, 861-882 (2008).</li><li>Zu, Y., 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=TALEN-mediated+precise+genome+modification+by+homologous+recombination+in+zebrafish.">TALEN-mediated precise genome modification by homologous recombination in zebrafish.</a> Nature Protocols. 10, 329-332 (2013).</li><li>Bedell, V. M., 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=In+vivo+genome+editing+using+a+high-efficiency+TALEN+system.">In vivo genome editing using a high-efficiency TALEN system.</a> Nature. 491, 114-118 (2012).</li><li>Kizil, C., IItzsche, A., Kaslin, J., Brand, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromanipulation+of+gene+expression+in+the+adult+zebrafish+brain+using+cerebroventricular+microinjection+of+morpholino+oligonucleotides.">Micromanipulation of gene expression in the adult zebrafish brain using cerebroventricular microinjection of morpholino oligonucleotides.</a> J Vis Exp. , (2013).</li><li>Hyde, D., Godwin, A., Thummel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+electroporation+of+morpholinos+into+the+regenerating+adult+zebrafish+tail+fin.">In vivo electroporation of morpholinos into the regenerating adult zebrafish tail fin.</a> J Vis Exp. , (2012).</li><li>Thummel, R., Bailey, T., Hyde, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+electroporation+of+morpholinos+into+the+adult+zebrafish+retina.">In vivo electroporation of morpholinos into the adult zebrafish retina.</a> J Vis Exp. , (2011).</li><li>Rieger, S., Kulkarni, R. P., Darcy, D., Fraser, S. E., K&#246;ster, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantum+dots+are+powerful+multipurose+vital+labeling+agents+in+zebrafish+embryos.">Quantum dots are powerful multipurose vital labeling agents in zebrafish embryos.</a> Developmental Dynamics. 234, 670-681 (2005).</li><li>Madhavan, M., 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=The+role+of+Pax-6+in+lens+regeneration.">The role of Pax-6 in lens regeneration.</a> Proceedings of the National Academy of Science U. 103, 14848-14853 (2006).</li><li>Gilmour, D. T., Jessen, J. R., Lin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Zebrafish: a practical approach. 261, 121-143 (2000).</li><li>Schindelin, J., 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9, 676-682 (2012).</li><li>Eisen, J. S., Smith, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+morpholino+experiments:+don't+stop+making+antisense.">Controlling morpholino experiments: don't stop making antisense.</a> Development. 135, 1735-1743 (2008).</li><li>Bill, B. R., Petzold, A. M., Clark, K. J., Schimmenti, L. A., Ekker, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+primer+for+morpholino+use+in+zebrafish.">A primer for morpholino use in zebrafish.</a> Zebrafish. 6, 69-77 (2009).</li><li>Hyatt, T. M., Ekker, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vectors+and+techniques+for+ectopic+gene+expression+in+zebrafish.">Vectors and techniques for ectopic gene expression in zebrafish.</a> Methods Cell Biol. 59, 117-126 (1999).</li><li>Hans, S., Kaslin, J., Freudenreich, D., Brand, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporally+controlled+site-specific+recombination+in+zebrafish.">Temporally controlled site-specific recombination in zebrafish.</a> PLoS One. 4, (2009).</li><li>Lee, Y., Grill, S., Sanchez, A., Murphy-Ryan, M., Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fgf+signaling+instructs+position-dependent+growth+rate+during+zebrafish+fin+regeneration.">Fgf signaling instructs position-dependent growth rate during zebrafish fin regeneration.</a> Development. 132, 5173-5183 (2005).</li><li>Liu, S., D, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+models+for+cancer.">Zebrafish models for cancer.</a> Annu. Re. Pathol. Mech. Dis. 6, 71-93 (2011).</li><li>Stoick-Cooper, C. L., 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=Distinct+Wnt+signaling+pathways+have+opposing+roles+in+appendage+regeneration.">Distinct Wnt signaling pathways have opposing roles in appendage regeneration.</a> Development. 134, 479-489 (2007).</li><li>Shen, S. P., Aleksic, J., Russell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+targets+of+the+Sox+domain+proteins+Dichaete+in+the+Drosophila+CNS+via+targeted+expression+of+dominant+negative+proteins.">Identifying targets of the Sox domain proteins Dichaete in the Drosophila CNS via targeted expression of dominant negative proteins.</a> BMC Dev. Biol. 13, (2013).</li><li>McNeish, J. D., Scott, W. J., Potter Jr, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Legless,+a+novel+mutation+found+in+PHT1-1+transgenic+mice.">Legless, a novel mutation found in PHT1-1 transgenic mice.</a> Science. 241, 837-839 (1988).</li><li>Covarrubias, L., Nishida, Y., Terao, M., D'Eustachio, P., Mintz, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+DNA+rearrangements+and+early+developmental+arrest+caused+by+DNA+insertion+in+transgenic+mouse+embryos.">Cellular DNA rearrangements and early developmental arrest caused by DNA insertion in transgenic mouse embryos.</a> Mol. Cell. Biol. 7, 2243-2247 (1987).</li></ol>

Los autores no tienen nada que revelar.

La inyección intraventricular proporciona un método de evaluación rápida y fiable para probar la función de genes en las etapas posteriores del desarrollo o de la homeostasis del cuerpo sin afectar a la función del gen durante la embriogénesis. Para asegurar el éxito de esta técnica, uno debe ser consciente de los cuatro puntos críticos: 1) Tamaño de la aguja, 2) la desecación de la aguja, 3) reducir al mínimo el volumen, y 4) el tiempo mínimo de exposición en la solución de la sedación. Agujas que son ...

La regeneración de órganos y apéndices es de fundamental importancia para la supervivencia y la aptitud; sin embargo, varios vertebrados, incluido el hombre han limitado las capacidades regenerativas. Aunque existen varios modelos animales que tienen una amplia capacidad de regeneración, las técnicas de genética inversa para evaluar la función de genes durante la regeneración de órganos y apéndices siguen siendo muy limitada o inexistente. Por lo tanto, se necesitan nuevos enfoques para diseccionar la biología molecular de la regeneración en estos organismos modelo. El pez cebra es un modelo bien establecido para la comprensión de la biología celular y molecular de la regeneración de órganos y apéndice 1, no sólo debido a su capacidad significativa para regenerar múltiples órganos, tejidos y apéndices, sino también porque existen varias líneas de peces transgénicos para rastrear las células y para sobreexpresar genes construye 2, 3. Sin embargo, la inhibición de genes en larvas de pez cebra se limita principalmente a la overexpression de las construcciones dominantes negativos, que no están disponibles para todos los genes de interés o cuyo producto transgénico puede adquirir con ganancia de función de los efectos que no reflejan la actividad endógena del gen. Por lo tanto, se necesita un método alternativo para eliminar específicamente la expresión de genes por knockout o knockdown para superar estos problemas. Específica la orientación de genes usando TALENS existe como un medio de genética inversa para octavos de final la función de genes; Sin embargo, esta estrategia eliminatoria es muy limitado con frecuencia a evaluaciones funcionales durante la embriogénesis temprana, porque los requisitos iniciales del gen impiden la progresión del desarrollo embrionario. Por lo tanto, el estudio de los fenómenos posteriores como la regeneración o la homeostasis de órganos después de desarrollo utilizando TALENS está impedido 4, 5. Por lo tanto, se necesita una estrategia alternativa de eliminación de genes que se dirige a la función de genes después del desarrollo temprano para evaluar los requisitos de genes en los órganos y estructuras completamente formados. </p&gt; Morfolino inyección se ha demostrado ser eficaz en la orientación de los genes en un par de órganos de adultos y el adulto regeneración de aleta 6-8, pero estos métodos requieren la electroporación y muchos órganos internos son difíciles de electroporar ya sea debido a su ubicación o debido a su sensibilidad a eléctrica interrupción. Por otra parte, algunos tejidos de la larva son difíciles de inyectar directamente, ya que la inyección directa puede alterar su integridad estructural o porque su tamaño es limitante. La aleta caudal de la larva es un tal estructura, debido a la inyección directa en el pliegue de la aleta no es posible. Por lo tanto, se necesita una alternativa a la electroporación y la inyección directa de los genes objetivo en los tejidos que son demasiado pequeña para inyectar o no puede ser a electroporación. Con el fin de dirigir e inhibir la función de genes específicos durante la regeneración de la aleta caudal de larvas, hemos modificado tecnologías de morfolino existentes permitiendo que el unVALUACIÓN de la función génica durante la regeneración de la aleta caudal en larva-etapas finales. Este método emplea la entrega intraventricular 9 de morfolinos fluoresceína etiquetado junto con Endo-Porter transfección reactivo 10. Una vez en el ventrículo, la mezcla morfolino-Endo-Porter se propaga rápidamente a través de la larva a través de la vasculatura y entra en los tejidos que han sido previamente imposible para apuntar. Este método de inyección puede ser modificado para los genes objetivo en tejidos específicos y muy posiblemente puede ser aplicado en otros modelos animales que actualmente carecen de métodos de genética inversa para inhibir la función del gen. Así, se ofrece un método rápido y fácil con el potencial de uso de gama amplia para estudiar de inmediato la función de genes durante la homeostasis de órganos en general y la regeneración en las etapas larvales.

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

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

El pez cebra es un modelo importante para comprender la biología celular y molecular de la regeneración de órganos y apéndice. Sin embargo, las estrategias moleculares para emplear genética inversa todavía no se han desarrollado adecuadamente para evaluar la función del gen en la regeneración o la homeostasis del tejido durante las etapas de larvas de pez cebra después de la embriogénesis, y varios tejidos dentro de la larva de pez cebra son difíciles de orientar. Inyecciones intraventriculares de morfolinos de genes específicos ofrecen un método alternativo para la incapacidad actual para apuntar genómicamente genes de pez cebra de una manera controlada temporalmente en estas etapas. Este método permite la dispersión completa y la posterior incorporación de la morfolino en varios tejidos de todo el cuerpo, incluyendo estructuras que antes eran imposibles de alcanzar, tales como los de la aleta caudal de larvas, una estructura a menudo utilizado para investigar de forma no invasiva la regeneración de tejidos. Varios genes activados durante la regeneración pliegue de la aleta larval también se Present en la regeneración de tejidos de vertebrados adultos, por lo que la larva es un modelo útil para entender la regeneración en adultos. Este método de dispersión morfolino permite la identificación rápida y fácil de los genes necesarios para la regeneración de los tejidos de las larvas, así como otros fenómenos fisiológicos que regulan la homeostasis del tejido después de la embriogénesis. Por lo tanto, este método de entrega proporciona una estrategia necesaria en la actualidad para el control temporal de la evaluación de la función de genes después de la embriogénesis.

La aguja de inyección agudo, biselado se coloca fácilmente en el ventrículo cardíaco de la larva de pez cebra cuando se acercó dorsalmente (Figura 3A). El corazón continúa bombeando y se mantiene el flujo de sangre a pesar de la presencia de la aguja (Figuras 3B y 3C). Inyecciones de cuidado no alteran la morfología del ventrículo o contracciones cardíacas (Figura 3D) a pesar de la inyección de la morfolino en el corazón (Figura 3E)....

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

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

Un método de genética inversa adaptable para el pez cebra para evaluar la función de genes durante las etapas posteriores del desarrollo y la homeostasis fisiológica, tales como la regeneración de tejidos usando inyecciones intraventriculares de morfolinos de genes específicos.

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 ...

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

Research

JoVE Journal

Biology

10.5K vistas.  Technische Universität Dresden.  Un método de genética inversa adaptable para el pez cebra para evaluar la función de genes durante las etapas posteriores del desarrollo y la homeostasis fisiológica, tales como la regeneración de tejidos usando inyecciones intraventriculares de morfolinos de genes específicos. 

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

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

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

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

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 ...

Investigación

Biología

Zebrafish Brain Ventricle Injection

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

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

Cerebro del pez cebra ventrículo inyección

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

Retro-orbital Injection in Adult Zebrafish

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

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

Retro-orbital de inyección en adultos de pez cebra

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

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

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

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

Utilizando el sistema de electroporación Gene Emisores MXcell para transfectar células primarias con alta eficiencia

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

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

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

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

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

Un enfoque práctico de elaboración de mapas genéticos inducible destino: una guía visual para marcar y rastrear las células En Vivo</em

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

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

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

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

Un enfoque de genética inversa para probar la redundancia funcional durante la embriogénesis

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

Intraperitoneal Injection into Adult Zebrafish

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

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

Inyección intraperitoneal en adultos de pez cebra

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

Gavaging Adult Zebrafish

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

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

Gavage adultos de pez cebra

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

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

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

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

Nefrotoxina Microinyección en pez cebra para modelar la lesión renal aguda

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

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

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

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

A Multi-agujero criovial Elimina congelación Artifacts cuando los tejidos musculares se sumergen directamente en nitrógeno líquido

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