Agradecemos a la Sra. Paula Roy para la cría de peces de excelente, y Steve Ekker para el tipo de regalo el plásmido pDB600. Estos estudios fueron financiados por una donación a San Francisco de NHGRI.

1. Selección y la clonación de secuencias conservadas para las pruebas <ol><li> Primero hay que identificar las posibles secuencias reguladoras de las pruebas funcionales. Contamos con la conservación de una secuencia evolutiva como un criterio para seleccionar las secuencias, y la mayoría utilizan a menudo el PhastCons algoritmo, que es accesible como una pista en la UCSC Genome Browser (http://genome.ucsc.edu/cgi-bin/hgGateway). Sin embargo, hay muchos otros algoritmos disponibles para evaluar la secuencia de conservación entre las especies. </li><li> Una vez que hayamos seleccionado a nuestro secuencias, diseño de cebadores para amplificar cada secuencia de ADN genómico. Cebadores deben ser seleccionados para amplificar la región conservada más 30 o más pares de bases a cada lado. </li><li> Para la amplificación de las secuencias seleccionadas se utiliza el sistema de Takara LA TaqTM. Otras polimerasas va a funcionar, pero es importante utilizar una mezcla que incluye una enzima con capacidad de corrección de pruebas, para reducir al mínimo las posibilidades de errores introducidos durante la amplificación. Verificamos el tamaño de amplificación por electroforesis en gel de agarosa. </li><li> Nosotros usamos la PCR 8/GW/TOPO TA Clonación Kit (Invitrogen) para clonar el producto de PCR en un vector de entrada que contiene los sitios attL recombinación, para generar el clon de la entrada de puerta de enlace. La reacción de la clonación es más eficiente con el nuevo producto de la PCR, lo que es mejor para llevar a cabo la clonación en un día de la PCR. Transformación de las cepas se realiza DH5a seguido por la selección resistencia a la espectinomicina. </li><li> Se verifica que el producto de la clonación TA por digestión de ~ 500 ng del plásmido con EcoRI para liberar la pieza, y confirmar el tamaño de la pieza por electroforesis en gel de agarosa. Se recomienda la secuenciación para verificar la secuencia de inserción y orientación; cebadores utilizados para la amplificación también puede ser utilizado para la secuenciación. </li><li> Desde el clon de la entrada, la secuencia no codificadora se conserva transportados por recombinación LR en nuestro vector de puerta de enlace destino listo, pGW_cfosGFP1, con puerta de enlace LR Clonase II de la enzima Mix (Invitrogen). Transformación de las cepas se realiza DH5a seguido por la selección de resistencia a ampicilina. </li><li> Se verifica que el producto de la recombinación LR por la digestión de ~ 500 ng del plásmido con EcoRV para liberar el inserto, y confirmar el tamaño de la pieza por electroforesis en gel de agarosa. Una vez que un clon exacto ha sido identificado, nos preparamos con el ADN del plásmido HiSpeed ​​Qiagen ® Kit Midi plásmido. Estamos más purificar el plásmido con el Kit QIAquick PCR Purificación, de acuerdo a los protocolos del fabricante, y eluir el ADN con 30 l RNasa libre de agua. El plásmido se diluye a una concentración de 125 ng / mL y almacenadas a 4 ° C antes de las inyecciones. </li><li> ARN que codifican funcional Tol2 enzima transposasa se ​​transcribe in vitro de la pDB600 plasmid2. Podemos purificar el plásmido con el Qiagen Midi-Prep kit, y luego alinear 10-20 mg con XbaI. La síntesis de ARNm se lleva a cabo siguiendo el protocolo mMESSAGE mMACHINE Kit (Ambion). </li><li> Nos purifica y precipitar el ARN de acuerdo con las instrucciones del kit. Nos resuspender ARN a una concentración final de ~ 1μg/μl, o 20 l para una sola reacción, en RNasa libre de agua, y cuantificar mediante espectrofotometría UV. Asimismo, se analizan ~ 1 g de ARN mediante electroforesis en gel de agarosa para verificar la transcripción de larga duración. A pesar de un estándar de TAE o TBE gel es adecuado para este análisis, la muestra de amortiguación desnaturalización incluido en el kit de transcripción se debe utilizar de acuerdo a las instrucciones del kit. </li><li> Probamos cada nuevo lote de transposasa de ARN para la eficacia mediante la realización de inyecciones con un conocido plásmido (Figura 1). Tras la verificación, el ARN se divide en pequeñas partes alícuotas y almacenados a -80 ° C, para evitar la descongelación repetida y volver a congelar de alícuotas individuales. <img src="/files/ftp_upload/1722/1722fig1.jpg" alt="Figura 1" /> Figura 1. Embrión de pez cebra inyectados con el potenciador del ratón SOX10 como control. (Arriba) imagen campo claro (inferior) de imágenes de fluorescencia de GFP. Cada nuevo lote de transposasa ARN es la prueba de la eficacia. </li></ol> 2. Microinyección de embriones de pez cebra para el análisis de mosaico transgénicos <ol><li> Tiramos las agujas para la microinyección de 1,2 mm de diámetro exterior de vidrio de filamento capilar en un Sutter P-97 Extractor Micropipeta, con un programa diseñado para dar un consejo fuerte, con una inclinación bastante agudo para penetrar coriones intacto. Rompemos las puntas a mano bajo un microscopio estereoscópico a un diámetro exterior de aproximadamente 15 micras, con una cuchilla limpia y un tobogán de micrómetro. Las agujas se puede hacer el día antes de las inyecciones, y se almacena en un plato de explotación objeto de mantener limpio. </li><li> Mantenemos nuestro pez cebra en un ciclo regular oscura luz, con 14 horas de luz, bajo la norma conditions3. El día antes de la realización de microinyecciones, hemos creado la pesca de apareamientos tiempo en los tanques de cría de pequeños, cada uno con un tanque de base, una inserción de ranuras, y una tapa de plástico. Ponemos tres hembras y dos machos por tank en filas paralelas. </li><li> En la mañana de la micro-inyecciones, poco después comienza el ciclo de luz, comenzamos la combinación de hombres y mujeres en la limpieza del sistema de agua tratada para la producción de huevos. Es importante comprobar el pescado con frecuencia, y recoger los huevos en unos pocos minutos de la puesta, para obtener lotes bien sincronizada. La producción de huevos se puede mantener durante dos o tres horas, al continuar combinar nuevos grupos de peces a lo largo de la mañana. </li><li> Con una amplia gama de diámetro, 5 1 / 4 &quot;pipeta Pasteur de vidrio provista de un bulbo de látex, que ordenar los embriones obtenidos en 60 x 15 mm platos Petri, parcialmente lleno de embriones Medium2, en grupos de 50 embriones, y marcar el tiempo de recogida de la tapa de cada plato. </li><li> Una vez que los peces han comenzado a sentar, preparamos una solución nueva inyección mediante la mezcla de lo siguiente en un tubo de microcentrífuga en hielo: <table border="1"><tbody><tr><td> Componente </td><td></td></tr><tr><td> Transposón plásmido de acciones (125ng/μl) </td><td> 1μl </td></tr><tr><td> Transposasa ARN de acciones (175ng/μl) </td><td> 1μl </td></tr><tr><td> Fenol sangre roja (2% en H 2 O) </td><td> 0.5μl </td></tr><tr><td> RNasa libre de agua </td><td> 2.5μl </td></tr></tbody></table> Las agujas de inyección se colocan en la celebración de los platos y cubiertos por pipeteo 500 gotas nl de solución inyectable en el extremo ancho de cada aguja. Después de que el líquido se extrae a la punta por capilaridad, la solución adicional puede ser añadido a un total de 1,5 a 2 l. Nos preparamos por lo menos dos agujas para cada solución de la inyección. </li><li> La aguja llena se carga en la mano porta-agujas de un neumático Pico-bomba o similares en la presión de inyección, configurada y conectada a un tanque de N2 por las instrucciones del fabricante. Nos calibrar el volumen de inyección, midiendo el diámetro de las gotas expulsadas al aceite mineral sobre un portaobjetos de micrómetro. Por lo general, un tiempo de inyección de 120 ms con una presión de 20 psi dará lugar a una gota de aproximadamente 1 nl, pero pequeñas variaciones en la aguja de diámetro afectará a estos parámetros y calibración puede ser necesaria entre las agujas. </li><li> Las inyecciones se realizan bajo un microscopio estereoscópico a las 6-10X de aumento. Nosotros usamos un par de pinzas finas para retener el embrión en su lugar, introducir la aguja de la inyección con una presión constante a través del corion y en la yema de un embrión en la celda de uno o dos primeras etapas de la célula. Lo ideal es que la posición de la punta de la aguja en la yema justo por debajo de los blastómeros. Aproximadamente 1 nl de solución inyectable es expulsado y se retira la aguja. Para construir cada uno se inyecta ≥ 200 embriones. También es muy importante, para cada nidada de embriones, para salvar a un plato de control de los embriones inyectados. </li><li> Después de las inyecciones que se han completado ordenar los embriones mediante la eliminación de huevos no fecundados, embriones dañados, y las inyecciones no (embriones sin rojo de fenol en blastómeros). A continuación, se incuban los embriones en un medio de embrión a 28,5 ° C hasta el momento apropiado para la observación. </li></ol> 3. Análisis de patrones de expresión del mosaico <ol><li> Después de un tiempo de incubación adecuado, se examinan los embriones inyectados por fluorescencia. El tiempo dependerá del patrón de expresión normal del gen del pez cebra endógena, pero generalmente se ven todos los días durante los primeros 5 días después de la fecundación. </li><li> Se examinan los embriones de fluorescencia en un macroscopio Olympus MVX10 equipado para epifluorescencia, pero cualquier microscopio capaz de obtener imágenes similares bajo consumo de energía va a funcionar. Si hay un gran número de embriones que mueren o un desarrollo anormal en el primer día, mira los controles no inyectados para que el embrague para ver si se ven normales. Si los controles de aspecto normal, el problema más común es la pureza insuficiente del ADN inyectado. Además, los embriones en las primeras etapas son sensibles a la cantidad total de plásmido se inyecta, por lo que es posible que la cuantificación es inexacta y el ADN se inyecta demasiado. </li><li> Al examinar los embriones, tenga en cuenta que todos ellos serán de mosaico para la construcción de inyección. Será necesario examinar cuidadosamente todos los embriones, sobre todo si el dominio esperado de expresión es pequeño. También recuerda que la expresión puede ser dinámico, fuerte fluorescencia en el día 1 podrían desaparecer para el día 2, y los embriones negativos para la expresión de los 3 primeros días podrían mostrar algo en el día 4. </li><li> Hemos escogido algunos embriones con la expresión más amplia, y el uso de estos para documentar el patrón de fluorescencia por photomicroscopy. Después de 5 días de observaciones, los embriones deben ser devueltos a las instalaciones y se crió en una población. En varios meses, los adultos pueden ser examinados para la transmisión de la línea germinal de los transgenes. </li></ol> 4. Resultados Vemos una gran variedad de los patrones y los niveles de expresión, según el elemento potenciador que se analiza. Por lo general estamos muy interesados ​​en los patrones de expresión específica de tejido, y con frecuencia se centra en los genes expresados ​​en el esqueleto. y con frecuencia se centra en los genes expresados ​​en el esqueleto. La figura 2 muestra ejemplos de los patrones de expresión de mosaico que hemos observado en nuestros embriones inyectados, con potenciadores de regulación de la expresión en el cartílago y el hueso. Finalmente, una parte sustancial de las secuencias de prueba no regulan la expresión específica de tejido. Para estos, la mayoría ven a menudo fluorescencia muy poco, tal vez unas pocas células esparcidas por embrión. Con menos frecuencia, podríamos ver la fluorescencia, pero sólo en dos sitios comunes para la expresión ectópica, la epidermis y el músculo esquelético (Figura 3). <img src="/files/ftp_upload/1722/1722fig2.jpg" alt="Figura 2" /> Figura 2. Ejemplo de patrones de expresión de cartílago y hueso observados en los embriones inyectados. <img src="/files/ftp_upload/1722/1722fig3.jpg" alt="Figura 3" /> Figura 3. Hay poca correlación entre la ubicación de la relación potenciador de la regulación de genes y de expresión. Una parte sustancial de las secuencias de prueba no regulan la expresión específica de tejido. Estos a menudo tienen fluorescencia muy poco, o tener dos sitios comunes para la expresión ectópica: la epidermis y el músculo esquelético. (Arriba) imagen campo claro (abajo) la fluorescencia de GFP de la imagen.

<ol>
	<li>Fisher, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+biological+relevance+of+putative+enhancers+using+Tol2+transposon-mediated+transgenesis+in+zebrafish.">Evaluating the biological relevance of putative enhancers using Tol2 transposon-mediated transgenesis in zebrafish.</a> Nat Protoc. 1 (3), 1297-12 (2006).</li><li>Balciunas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+a+high+cargo-capacity+transposon+for+genetic+applications+in+vertebrates.">Harnessing a high cargo-capacity transposon for genetic applications in vertebrates.</a> PLoS Genet. 2 (11), e169-169 (2006).</li><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. , (1995).</li></ol>

Hemos demostrado el método utilizado en nuestro laboratorio para el análisis eficiente de los potenciadores del potencial uso de la transgénesis de pez cebra. Muy a menudo, se utiliza este método para buscar tejido-específica potenciadores asociados con genes humanos. A pesar de la falta de homología de secuencia más evidente de la época entre los humanos y los peces secuencias no codificantes, nos encontramos con que este enfoque suele tener éxito en la identificación de los potenciadores de los genes de interés para nosotros. Los factores críticos de éxito son el cuidado en la preparación del plásmido de ADN y el ARN transposasa, y la inyección y el examen de un número suficiente de embriones para cada construcción. Los métodos generales de la construcción transgénica, microinyecciones de embriones, y análisis de la expresión del mosaico sería aplicable a la generación de líneas transgénicas de pez cebra para muchos otros fines.

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		<div>
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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Takara LA Taq</td>
<td>&nbsp;</td>
<td>Takara Bio Inc</td>
<td>RR02M</td>
<td>&nbsp;</td>
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<td>pCR8/GW/TOPO TA Cloning Kit</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>K2500-20</td>
<td>&nbsp;</td>
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<td>Gateway LR Clonase II enzyme Mix</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11791-100</td>
<td>&nbsp;</td>
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<td>Library efficiency competent cells DH5&alpha;</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12535-019</td>
<td>&nbsp;</td>
<tr>
<td>Library efficiency competent cells DB3.1</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11782-018</td>
<td>&nbsp;</td>
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<td>mMESSAGE mMACHINE Kit</td>
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<td>12643</td>
<td>&nbsp;</td>
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<td>QIAquick PCR Purification Kit </td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>28104</td>
<td>&nbsp;</td>
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<td>Flaming/Brown Micropipette Puller</td>
<td>&nbsp;</td>
<td>Sutter Instrument</td>
<td>P-97</td>
<td>&nbsp;</td>
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<td>Pneumatic PicoPump</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>SYS-PV820</td>
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mosaic zebrafish transgenesis for evaluating enhancer sequences

La finalización de la secuencia del genoma humano, junto con la de muchas otras especies, ha puesto de relieve el desafío de atribuir funciones específicas a la no codificación de secuencias. Una de las funciones destacadas llevadas a cabo por la fracción no codificantes del genoma es la de regular la transcripción de genes, sin embargo, no existe ningún método eficaz para predecir ampliamente cis-elementos reguladores de la secuencia primaria del ADN. Hemos desarrollado un protocolo eficaz para evaluar funcionalmente posibles elementos reguladores cis-a través de la transgénesis de pez cebra. Nuestro enfoque ofrece ventajas significativas sobre las técnicas de cultivo celular basada en los genes de desarrollo importantes, ya que proporciona información sobre la regulación genética espacial y temporal. Por el contrario, es más rápido y menos costoso que experimentos similares en ratones transgénicos, y que habitualmente se aplica a las secuencias aisladas del genoma humano. Aquí demostramos nuestro enfoque a la selección de los elementos de prueba sobre la base de la secuencia de conservación y el protocolo para las secuencias de la clonación y la microinyección en embriones de pez cebra.

Watch this Scientific Journal Video about Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences at JoVE.com

Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences

Demostramos nuestro enfoque a la búsqueda de posibles elementos potenciadores de los genes de desarrollo regulado y la evaluación de su función a través de la transgénesis mosaico de pez cebra.

Mosaico transgénesis pez cebra para la evaluación de secuencias potenciadoras

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non ...

mosaic-zebrafish-transgenesis-for-evaluating-enhancer-sequences

Universidad San Sebastian

Research

JoVE Journal

Biology

13.9K vistas.  University of Pennsylvania. Demostramos nuestro enfoque a la búsqueda de posibles elementos potenciadores de los genes de desarrollo regulado y la evaluación de su función a través de la transgénesis mosaico de pez cebra.

Video: Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences

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 ...

Investigación

Biología

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 ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

En electroporación in vivo de Morpholinos en la regeneración de adultos de pez cebra Aleta de cola

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Blood Collection for Biochemical Analysis in Adult Zebrafish

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies1. 
There are relatively few publications related to adult zebrafish physiology of organs and systems2, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking3. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues4 who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta5. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year6. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish7. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al.8. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. 
Recently, Gupta et al.9 described how to dissect adult zebrafish organs, Kinkle et al.10 described how to perform intraperitoneal injections, and Pugach et al.11 described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish.
The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.

This paper presents a technique for collection of blood from the dorsal aorta of Zebrafish. It also provides instructions for obtaining serum for use in biochemical analyses, such as tests for determining cholesterol and triglyceride levels.

Extracción de sangre para análisis bioquímicos en adultos de pez cebra

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

Celular seguimiento mediante fotoconvertible proteínas durante el desarrollo de pez cebra

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

Laser-infligido lesiones de músculo esquelético embrionario de pez cebra

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

El tomate / GFP-FLP / FRT Método para imágenes en vivo del mosaico adultos<em&gt; Drosophila</em&gt; Las células fotorreceptoras

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation

Mammalian spermatogenesis is a complex differentiation process that occurs in several stages in the seminiferous tubules of the testes. Currently, there is no reliable cell culture system allowing for spermatogenic differentiation in vitro, and most biological studies of spermatogenic cells require tissue harvest from animal models like the mouse and rat. Because the testis contains numerous cell types - both non-spermatogenic (Leydig, Sertoli, myeloid, and epithelial cells) and spermatogenic (spermatogonia, spermatocytes, round spermatids, condensing spermatids and spermatozoa) - studies of the biological mechanisms involved in spermatogenesis require the isolation and enrichment of these different cell types. The STA-PUT method allows for the separation of a heterogeneous population of cells - in this case, from the testes - through a linear BSA gradient. Individual cell types sediment with different sedimentation velocity according to cell size, and fractions enriched for different cell types can be collected and utilized in further analyses. While the STA-PUT method does not result in highly pure fractions of cell types, e.g. as can be obtained with certain cell sorting methods, it does provide a much higher yield of total cells in each fraction (~1 x 108 cells/spermatogenic cell type from a starting population of 7-8 x 108 cells). This high yield method requires only specialized glassware and can be performed in any cold room or large refrigerator, making it an ideal method for labs that have limited access to specialized equipment like a fluorescence activated cell sorter (FACS) or elutriator.

The STA-PUT method allows for the separation of different populations of spermatogenic cells based on size and density.

La separación de los tipos de células de espermatogénesis Usando STA-PUT Sedimentación Velocity

Mammalian spermatogenesis is a complex  differentiation process that occurs in several stages in the seminiferous  tubules of the testes. Currently, there ...

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca2+ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca2+ signaling.

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.

Análisis automatizado de Dinámica Ca<sup&gt; 2 +</sup&gt; Las señales en secuencias de imágenes

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity greatly impacts cell behavior to regulate normal and pathologic processes including cell proliferation, differentiation, adhesion signaling and directional migration. In this regard, the innate ability of certain cell types to migrate towards a stiffer, or less compliant matrix substrate is referred to as durotaxis. This phenomenon plays an important role during embryonic development, wound repair and cancer cell invasion. Here, we describe a straightforward assay to study durotaxis, in vitro, using polydimethylsiloxane (PDMS) substrates. Preparation of the described durotaxis chambers creates a rigidity interface between the relatively soft PDMS gel and a rigid glass coverslip. In the example provided, we have used these durotaxis chambers to demonstrate a role for the cdc42/Rac1 GTPase activating protein, cdGAP, in mechanosensing and durotaxis regulation in human U2OS osteosarcoma cells. This assay is readily adaptable to other cell types and/or knockdown of other proteins of interests to explore their respective roles in mechanosignaling and durotaxis.

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

Many mammalian cells preferentially migrate towards a more rigid matrix or substrate through durotaxis. The goal of this protocol is to provide a simple in vitro system that can be used to study and manipulate cell durotaxis behaviors by incorporating polydimethylsiloxane (PDMS) substrates of defined rigidity, interfacing with glass coverslips.

Un sistema simplificado para la evaluación de la célula Mechanosensing y Durotaxis<em&gt; In Vitro</em

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity ...