Este procedimiento sigue en gran medida la técnica de trasplante desarrolladas para peces de las cavernas por el laboratorio de Bill Jeffrey.

Parte 1: Preparación de los embriones En este protocolo, vamos a utilizar el símbolo jj xy para referirse a un pez cebra mutante objetivo hipotético. <ol><li> Cepas de pez cebra se mantienen en condiciones estándar de instalación de los peces a 28,5 C en un ciclo de 14h light/10h oscuro. </li><li> Por la noche, los hombres y las mujeres el lugar del pez cebra cepa xy jj: AB / TU, tg (mGFP) en un tanque con un divisor para separar el uno del otro. La descendencia de este cruce se expresa el transgén GFP en todos los tejidos, y se utilizarán como donantes. Al mismo tiempo, utilizar el mismo procedimiento para establecer los cruces entre los no transgénicos animales de tipo salvaje. Dependiendo del propósito de este experimento, uno puede cruzar animales de tipo salvaje en el locus jj xy como donantes o los ejércitos. </li><li> Una hora después se enciende la luz de la mañana, quitar los separadores que permiten a los peces a su compañero. </li><li> Después de 15-30 minutos recolectar embriones de 100 x 15 mm placas de Petri, limpios, y el cambio del medio (agua de huevo). </li><li> Mantener los embriones en una incubadora de 28,5 C hasta el momento deseado. </li><li> Cuando esté listo para el trasplante, retire el corion manualmente con dos pares de pinzas afiladas, y se incuban los embriones en un 0,2% en EDTA libre de calcio ZFR durante 30 minutos. </li></ol> Parte 2: Preparación de las agujas de disección <ol><li> El procedimiento de trasplante requiere dos tipos de agujas: una afilada, que se utiliza para cortar los tejidos que rodean el lente, quite el corion, y los embriones liberación de agarosa, y una aguja de punta roma para mover la lente de los donantes y la inserta en el host. Aquí le mostraremos cómo hacer que estas agujas. </li><li> En primer lugar, la punta de una pipeta Pasteur se debe cortar con un cuchillo de diamante. A continuación, inserte un capilar de vidrio en la punta de una pipeta Pasteur a fin de que aproximadamente la mitad de su longitud está dentro de él. Esto hará que sea más fácil para inmovilizar el alambre de tungsteno por la fusión en el tarro en el siguiente paso. </li><li> A continuación, inserte un alambre de tungsteno delgado en el extremo abierto del capilar. Fundir el vidrio sobre el alambre con un mechero de Bunsen. Con unas pinzas, se mantienen constantes al final de cristal con el alambre de metal retorcido, mientras que el otro extremo de la aguja con la mano. El vidrio debe suavizado en espiral alrededor del metal se agarre en su lugar. </li><li> Si usted está haciendo una aguja punta roma, entonces este es el final del procedimiento. Para crear una aguja afilada, sujete el alambre de tungsteno sobre un mechero Bunsen durante 1-1,5 minutos, quemando el metal por lo que una punta muy fina se crea. Comprobar la calidad de la punta por microscropy.It es una buena idea para esterilizar las agujas, tanto en la llama durante unos segundos inmediatamente antes del trasplante. </li></ol> Parte 3: Preparación de los reactivos <ol><li> Libre de calcio pez cebra Ringer (ZFR) Soluciones (116 mM NaCl, 2,9 mM KCl, 5 mM HEPES, pH 7,2) </li><li> 0,2% en EDTA libre de calcio pez cebra Ringer Solutions (pH 7,2) </li><li> Agarosa al 1,2% (bajo punto de fusión) en el calcio libre ZFR </li><li> 0,2% de agarosa (bajo punto de fusión) en el calcio libre ZFR </li><li> Medio de embriones (pH 7.0, contiene por litro: 10 ml solución Hanks # 1, 1 ml de solución de Hanks # 2, 10 ml de solución de Hanks # 4, 10 ml de solución de Hanks # 5, 0,35 g de bicarbonato sódico, 300 l de HCl 2 M, penicilina-estreptomicina 500.000 U) Como en el libro El pez cebra (Universidad de Oregon Press, 2000). </li><li> Alambre de tungsteno, chapado en oro de diámetro, 0,1 mm, Alfa Aesar </li><li> Tubo capilar, el diámetro de 1,0 mm, instrumentos de precisión Mundial </li></ol> Parte 4: procedimiento de trasplante <ol><li> En un tubo de centrífuga de plástico, prepare un 1,2% de agarosa de bajo punto de fusión en el calcio libre ZFR precalentado a 40 º C en un baño de agua. </li><li> En la etapa deseada (30 HPF, por ejemplo), ocupan los embriones en una pipeta Pasteur, poco a poco su expulsión en el tubo de centrífuga, y poco a poco su expulsión en el tubo de centrífuga y se incuba durante unos 5 segundos. Los donantes y los anfitriones deben ser incubados por separado. </li><li> Con una pipeta, tomar a los embriones en agarosa al 1,2% de la centrífuga y colocarlos en dos filas paralelas de poliestireno de 100 mm en una placa de Petri, manteniendo los anfitriones y los donantes por separado. Pipeteando hacia fuera despacio y con cuidado, la mayoría de los embriones se encuentran en los lados de la agarosa. Usted tendrá que reorientar las que no se encuentran en esta posición con la aguja de punta roma para que el ojo está mirando hacia arriba antes de la agarosa solidifica. </li><li> Espere a que el 1,2% de agarosa para consolidar, a continuación, superposición con bajo punto de fusión de una solución 0,2% de agarosa. </li><li> Con la aguja afilada, eliminar cualquier agarosa alrededor de los ojos, y corte con cuidado la lente de la Hostia y embriones de donantes con pequeñas pinceladas. Asegúrese de cortar lo suficientemente cerca de la lente para no destruirlo, sino también por lo que no quita exceso de tejido del resto del ojo receptor. </li><li> Una vez libre, lentes de flotar en el medio. Utilizando la aguja de punta roma, empuje con cuidado el donantelente justo por encima de la ubicación de donde la lente de acogida sería normalmente, y luego lo empuja hacia abajo en el ojo con la aguja de punta roma. La lente de acogida puede ser descartado. </li><li> Deja embriones de donantes y de acogida en agarosa al 1,2% durante 30-60 minutos, y luego los libera de la agarosa utilizando la aguja, y transferirlos a una placa de 24 pozos con medio embrión. Cada embrión debe ocupar un bien por separado. Asegúrese de marcar los pozos correctamente de modo que es evidente que corresponde a cada huésped de los donantes. </li><li> Se incuba a 28.5 C. El derivado del donante lente se puede distinguir de la lente de acogida en el lado no operado del mismo animal basado en la fluorescencia de GFP. Las secciones también se puede hacer para medir el tamaño del objetivo y para determinar si se distingue adecuadamente. </li></ol><img src="/files/ftp_upload/1258/1258fig1.jpg" alt="la figura 1" /> Fig. Una imagen de bajo aumento de una aguja afilada utilizada para la lente de un trasplante. Un vaso capilar (B) se inserta en la pipeta Pasteur (A), y un fino alambre de tungsteno (C) se inserta en el extremo abierto del capilar. El vidrio a través del cable se suaviza con un mechero de Bunsen. Con unas pinzas, se mantienen constantes al final de cristal con el alambre de metal mientras gira el otro extremo de la pipeta Pasteur con la mano. El vidrio debe suavizado en espiral alrededor del metal, agarrándolo en su lugar. <img src="/files/ftp_upload/1258/fig2 1258.png" alt="" width="600" height="204" /> Fig. 2 Las puntas de los dos tipos de agujas que se usan para la lente de un trasplante. <img src="/files/ftp_upload/1258/1258fig3.jpg" alt="la figura 3" /> Fig. 3 embriones sometidos a trasplante de lentes en 30hpf. Se muestran un embrión donante (A, B), un ojo con lente acogida en trasplantados (C, D), y el lado del control del embrión de acogida (E, F). Cada ojo se muestra tanto en la luz transmitida y luz UV, como se indica. Las fotografías fueron tomadas a las 48 HPF. El Q01 [9] línea transgénica se utiliza en este experimento.

<ol>
	<li>Thut, C. J., Rountree, R. B., Hwa, M., Kingsley, D. 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+large-scale+in+situ+screen+provides+molecular+evidence+for+the+induction+of+eye+anterior+segment+structures+by+the+developing+lens.">A large-scale in situ screen provides molecular evidence for the induction of eye anterior segment structures by the developing lens.</a> Dev Biol. 231, 63-76 (2001).</li><li>Yamamoto, Y., Jeffery, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+role+for+the+lens+in+cave+fish+eye+degeneration.">Central role for the lens in cave fish eye degeneration.</a> Science. 289, 631-633 (2000).</li><li>Easter, S. S., Nicola, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+vision+in+the+zebrafish(Danio+rerio).">The development of vision in the zebrafish(Danio rerio).</a> Dev Biol. 180, 646-663 (1996).</li><li>Schmitt, E., Dowling, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+eye+morphogenesis+in+the+zebrafish.+Brachydanio+rerio.">Early eye morphogenesis in the zebrafish. Brachydanio rerio.</a> J.comp. Neuro. 344 (4), 532-542 (1994).</li><li>Malicki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+the+power+of+forward+genetics--analysis+of+neuronal+diversity+and+patterning+in+the+zebrafish+retina.">Harnessing the power of forward genetics--analysis of neuronal diversity and patterning in the zebrafish retina.</a> Trends Neurosci. 23 (11), 531-541 (2000).</li><li>Malicki, J., Pujic, Z., Thisse, C., Thisse, B., Wei, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forward+and+reverse+genetic+approaches+to+the+analysis+of+eye+development+in+zebrafish.">Forward and reverse genetic approaches to the analysis of eye development in zebrafish.</a> Vision Res. 42 (4), 527-533 (2002).</li><li>Avanesov, A., Malicki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approaches+to+study+neurogenesis+in+the+zebrafish+retina.">Approaches to study neurogenesis in the zebrafish retina.</a> Methods Cell Biol. 76, 333-384 (2004).</li><li>Vihtelic, T. S., Hyde, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+mutagenesis+yields+eye+morphological+mutants+with+retinal+and+lens+defects.">Zebrafish mutagenesis yields eye morphological mutants with retinal and lens defects.</a> Vision Res. 42, 535-540 (2002).</li><li>Godinho, L., Mumm, J. S., Williams, P. R., Schroeter, E. H., Koerber, A., Park, S. W., Leach, S. D., Wong, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+amacrine+cell+neurites+to+appropriate+synaptic+laminae+in+the+developing+zebrafish+retina.">Targeting of amacrine cell neurites to appropriate synaptic laminae in the developing zebrafish retina.</a> Development. Nov 132, 5069-5079 (2005).</li></ol>

Varios pasos requieren una atención especial durante el trasplante de la lente. <ol><li> Afiladas agujas: Es mejor preparar varias agujas antes de la realización del objetivo del trasplante, y la punta de la aguja debe ser lo más fina posible. Una aguja más gruesa lágrima más tejido debido a su mayor diámetro, provocando una falla en el ojo para curar. </li><li> La organización de los embriones: Justo antes de que el trasplante debe colocar los embriones manera que se acueste de costado. Cuando el posicionam...

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<td>0.1mm diameter tungsten wire</td>
<td>&nbsp;</td>
<td>A Johnson Matthey</td>
<td>45086</td>
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<td>Agarose(low melting point)</td>
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<td>39346</td>
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<td>Forceps</td>
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lens transplantation in zebrafish and its application in the analysis of eye mutants

La lente tiene un papel importante en el desarrollo de la copa óptica [1,2]. Utilizando el pez cebra como modelo, cuestiones relacionadas con el desarrollo del objetivo se puede abordar. El pez cebra es útil para estudios genéticos, debido a varias características ventajosas, como el tamaño pequeño, alta fecundidad, el ciclo de vida corto, y la facilidad de cuidado. El desarrollo de la lente se produce con rapidez en el pez cebra. Por 72 HPF, la lente de pez cebra es funcionalmente maduro [3]. La abundancia de recursos genéticos y moleculares están disponibles para apoyar la investigación en el pez cebra. Además, la similitud de los ojos de pez cebra a los de otros vertebrados, proporciona la base para su uso como un excelente modelo animal de los defectos humanos [4-7]. Varios mutantes del pez cebra presentan anormalidades del objetivo, incluyendo altos niveles de muerte celular, que en algunos casos conduce a una completa degeneración de los tejidos del cristalino [8]. Para determinar si las anomalías de lentes se deben a causas intrínsecas o defectuosa la interacción con los tejidos circundantes, el trasplante de una lente en un ojo mutante de tipo salvaje se lleva a cabo. El uso del fuego pulido agujas de metal, lentes de mutantes o de tipo salvaje son cuidadosamente disecados de los animales donantes, y se introducen en la máquina. Para distinguir los tejidos de tipo salvaje y mutante, una línea transgénica es utilizada como donante. Esta línea expresa de membrana buenas prácticas agrarias en todos los tejidos, incluyendo la lente. Esta técnica de trasplante es una herramienta esencial en los estudios de los mutantes de la lente de pez cebra.

Watch this Scientific Journal Video about Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants at JoVE.com

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

Lente del desarrollo implica interacciones con otros tejidos. Varios mutantes ojo de pez cebra se caracteriza por un tamaño anormalmente pequeño lente. Aquí se demuestra un experimento de trasplante de lentes para determinar si este fenotipo se debe a causas intrínsecas o interacciones defectuosas con los tejidos que rodean a la lente.

Trasplante de lentes en el pez cebra y su aplicación en el análisis de mutantes de ojos

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

lens-transplantation-zebrafish-its-application-analysis-eye

Research

JoVE Journal

Biology

11.0K vistas.  The Second Teaching Hospital of Jilin University. Lente del desarrollo implica interacciones con otros tejidos. Varios mutantes ojo de pez cebra se caracteriza por un tamaño anormalmente pequeño lente. Aquí se demuestra un experimento de trasplante de lentes para determinar si este fenotipo se debe a causas intrínsecas o interacciones defectuosas con los tejidos que rodean a la lente.

Video: Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

El trasplante de médula del riñón entero en adultos de pez cebra

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

Investigación

Biología

The Ladder Rung Walking Task: A Scoring System and its Practical Application.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to elaborate distinct aspects of motor function and to determine even subtle loss of movement capacity. To enhance efficacy and resolution of testing, tests should permit qualitative and quantitative measures of motor function and be sensitive to changes in performance during recovery periods. The present study describes a new task to assess skilled walking in the rat to measure both forelimb and hindlimb function at the same time. Animals are required to walk along a horizontal ladder on which the spacing of the rungs is variable and is periodically changed. Changes in rung spacing prevent animals from learning the absolute and relative location of the rungs and so minimize the ability of the animals to compensate for impairments through learning. In addition, changing the spacing between the rungs allows the test to be used repeatedly in long-term studies. Methods are described for both quantitative and qualitative description of both fore- and hindlimb performance, including limb placing, stepping, co-ordination. Furthermore, use of compensatory strategies is indicated by missteps or compensatory steps in response to another limb&#x2019;s misplacement. 

The ladder rung walking task is a new test to assess skilled walking and measure both forelimb and hindlimb placing, stepping, and inter-limb co-ordination.

El peldaño de la escalera a pie de la tarea: un sistema de puntuación y su aplicación práctica.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to ...

Preparation and Culture of Rat Lens Epithelial Explants for Studying Terminal Differentiation

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  Following division, these cells migrate posteriorly, where FGF diffusing from the retina induces them to differentiate into a posterior array of elongated lens fiber cells, which compose the bulk of the lens.  Differentiation of lens epithelial cells into lens fibers can be induced in vitro by culturing explants of the central region of the anterior epithelium in the presence of FGF-2.  Explants are prepared from lenses of neonatal rats by removing the lens from the eye and grasping the lens capsule on the posterior side with dissecting tweezers.  The posterior capsule is then gently torn open and pressed down into the plastic bottom of a tissue culture dish. The peripheral regions of the explant are removed with a scalpel and the central area is then cultured in the presence of 100ng/ml FGF-2 for as long as 2-3 weeks, depending on the parameters to be studied.  Since epithelial cells in cultured explants differentiate in approximate synchrony over a period of days to weeks, the time course of signaling and gene expression can be determined using molecular, biochemical, and pharmacological techniques.  Immunofluorescence microscopy is a powerful adjunct to these methods as it demonstrates the subcellular localization of proteins of interest and can reveal the physiological consequences of experimental manipulations of signaling pathways.

Explants of the central region of rat lens epithelia differentiate synchronously when cultured in the presence of FGF-2. Immunofluorescence microscopy of such cultures can provides novel information about gene expression and signaling events associated with terminal differentiation.

Preparación y Cultura de la lente explantes Rata epiteliales para el estudio de la diferenciación terminal

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

Cultura primaria y electroporación plásmido del órgano de Corti murino.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

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

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

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 ...

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here&#160;we describe the design and analysis of TPR in Drosophila.

Diseño y Análisis Del Comportamiento De Preferencia De Temperatura Y Su Ritmo Circadiano En Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.

The zebrafish adult kidney is an excellent system for renal regeneration and disease studies. An essential aspect of such research is the assessment of nephron structure and function. This protocol describes several methodologies that can be implemented to assess nephron tubule composition and to evaluate renal reabsorption.

Análisis de Composición Nephron y función en el riñón adultos de pez cebra

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys ...

Sequential Application of Glass Coverslips to Assess the Compressive Stiffness of the Mouse Lens: Strain and Morphometric Analyses

The eye lens is a transparent organ that refracts and focuses light to form a clear image on the retina. In humans, ciliary muscles contract to deform the lens, leading to an increase in the lens&#39; optical power to focus on nearby objects, a process known as accommodation. Age-related changes in lens stiffness have been linked to presbyopia, a reduction in the lens&#39; ability to accommodate, and, by extension, the need for reading glasses. Even though mouse lenses do not accommodate or develop presbyopia, mouse models can provide an invaluable genetic tool for understanding lens pathologies, and the accelerated aging observed in mice enables the study of age-related changes in the lens. This protocol demonstrates a simple, precise, and cost-effective method for determining mouse lens stiffness, using glass coverslips to apply sequentially increasing compressive loads onto the lens. Representative data confirm that mouse lenses become stiffer with age, like human lenses. This method is highly reproducible and can potentially be scaled up to mechanically test lenses from larger animals.

Age-related increases in eye lens stiffness are linked to presbyopia. This protocol describes a simple, cost-effective method for measuring mouse lens stiffness. Mouse lenses, like human lenses, become stiffer with age. This method is precise and can be adapted for lenses from larger animals.

La aplicación secuencial de cubreobjetos de vidrio para evaluar la compresión Rigidez de la lente del ratón: tensión y el análisis morfométricos

The eye lens is a transparent organ that refracts and focuses light to form a clear image on the retina. In humans, ciliary muscles contract to deform the ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

-Alta resolución de imagen de lapso de tiempo y análisis automatizado de los microtúbulos en la dinámica de estar umbilical humana Las células endoteliales de vena

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...