The authors have nothing to disclose.

Todos los procedimientos se llevaron a cabo siguiendo las directrices que se describen en &quot;Principles of Laboratory Animal Care&quot; (Institutos Nacionales de la Salud de la publicación no. 85-23, revisado 1985) y el protocolo de los animales Animal Comité Institucional de Cuidado y Uso de Rosalind Franklin University aprobada 08-19. 1. Gentamicina-inducción de la necrosis de las células de pelo <ol><li> Preparar sulfato de gentamicina en solución salina normal a una concentración final de 0,004% (4,32 mM). </li><li> Coloque el pescado adulto (D. rerio, de 4-6 meses de edad) en un recipiente que contiene el 0,004% (4,32 mM) solución de gentamicina. Cualquier recipiente puede ser utilizado, pero se utiliza un recipiente de pescado a partir de un sistema acuático Pharmacal que es 7 de ancho, 6 de alto y 7 de largo. Coloque el recipiente con los peces en una incubadora a 28 º C durante 24 horas. Ajuste el volumen total de fluido en el depósito a un nivel suficiente para mantener los peces en un estado viable para el período de 24 horas. Nota: La aireación del fluido de gentamicina no es necesario Si hay volumen suficiente se utiliza para el número de peces que se está tratando. </li></ol> 2. Tinción vital de las células ciliadas <ol><li> Preparar una concentración de 0,08% (en solución salina normal) del colorante vital fluorescente [4-4-diethylaminostyryl yoduro)-N-metilpiridinio (485 nm λ de excitación y 603 nm de emisión λ en metanol) a partir de una solución madre de trabajo de 15 mg / ml en etanol. </li><li> Para determinar si el tratamiento gentamicina fue eficaz un subconjunto de control y gentamicina peces tratados se tiñeron inmediatamente mediante la colocación de los peces en el pocillo de una placa de cultivo de 6 pocillos que contiene el colorante vital. Utilice un número suficiente de peces (y placas de cultivo como se requiere para la significación estadística que deben alcanzarse. Sobre la base de la velocidad del examinador de contar neuromast, coloque el pescado en los platos de forma escalonada en el tiempo para que los peces no se tiñen durante más de 75 min como se describe en el paso 2.3. </li><li> Coloque las placas desde el paso 2.2 en un cajón banco por el microscopio de fluorescencia paraser utilizado para el examen de neuromastos manchados. Apagar las luces de la habitación para evitar la extinción del colorante vital durante el periodo de tinción de 1 hora a temperatura ambiente. </li><li> Preparar tanto tinte de lavado y depósitos de agua anestésicos. Tinte de agua de lavado de agua normal de los peces y de agua anestésico, agregue suficiente 2-fenoxietanol manera que se consigue una dilución 1:1000 en agua normal de los peces. </li><li> Coloque el pescado en exceso de agua normal de los peces para enjuagar el exceso de colorante vital y vaya al paso 3.1 para la observación de tinte manchado pescados vital. </li><li> Para examinar la regeneración de neuromastos, transferir peces gentamicina tratados que fueron lavados en agua normal de los peces a una incubadora de entre 8 a 16 horas a 28 ° C. </li><li> En diversos momentos entre 8-16 hr, pescado se retiran de la incubadora, se lavaron y se tiñeron como se indica en los pasos 2.1 a 2.4. Continúe en el paso 3.1 para la observación de tinte manchado pescados vital. </li></ol> 3. Anestesiar Fish and Counting fluorescente de neuromastos <ol><li&gt; Seque cada pez sobre una toalla de papel para eliminar el exceso de líquido y luego se coloca en un pedazo de papel de filtro humedecido que se centra en la tapa de una placa Petri de plástico. </li><li> Coloque la tapa en la etapa de un microscopio estereoscópico de fluorescencia para obtener una imagen digital del colorante vital neuromastos manchadas de los puntos de sutura del cuerpo mediados. </li><li> Utilizar una cámara digital se coloca en el microscopio fluorescente estéreo establece un aumento de 2X para capturar imágenes para el análisis cuantitativo posterior. Nota: La configuración de la ampliación del microscopio estereoscópico puede depender de la marca del microscopio usado, pero el ajuste debe permitir una fácil visualización y recuento de neuromastos individuales dentro de los puntos a mediados del cuerpo. </li><li> Determinar la cantidad de regeneración mediante el recuento del número de neuromastos visibles dentro de los cuatro puntos de sutura designadas en el lado inferior más ventral de los peces justo proximal a la aleta pectoral derecha (véase la Figura 1). Para el análisis estadístico usar una prueba adecuada, como ANOVA or T-test de Student. Los experimentos deberían utilizar un mínimo de 5 peces por punto de tiempo y todos los experimentos deben repetirse un mínimo de 3 aumentos. </li><li> Sobre la base de la curva de tiempo de regeneración neuromast (véase la Figura 3), contar neuromastos entre 8-16 h después de gentamicina lavar de salida para estar dentro de la fase lineal de la curva de la regeneración. Nota: El uso de la fase de tiempo lineal permite el análisis cuantitativo adecuado entre el control y los grupos experimentales. </li></ol> 4. Conteo fluorescente de las células ciliadas individual para la obtención de mayor resolución del Análisis Cuantitativo si el análisis neuromast no es estadísticamente significativo <ol><li> Si el análisis cuantitativo en el nivel de neuromastos no es significativa, el análisis a nivel de la célula individual del pelo también se puede utilizar para obtener un mayor grado de resolución. Seleccione peces en un momento posterior gentamicina en particular lavar Salida (punto en el tiempo sobre la base de los estudios neuromast anteriores), colorante vital stain el pescado tal como se describe en el Protocolo 2 y, a continuación, la eutanasia a los peces utilizando 2-fenoxietanol a una dilución 1:500 de 1-5 min. </li><li> A la luz tenue para evitar enfriamiento rápido, hacer cuatro incisiones para que una preparación de colgajo de piel cuadrado se hace de la siguiente manera. Hacer una incisión a lo largo de las costillas superiores del pescado hasta que esté alineado con las aletas anales, a continuación, hacer una incisión en el vientre, y, finalmente, hacer dos incisiones verticales a cada lado de estas incisiones por lo que se crea el colgajo de piel cuadrado. Nota: Esta preparación de la piel incorporará los puntos de sutura del cuerpo mediados utilizados en los experimentos neuromast. </li><li> Coloque la muestra de la piel en un portaobjetos de vidrio y luego se coloca una hoja de cubierta de vidrio circular sobre la muestra de piel extirpadas para ayudar ancla y aplanar el tejido para la imagen digital posterior. </li><li> El uso de las muestras de piel desde el paso 4.3, obtener imágenes digitales de las células ciliadas dentro de cada neuromast de los puntos de sutura del cuerpo mediados. Tome imágenes con un aumento de por lo menos 60X y luego contar el ce pelolls dentro neuromastos individuales para el análisis cuantitativo comparativo de los grupos control y experimentales (ver Figura 4). </li></ol>

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This work was supported by a research grant from the Iacocca Family Foundation, National Institutes of Health Grant DK092721 (to R.V.I.), and Rosalind Franklin University start-up funds. No potential conflicts of interest relevant to this article were reported. G.C.P., S.M.M, and N.D. researched data. M.P.S. Jr. and RI oversaw the project, contributed to the discussion of the data, and oversaw the writing and editing of the manuscript.

Basado en el extenso cuerpo de literatura que se ha establecido para el análisis de la línea lateral de regeneración (LL) en embriones y larvas de pez cebra 8,24,25, el objetivo de nuestro estudio fue desarrollar un ensayo cuantitativo para la regeneración de la línea lateral en el pez cebra que podrían ser aplicada a modelos de enfermedades que se estudian mejor en los peces adultos. Hemos encontrado que ciertos puntos críticos son importantes en la aplicación de los procedimientos desarrollados para...

La línea lateral del sistema (SD) es un órgano mecanosensorial encontrado en ambos peces y anfibios que se encarga de la audición, el equilibrio, rheotaxis y conductas mediadoras como la escolarización y la evitación de depredadores 1-5. Se compone de grupos de células de pelo rodeado de células de soporte, ambos de los cuales están situados en estructuras llamadas neuromastos 6. Estos neuromastos están organizados típicamente en líneas verticales (llamado puntos) a lo largo del eje longitudinal del cuerpo y la cola con algunos puntos de sutura horizontales observados en la cabeza del pescado. En el adulto, neuromastos son significativamente mayores en número dentro de los puntos de sutura en comparación con los peces de embriones o larvas 6. Estudios biomédicos en el pez cebra se han centrado en el efecto del tratamiento antibiótico, el trauma a causa del ruido, la infección crónica, etc. en células ciliadas 7,8 en un intento de comprender mejor sus efectos en los seres humanos. A diferencia de la mayoría de los vertebrados, TEleosts, como el pez cebra (Danio rerio), tienen la capacidad de regenerar las células pilosas perdidas. El pez cebra son particularmente útiles debido a su tiempo de desarrollo rápido y de alta capacidad de regeneración. Hasta la fecha, sin embargo; estudios sobre el desarrollo de pez cebra de la línea lateral y / o regeneración han utilizado principalmente el pescado etapa embrionario y larval debido a la reducción del número de neuromastos la línea lateral que permite para el recuento más fácil y análisis 6,9,10. Sin embargo, se estudian como muchos modelos de pez cebra de las enfermedades neurológicas y no neurológicas 11-16 en el pez adulto y no las larvas, que se centró en el desarrollo de un ensayo de regeneración de la línea lateral en el pez cebra adultos usando gentamicina (un aminoglucósido utilizado anteriormente en larvas de pez cebra y más recientemente utilizado con peces adultos 17) de manera que un ensayo estaba disponible que podría aplicarse a modelos de enfermedad de pez cebra adultos actuales. Mientras que previamente publicado procedimientos de Van Trump et al. 17 establece las condiciones para la ablación de las células ciliadas en los peces adultos, no establecieron una curva estándar para la regeneración neuromast que se requiere para la comparación cuantitativa entre el control y los grupos experimentales, como al utilizar líneas de pez cebra transgénico o estados de enfermedad inducidos farmacológicamente- en el pez cebra 18. Por lo tanto, seguimos los procedimientos de Van Trump et al. 17 para la ablación de las células ciliadas, pero construido sobre sus actividades para crear una curva estándar de la regeneración neuromast para permitir a los investigadores a utilizar nuestros datos cuando se comparan grupos de control y experimentales, como con modelos de enfermedades de pez cebra adultos . El ensayo también fue diseñado para permitir la extensión del análisis a la célula individual del pelo cuando se requiere un mayor nivel de resolución.

<table border="0" cellpadding="0" cellspacing="0" width="431">
	<tbody>
		<tr>
			<td>Gentamicin sulfate solution (50mg/ml)</td>
			<td>Sigma Aldrich</td>
			<td>G1397</td>
			<td></td>
		</tr>
		<tr>
			<td>2 Phenoxyethanol</td>
			<td>Sigma Aldrich</td>
			<td>P1126</td>
			<td></td>
		</tr>
		<tr>
			<td>4-4-diethylaminostryryl-N-methylpyridinium iodide (4-Di-2-Asp)</td>
			<td>Aldrich</td>
			<td>D-3418</td>
			<td>485 nm excitation &#955; and 603 nm emission &#955;</td>
		</tr>
		<tr>
			<td>in methanol</td>
		</tr>
		<tr>
			<td>6 well plates</td>
			<td>Mid Sci</td>
			<td>TP92006</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes</td>
			<td>Fisher Scientific</td>
			<td>08-757-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Bottom Microwell Dishes</td>
			<td>Matek Corporation</td>
			<td>P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting&#160; Microscope</td>
			<td>Nikon</td>
			<td>TMZ-1500</td>
			<td>Any dissecting microscope is fine.</td>
		</tr>
		<tr>
			<td>Camera for Imaging</td>
			<td>Nikon</td>
			<td>Q imaging</td>
			<td>Any camera is suitable.</td>
		</tr>
		<tr>
			<td>Image J software</td>
			<td>National Institutes of Health</td>
			<td>NIH Image</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS Elements</td>
			<td>Nikon</td>
			<td></td>
			<td>Any imaging software is suitable.</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Olympus</td>
			<td>FV10i</td>
			<td>Any high resolution fluorescent microscope is suitable</td>
		</tr>
		<tr>
			<td>Aquatic System</td>
			<td>&#160;KG Aquatics ZFS Rack System.</td>
			<td></td>
			<td>Any aquatic system can be used</td>
		</tr>
	</tbody>
</table>

an assay for lateral line regeneration in adult zebrafish

Debido a la importancia clínica de la audición y trastornos del equilibrio en el ser humano, los organismos modelo, como el pez cebra se han utilizado para estudiar el desarrollo de la línea lateral y la regeneración. El pez cebra es particularmente atractivo para estos estudios debido a su tiempo de desarrollo rápido y su alta capacidad de regeneración. Hasta la fecha, los estudios de pez cebra de la regeneración de la línea lateral se han utilizado principalmente pescado de las etapas embrionarias y larvarias debido al menor número de neuromastos en estas etapas. Esto ha hecho que el análisis cuantitativo de la línea de la regeneración y / o el desarrollo más fácil en las etapas anteriores del desarrollo lateral. Debido a que muchos modelos de pez cebra de las enfermedades neurológicas y no neurológicas se estudian en los peces adultos y no en el embrión / larvas, nos centramos en el desarrollo de un ensayo de regeneración de la línea lateral cuantitativa en adultos de pez cebra para que un ensayo estaba disponible que podría ser aplicado a la corriente modelos de enfermedad de pez cebra adultos. Sobre la base de estudios previos de Van Trump et al 17. que describe los procedimientos para la ablación de las células ciliadas en peces adultos mexicana ciega cueva y el pez cebra (Danio rerio), nuestro ensayo fue diseñado para permitir la comparación cuantitativa entre el control y los grupos experimentales. Esto se logró mediante el desarrollo de una curva estándar neuromast regenerativa basado en el porcentaje de reaparición neuromast durante un período de tiempo de 24 horas después de la necrosis inducida por la gentamicina de las células ciliadas en una región definida de la línea lateral. El ensayo también fue diseñado para permitir la extensión del análisis a nivel de la célula individual del pelo cuando se requiere un mayor nivel de resolución.

Optimización de los procedimientos para cuantificar neuromast regeneración de la línea lateral en el pez cebra adulto. Los neuromastos de larvas de pez cebra son fácilmente cuantificables; Sin embargo, la línea lateral del pez cebra adulto tiene un número mucho mayor de neuromastos por puntadas que hacen análisis cuantitativos más difícil 6,17,19,20. Como se ve en la Figura 1A, la cabeza tiene un número significativamente mayor de neurom...

Watch this Scientific Journal Video about An Assay for Lateral Line Regeneration in Adult Zebrafish at JoVE.com

An Assay for Lateral Line Regeneration in Adult Zebrafish

Debido a que muchos modelos de pez cebra de enfermedades neurológicas y no neurológicas se estudian en los peces adultos en lugar de el embrión / larvas, hemos desarrollado un ensayo cuantitativo regenerativa línea lateral que se puede aplicar a modelos de enfermedad de pez cebra adultos. El ensayo implicó resolución en el neuromast 1) y 2) los niveles de células cabello individual.

Un ensayo para la línea lateral de regeneración en adultos de pez cebra

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

an-assay-for-lateral-line-regeneration-in-adult-zebrafish

Research

JoVE Journal

Biology

12.5K vistas.  Dr. William M Scholl College of Podiatric Medicine, Rosalind Franklin University of Medicine and Science.  Debido a que muchos modelos de pez cebra de enfermedades neurológicas y no neurológicas se estudian en los peces adultos en lugar de el embrión / larvas, hemos desarrollado un ensayo cuantitativo regenerativa línea lateral que se puede aplicar a modelos de enfermedad de pez cebra adultos. El ensayo implicó resolución en el neuromast 1) y 2) los niveles de células cabello individual. 

Video: An Assay for Lateral Line Regeneration in Adult Zebrafish

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

Cellular Toxicity of Nanogenomedicine in MCF-7 Cell Line: MTT assay

Cytotoxicity of the futuristic nanogenomedicine (e.g., short interfering RNA and antisense) may hamper its clinical development. Of these, the gene-based medicine and/or its carrier may elicit cellular toxicity. For assessment of such cytotoxicity, a common methodology is largely dependent upon utilization of the 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay which has been widely used as a colorimetric approach based on the activity of mitochondrial dehydrogenase enzymes in cells. In this current investigation, MCF-7 cells were inoculated in 96-well plate and at 50% confluency they were treated with different nanopolyplexes and subjected to MTT assay after 24 hours. Water soluble yellow MTT is metabolized by the metabolically active cells to the water insoluble purple formazan, which is further dissolved in dimethylsulfoxide and Sornson s buffer pH 10.5. The resultant product can be quantified by spectrophotometry using a plate reader at 570 nm.

The MTT assay is an easy and reproducible colorimetric assay for evaluation of cell viability based on reduction of yellow MTT and production of water insoluble purple formazan. Here, the viability of MCF-7 cells upon treatment of nanogenomedicine has been evaluated.

La toxicidad celular de Nanogenomedicine en la línea celular MCF-7: ensayo de MTT

Cytotoxicity of the futuristic nanogenomedicine (e.g., short interfering RNA and antisense) may hamper its clinical development. Of these, the gene-based ...

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 ...

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, ...

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 ...

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 ...

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from zebrafish has not been established. The previous methods for blood collection from zebrafish are lethal, such as lateral incision, decapitation and tail ablation. Thus we have developed a novel &#8220;repeated&#8221; blood collection method, and present here a detailed protocol outlining this procedure. This method is minimally invasive and results in a very low mortality rate (2.3%) for zebrafish, thus enabling repeated blood sampling from the same individual. The maximum volume of blood sampling is dependent on body weight of the fish. The volume for repeated blood sampling at intervals should be&#160;&#8804;0.4% of body weight every week or &#8804;1% every 2 weeks, which were evaluated by measurements of blood hemoglobin. Additionally, hemoglobin, fasting blood glucose, plasma triacylglycerol (TG) and total cholesterol levels in male and female adult zebrafish were measured. We also applied this method to investigate the dysregulation of glucose metabolism in diet-induced obesity. This blood collection method will allow many applications, including glucose and lipid metabolism and hematological studies, which will increase the use of zebrafish as a human disease model organism.

Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

Repetida extracción de sangre para pruebas de sangre en adultos de pez cebra

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from ...

In Vivo Surface Electrocardiography for Adult Zebrafish

The electrocardiogram waveforms of adult zebrafish and those of humans are remarkably similar. These electrocardiogram similarities enhance the value of zebrafish not only as a research model for human cardiac electrophysiology and myopathies but also as a surrogate model in high throughput pharmaceutical screening for potential cardiotoxicities to humans, such as QT prolongation. As such, in vivo electrocardiography for adult zebrafish is an electrical phenotyping tool that is necessary, if not indispensable, for cross-sectional or longitudinal in vivo electrophysiological characterizations. However, too often, the lack of a reliable, practical, and cost-effective recording method remains a major challenge preventing this in vivo diagnostic tool from becoming more readily accessible. Here, we describe a practical, straightforward approach to in vivo electrocardiography for adult zebrafish using a low-maintenance, cost-effective, and comprehensive system that yields consistent, reliable recordings. We illustrate our protocol using healthy adult male zebrafish of 12-18 months of age. We also introduce a rapid real-time interpretation strategy for quality validation to ensure data accuracy and robustness early in the electrocardiogram recording process.

Here, we present a reliable, minimally invasive, and cost-effective method to record and interpret electrocardiograms in live anesthetized adult zebrafish.

Electrocardiografía de superficie In Vivo para pez cebra adulto

The electrocardiogram waveforms of adult zebrafish and those of humans are remarkably similar. These electrocardiogram similarities enhance the value of ...