Damos las gracias a Liao CE para la ayuda con la sutura, y R. Ethier y L. Gyr para el cuidado de pez cebra. AJD fue apoyada por el Harvard Stem Cell Institute, la American Society of Nephrology, y NIH / NIDDK (P50DK074030). CQD fue apoyada por el Massachusetts General Hospital y el Fondo para el descubrimiento médico.

1. El acondicionamiento de los peces receptores no transgénicos. <ol><li> Seleccione el pez más grande hombre en el sistema para el trasplante. </li><li> Coloque el pescado en un 0,02% tricaína (pH 7) durante aproximadamente 3 minutos o hasta que deja de nadar, pero no tan largo que el corazón deja de latir. </li><li> Ponga el pescado anestesiado sobre una toalla de papel húmeda en una mesa de trabajo. </li><li> Use una aguja de la jeringa de insulina para inyectar 40 ug de gentamicina en 20 l de agua en la cavidad intraperitoneal de los peces. Para los estudios de regeneración renal, este paso es importante para la inducción de un ambiente favorable para el injerto mejorado. </li><li> Poner el pez en el agua del sistema para la recuperación. </li><li> El pescado puede ser dócil y muy sensible a los estímulos externos como la vibración. Después de 3 horas de recuperación, el tratamiento de los peces con 25 grises de rayos gamma. </li><li> Ponga el pescado de nuevo en el sistema durante 3 días sin comer antes del trasplante. </li></ol> 2. Preparación de las células progenitoras de donantes genéticamente etiquetados con un marcador fluorescente (EGFP o mCherry). <ol><li> Justo antes del procedimiento quirúrgico, preparar las células del donante para ser trasplantadas y mantener en hielo. </li><li> Para los estudios renales, se utiliza toda células de la médula del riñón de la Tg (cdh17: EGFP) línea transgénica, que contiene células progenitoras hematopoyéticas y renal. Estas células progenitoras son genéticamente etiquetados y sólo se expresan EGFP si se injertan y se diferencian en células epiteliales renales. Otros tipos de células progenitoras de la etiqueta también se puede utilizar, como Tg (SCL: EGFP) de células madre hematopoyéticas o Tg (fli1a: EGFP) para progenitores angioblast trasplantes. </li><li> Eutanasia tres peces adultos transgénicos, colocándolos en un 0,2% tricaína durante 5 minutos. </li><li> Use una hoja de afeitar esterilizada para quitar de la cabeza y la cola, y tijeras de disección para realizar una incisión a lo largo de la parte ventral. </li><li> Bajo un estereoscopio, eliminar todos los órganos internos en la cavidad del cuerpo como la vejiga natatoria con pinzas. Tenga cuidado de no extraer el riñón, que es un órgano plano y pigmentadas pegado a la pared dorsal de la cavidad. </li><li> A continuación, diseccionar el riñón de la pared dorsal y colocarlo en un tubo centrífugo de 1,5 ml que contiene 300 l de solución salina fría tampón fosfato suplementado con 2% de suero fetal bovino (PBS 1X / 2% de FCS). </li><li> Use un mazo para cortar el riñón hasta que se homogeniza. </li><li> Colocación de un filtro 40 uM de células dentro de un tubo Falcon de 50 ml y la transferencia de la solución de células en la parte superior del filtro de la célula, permitiendo que las células sueltas de filtrar a través de en el tubo Falcon. </li><li> Use la mano del mortero con suavidad frotis las grandes piezas de tejido que se quedan en el colador para romper aún más a las células. </li><li> Deje la solución de células en el tubo Falcon y lavar el filtro con 1,2 ml de 1X PBS / FCS al 2% para recoger las células residuales de la coladera. </li><li> La transferencia de la solución de células en un tubo de 1,5 mL nuevo y se centrifuga a 4 ° C a 300 rcf durante 5 minutos. </li><li> Lavar el pellet de células con 1,5 ml de PBS 1X / 2% de FCS y pasarlo por un colador para filtrar nuevos grupos de células pegajosas y centrifugar de nuevo. </li><li> Resuspender el botón celular en 200 l de PBS 1X / 2% de FCS y la transferencia a un tubo de PCR. </li><li> Centrifugar el tubo de PCR (4 ° C, 300 fcr, 1 min) y eliminar la mayor parte del sobrenadante. </li><li> Resuspender las células en el sobrenadante residual para obtener un volumen final de 2.5 uL y lo almacena en hielo. </li><li> Rendimiento celular final debe ser de aproximadamente 2 millones de células por los riñones. </li><li> Cuente el número de células con un hemocitómetro y ajustar la concentración de 5 x 10 5 células por ul. </li></ol> 3. Cargue la solución de células en una jeringa Hamilton. <ol><li> Enjuague la jeringa Hamilton con agua estéril tres veces. </li><li> Enjuague la jeringa con etanol al 75% 3 veces. </li><li> Enjuague la jeringa de nuevo con 1X PBS / FCS al 2% 3 veces. </li><li> A continuación, cargue la jeringa con 1 l de solución de células contienen aproximadamente 5 x 10 5 células de la misma antes de la inyección. </li></ol> 4. El trasplante de células progenitoras directamente en el riñón. <ol><li> Anestesiar a los peces receptores acondicionado en un 0,02% tricaína durante unos 3 minutos, o hasta que el pescado ha dejado de moverse, pero no demasiado largo que el corazón deja de latir. </li><li> Bajo un microscopio estereoscópico, ponga el pez en una toalla de papel con la cabeza hacia el lado izquierdo, ventral hacia arriba, con el fin de secar la cara trasplantada. </li><li> Extender encima el pescado para dejar al descubierto la parte seca. </li><li> Con unas pinzas esterilizadas, extraiga las escalas en la región inmediatamente posterior a las branquias. </li><li> Haga una incisión de 1 cm lateral en esta área en la línea media con un bisturí mientras se estabiliza el tejido con pinzas. </li><li> Usando las pinzas, exponer el tejido sin dejar de hacer la incisión lo suficientemente profundapara exponer el riñón con la cabeza. </li><li> Si hay una gran cantidad de sangrado que impide la visualización de los riñones, euthenize los peces y empezar de nuevo con otro pez receptor. </li><li> Mientras curiosos del tejido para exponer el riñón cabeza, inyectar 1 l de la solución de células directamente en el riñón con la cabeza. </li><li> Luego, en el mismo tiempo que la aguja de la jeringa se está recuperando, la liberación de las pinzas para que el tejido vuelve a caer en su lugar para evitar fugas de las células inyectadas. </li><li> Mantenga una aguja de sutura con un controlador de la aguja y la traspasará a través del músculo en cada lado de la incisión. </li><li> Tie-2 nudos y cortar el exceso de sutura. Sólo una única sutura que se necesita para una incisión de 1 cm. </li><li> Coloque el pescado en los peces de agua que contiene 10 partes por millón de tricaína durante 5 horas para proporcionar a los peces con un efecto analgésico que calma. </li><li> Coloque el pescado de nuevo en el sistema de recuperación con una alimentación normal. </li><li> Después de 7-18 días de recuperación, diseccionar y analizar los peces injerto renal de células progenitoras de la fluorescencia en vivo EGFP. </li></ol> 5. Los resultados representativos: La estrategia global del trasplante se ilustra en la Figura 1. Para nuestros estudios renal, el riñón es diseccionado y analizado por EGFP fluorescencia. Injerto y la diferenciación de las células progenitoras renal se detecta tan pronto como siete días después del trasplante. Esto es evidente por la presencia de varios grupos de EGFP + células epiteliales renales (Figura 2). A los 18 días después del trasplante, se detecta un promedio de 24 EGFP + nefronas, lo que indica que las células del donante se han formado de tejido nuevo riñón (Figura 3). Para demostrar a largo plazo del injerto, se diseca un pez receptor de un trasplante después de 59 días (Figura 4). Este pez tenía muchas nefronas en el sitio de inyección (punta de flecha de color rojo), además de varias nefronas en sitios distantes (puntas de flecha blanca), lo que sugiere que las células progenitoras renal son migratorias. Esta técnica es más rápida y más eficiente en comparación a cuando las células se inyectan en la circulación a través del corazón, que dura aproximadamente tres meses para detectar el injerto. <img src="/files/ftp_upload/2725/2725fig1.jpg" alt="Figura 1"/> Figura 1. Esquema general del trasplante. Peces destinatario no transgénicos son irradiados para evitar el rechazo inmunológico de las células trasplantadas. Tres días más tarde, se hace una incisión en el flanco de los peces y las células progenitoras de los donantes se inyectan en el riñón la cabeza al descubierto. La incisión se cierra con una sutura y el pescado se coloca de nuevo en el sistema para la recuperación. Los riñones de los peces receptores se diseccionan en momentos posteriores y se analizaron para el injerto y la actividad de las células progenitoras. <img src="/files/ftp_upload/2725/2725fig2.jpg" alt="Figura 2"/> Figura 2. El injerto de células madre después de siete días. El injerto de células progenitoras renal se detecta en el lado de inyección del riñón cabeza a los siete días después del trasplante, según lo determinado por la presencia de grupos de EGFP + células renales epiteliales (recuadro - vista ampliada). <img src="/files/ftp_upload/2725/2725fig3.jpg" alt="Figura 3"/> Figura 3. El injerto de células madre después de 18 días Después de 18 días después del trasplante, se detecta un promedio de 24 EGFP + derivadas del donante nefronas (insertar - imagen ampliada de cada uno de EGFP + nefronas).. <img src="/files/ftp_upload/2725/2725fig4.jpg" alt="Figura 4"/> Figura 4. Injerto y la migración de las células progenitoras después de 59 días. Continuamos para detectar el injerto de las células progenitoras de largo después del trasplante, incluso después de 59 días (cabeza de flecha roja). En este punto más adelante, derivadas del donante nefronas se detectan en sitios fuera de la zona de inyección (puntas de flecha blanca), de conformidad con la migración de células progenitoras.

<ol>
	<li>Hopkins, C., Li, J., Rae, F., Little, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+options+for+kidney+disease.">Stem cell options for kidney disease.</a> The Journal of Pathology. 217, 265-281 (2009).</li><li>Brittijn, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+development+and+regeneration:+new+tools+for+biomedical+research.">Zebrafish development and regeneration: new tools for biomedical research.</a> Int J Dev Biol. 53, 835-850 (2009).</li><li>Traver, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+lethal+irradiation+in+zebrafish+and+rescue+by+hematopoietic+cell+transplantation.">Effects of lethal irradiation in zebrafish and rescue by hematopoietic cell transplantation.</a> Blood. 104, 1298-1305 (2004).</li></ol>

Las técnicas de trasplante de células son cruciales para los estudios de regeneración y son la prueba estándar de oro para medir la actividad de las células madre. Aquí, se presenta un método para el trasplante de células directamente en el riñón de los adultos de pez cebra para determinar madre y actividad de las células progenitoras. Este procedimiento consiste en realizar una incisión a través del músculo para exponer el riñón cabeza, seguida de la inyección de las células directamente en el riñón. La incisión se cierra con una sutura simple y el pescado que se operen tengan un índice de supervivencia del 90-100%. En nuestros experimentos, las células progenitoras injertadas renal se detecta en el 80-100% de los peces trasplantados por siete días después del trasplante. Una limitación de esta técnica es que la incisión está cerca de la aorta dorsal, que puede ser fácilmente roto por el bisturí. Sin embargo, una vez dominado, la tasa de sangrado puede ser reducido a menos del uno por ciento. Nuestro método podría ser adaptado para ofrecer otros tipos de células (sangre, estroma o vascular), así como las moléculas pequeñas en el riñón. El desarrollo de esta técnica ayudará a los estudios avanzados de regeneración y permitirá madre y el potencial de células progenitoras para evaluar in vivo.

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th></tr><tr><td> Aguja de la jeringa de insulina (calibre 28) </td><td> Becton Dickinson </td><td> 329461 </td></tr><tr><td> Células colador (40 uM) </td><td> Becton Dickinson </td><td> 352340 </td></tr><tr><td> Tricaína </td><td> Sigma </td><td> A5040 </td></tr><tr><td> Hamilton jeringa (calibre 26) </td><td> Sigma </td><td> 20734 </td></tr><tr><td> Ethilon sutura (tamaño 9-0) </td><td> Ethicon </td><td> 2819G </td></tr></tbody></table>

transplantation of cells directly into the kidney of adult zebrafish

La medicina regenerativa basada en el trasplante de células madre o progenitoras en los tejidos dañados tiene el potencial para tratar una amplia gama de enfermedades crónicas 1. Sin embargo, la mayoría de los órganos no son fácilmente accesibles, lo que exige la necesidad de desarrollar métodos quirúrgicos para obtener acceso a estas estructuras. En este artículo de vídeo, se describe un método para el trasplante de células directamente en el riñón de un adulto el pez cebra, un modelo popular para estudiar la regeneración y la enfermedad 2. Peces receptores están pre-condicionada por la irradiación para suprimir el rechazo inmune de las células inyectadas 3. Demostramos cómo el riñón cabeza pueden estar expuestos por una incisión lateral en el costado del pescado, seguido por la inyección de células directamente en el órgano. Usando la etiqueta fluorescente las células de la médula renal que comprende toda una población mixta de precursores hematopoyéticos y renal, que demuestran que los progenitores de nefronas puede injertar y se diferencian en tejido renal nuevo - el estándar de oro de cualquier terapia regenerativa basada en células. Esta técnica se puede adaptar para entregar purificada células madre o progenitoras y / o moléculas pequeñas en el riñón, así como otros órganos internos y mejora aún más el pez cebra como modelo versátil para estudiar medicina regenerativa.

Watch this Scientific Journal Video about Transplantation of Cells Directly into the Kidney of Adult Zebrafish at JoVE.com

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

El trasplante de células es una técnica fundamental para el estudio de la regeneración de tejidos y para el desarrollo de terapias basadas en células de la enfermedad. Se demuestra aquí una técnica de microcirugía que permite el trasplante de células genéticamente etiquetados directamente en el riñón de peces adultos de pez cebra.

El trasplante de células directamente en el riñón de adultos de pez cebra

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

transplantation-of-cells-directly-into-the-kidney-of-adult-zebrafish

Research

JoVE Journal

Biology

14.9K vistas.  Massachusetts General Hospital. El trasplante de células es una técnica fundamental para el estudio de la regeneración de tejidos y para el desarrollo de terapias basadas en células de la enfermedad. Se demuestra aquí una técnica de microcirugía que permite el trasplante de células genéticamente etiquetados directamente en el riñón de peces adultos de pez cebra.

Video: Transplantation of Cells Directly into the Kidney of 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

Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice. We found that it was easier to concentrate the islets or cells into pellets in the final delivery tubing (PE50) used to transplant the cells under the kidney capsule. This technique provides both speed and ease while reducing any undue stress to the cells or to the mouse.

Loading: Settled, hand picked, islets or pelleted cells are carefully aspirated off the bottom of a 1.5 mL microcentrifuge tube using a p200 pipetteman and a straight, thin-wall pipette tip. A length of PE50 tubing is attached to the pipette tip using a small silicone adapter tubing. Cells are allowed to settle, in the tip, and then are transferred to the PE50 tubing by slowly dialing the pipetteman. Once the cells are near the end of the PE50 tubing, a kink is made and the silicone adaptor tubing is placed over the kink. The PE50 tubing is transferred to a 15 mL conical containing a cut 5 mL pipet, and the PE50 tubing is taped over the side of the 5 mL pipet to prevent curling during centrifuging. Cells are allowed to reach 1,000 rpm and stopped.
Transplantation: Recipient mice are anesthetized, shaved, and cleaned. A small incision is made on the left flank of the mouse and the kidney is exposed. The kidney, fat, and tissue are kept moist with normal saline swab. The distal end of the PE50 is attached to a Hamilton screw drive syringe, containing a pipette tip, using the silicone adaptor tubing. A small nick is made on the right flank side of the kidney, not too large nor too deep. The beveled end of the PE50 tubing, nearest the cells, is carefully placed under the capsule, the tubing is moved around gently to make space while swabbing normal saline; a dry capsule can tear easily. A small air bubble is delivered under the capsule by slowly dialing the syringe screw drive. Islets are then slowly delivered behind the air bubble. Once the islets have been delivered kidney homeostasis is maintained and the knick is cauterized with low heat. The kidney is placed back into the cavity and the peritoneum and skin are sutured and stapled. Mice are immediately treated with Flunixin and Buprenorphine s.q. and placed in a cage on a heating pad.

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of mice. Cells are concentrated into pellets in the final tubing used for transplanting the cells under the kidney capsule. The ease of this technique reduces stress to the cells and the mouse.

El trasplante de islotes pancreáticos en la cápsula renal de ratones diabéticos

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice.  We found that it was easier ...

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

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 can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

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 ...

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

La disección de los órganos del pez cebra adultos

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

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, ...

Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as tumor-propagating cells, or tumor-initiating cells. It is thought that targeting these self-renewing cells for destruction will block tumor progression and stop relapse, greatly improving patient prognosis1. The most common way to determine the frequency of self-renewing cells within a tumor is a limiting dilution cell transplantation assay, in which tumor cells are transplanted into recipient animals at increasing doses; the proportion of animals that develop tumors is used the calculate the number of self-renewing cells within the original tumor sample2, 3. Ideally, a large number of animals would be used in each limiting dilution experiment to accurately determine the frequency of tumor-propagating cells. However, large scale experiments involving mice are costly, and most limiting dilution assays use only 10-15 mice per experiment. 
Zebrafish have gained prominence as a cancer model, in large part due to their ease of genetic manipulation and the economy by which large scale experiments can be performed. Additionally, the cancer types modeled in zebrafish have been found to closely mimic their counterpart human disease4. While it is possible to transplant tumor cells from one fish to another by sub-lethal irradiation of recipient animals, the regeneration of the immune system after 21 days often causes tumor regression5. The recent creation of syngeneic zebrafish has greatly facilitated tumor transplantation studies 6-8. Because these animals are genetically identical, transplanted tumor cells engraft robustly into recipient fish, and tumor growth can be monitored over long periods of time. Syngeneic zebrafish are ideal for limiting dilution transplantation assays in that tumor cells do not have to adapt to growth in a foreign microenvironment, which may underestimate self-renewing cell frequency9, 10. Additionally, one-cell transplants have been successfully completed using syngeneic zebrafish8 and several hundred animals can be easily and economically transplanted at one time, both of which serve to provide a more accurate estimate of self-renewing cell frequency.
Here, a method is presented for creating primary, fluorescently-labeled T-cell acute lymphoblastic leukemia (T-ALL) in syngeneic zebrafish, and transplanting these tumors at limiting dilution into adult fish to determine self-renewing cell frequency. While leukemia is provided as an example, this protocol is suitable to determine the frequency of tumor-propagating cells using any cancer model in the zebrafish.

Limiting dilution cell transplantation assays are used to determine the frequency of tumor-propagating cells. This protocol describes a method for generating syngeneic zebrafish that develop fluorescently-labeled leukemia and details how to isolate and transplant these leukemia cells at limiting dilution into the peritoneal cavity of adult zebrafish.

Cuantificar la frecuencia de tumores se propagan las células usando trasplante de células limitación de dilución en el pez cebra singénico

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

La disección del riñón adultos de pez cebra

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

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 ...

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 ...