Agradecemos a CE Liao para ajudar com sutura, e R. e L. Ethier Gyr para o cuidado zebrafish. AJD foi apoiada pela Harvard Stem celular Institute, da Sociedade Americana de Nefrologia e NIH / NIDDK (P50DK074030). CQD foi apoiado pelo Hospital Geral de Massachusetts-Fundo para a descoberta de Medicina.

1. Condicionamento da não-transgênicas peixes destinatário. <ol><li> Selecione o maior peixe do sexo masculino no sistema para transplante. </li><li> Coloque o peixe em tricaina 0,02% (pH 7) por aproximadamente 3 minutos ou até que ele pára de nadar, mas não tanto tempo que o coração pára de bater. </li><li> Coloque o peixe anestesiados em uma toalha de papel úmida no topo de uma bancada. </li><li> Use uma agulha de seringa de insulina para injetar 40 ug de gentamicina em 20 uL de água na cavidade intraperitoneal dos peixes. Para estudos de regeneração renal, esta etapa é importante para induzir um ambiente favorável para o enxerto reforçada. </li><li> Coloque o peixe de volta à água do sistema para a recuperação. </li><li> O peixe pode ser dócil e muito sensível a estímulos externos, tais como vibração. Após 3 horas de recuperação, tratar os peixes com 25 cinza da radiação gama. </li><li> Coloque o peixe novamente no sistema, durante 3 dias sem comer antes do transplante. </li></ol> 2. Preparação de células progenitoras do doador geneticamente marcado com um marcador fluorescente (EGFP ou mCherry). <ol><li> Pouco antes do procedimento cirúrgico, prepare as células dos doadores para serem transplantados e mantê-los no gelo. </li><li> Para estudos renal, usamos células de rim inteiro a partir da medula (cdh17: EGFP) Tg linhagem transgênica, que contém células progenitoras hematopoéticas e renal. Estas células progenitoras são geneticamente marcado e só vai expressar EGFP se enxertar e se diferenciar em células epiteliais renais. Outros tipos de células progenitoras rotulados também pode ser usado, como Tg (SCL: EGFP) para a célula-tronco hematopoiéticas ou Tg (fli1a: EGFP) para transplantes de células progenitoras angioblast. </li><li> Euthanize 3 peixes adultos transgênico, colocando-os em tricaina 0,2% por 5 minutos. </li><li> Use uma lâmina de barbear esterilizada para remover a cabeça ea cauda, ​​e tesouras de dissecação para fazer uma incisão mediana ao longo do lado ventral. </li><li> Sob um estereoscópio, remover todos os órgãos internos na cavidade do corpo, incluindo a bexiga natatória com pinças. Tenha cuidado para não remover o rim, que é um órgão plana e pigmentada fixados à parede dorsal da cavidade. </li><li> Em seguida, dissecar o rim da parede dorsal e colocá-lo em um tubo de centrífuga de 1,5 mL contendo 300 uL de solução salina fria tampão fosfato suplementado com 2% de soro fetal bovino (FCS 1X PBS / 2%). </li><li> Use um pilão para o rim de corte até que se torne homogêneo. </li><li> Coloque um filtro de 40 uM de células dentro de um tubo de Falcon 50 mL e transferir a solução da célula na parte superior do filtro de celular, permitindo que as células soltas para filtrar para dentro do tubo Falcon. </li><li> Use o pilão para gentilmente esfregaço grandes peças de tecido que são deixados no filtro para continuar a quebrar as células. </li><li> Deixe a solução da célula no tubo Falcon e lavar o filtro com 1,2 ml de 1X PBS FCS / 2% para coletar células residuais do filtro. </li><li> Transferir a solução de células em um tubo de 1,5 mL novo e centrifugar a 4 ° C a 300 rcf durante 5 minutos. </li><li> Lave o pellet celular com 1,5 ml de 1X PBS FCS / 2% e passá-lo através de um filtro novo para filtrar aglomerados de células pegajosa e centrifugar novamente. </li><li> Ressuspender o pellet celular em 200 uL de PBS 1X da FCS / 2% e transferir para um tubo de PCR. </li><li> Centrifugar o tubo de PCR (4 ° C, a 300 rcf, 1 min) e remover a maioria do sobrenadante. </li><li> Ressuspender as células no sobrenadante residual para obter um volume de 2-5 uL final e armazená-lo em gelo. </li><li> Rendimento celular final deve ser de aproximadamente 2 milhões de células por rim. </li><li> Contar o número de células com um hemocitômetro e ajustar a concentração de 5 x 10 5 células por uL. </li></ol> 3. Carregue a solução de célula em uma seringa de Hamilton. <ol><li> Enxágüe a seringa Hamilton com água estéril 3 vezes. </li><li> Enxágüe a seringa com 75% de etanol três vezes. </li><li> Enxágüe a seringa novamente com 1X PBS / 2% FCS 3 vezes. </li><li> Em seguida, carregar a seringa com uma uL de solução de células contêm cerca de 5 x 10 5 células pouco antes da injeção. </li></ol> 4. O transplante de células progenitoras diretamente no rim. <ol><li> Anestesiar os peixes acondicionados em recipiente tricaina 0,02% para cerca de 3 minutos, ou até o peixe parou de se mover, mas não muito tempo que o coração pára de bater. </li><li> Sob microscópio estereoscópico, coloque o peixe sobre uma toalha de papel com a cabeça para o lado, à esquerda ventral para cima, a fim de secar o lado transplantado. </li><li> Em seguida, rola o peixe mais para expor o lado seco. </li><li> Usando uma pinça esterilizada, retire as escamas na região imediatamente posterior às brânquias. </li><li> Faça uma incisão de 1 cm laterais nesta área na linha média com um bisturi, enquanto estabilizar o tecido com pinças. </li><li> Usando a pinça, expor o tecido enquanto continua a fazer a incisão profunda o suficientepara expor o rim cabeça. </li><li> Se existe uma grande quantidade de sangramento que impede a visualização do rim, euthenize o peixe e começar de novo com outro peixe destinatário. </li><li> Enquanto curiosos do tecido aberto para expor o rim cabeça, injectar 1 uL da solução de células diretamente no rim cabeça. </li><li> Então, ao mesmo tempo, como a agulha da seringa está sendo recuperado, solte a pinça de modo que o tecido cai de volta no lugar para evitar vazamento das células injetadas. </li><li> Segurar uma agulha de sutura com um motorista de agulha e fure-o através do músculo em cada lado da incisão. </li><li> Amarre dois nós e corte o excesso de sutura. Apenas uma única sutura é necessário para uma incisão de 1 cm. </li><li> Coloque o peixe em água de peixes contendo 10 partes por milhão de tricaina por 5 horas para fornecer peixe com um efeito calmante analgésico. </li><li> Coloque o peixe de volta ao sistema de recuperação com a alimentação normal. </li><li> Após 7-18 dias de recuperação, dissecamos o peixe e analisar o enxerto de células progenitoras renal por viver EGFP fluorescência. </li></ol> 5. Resultados representativos: A estratégia global do transplante é ilustrada na Figura 1. Para os nossos estudos renal, o rim é dissecado e analisado por fluorescência de EGFP. Enxerto e diferenciação de células progenitoras renal é detectado logo aos sete dias após o transplante. Isto é evidente pela presença de vários clusters de EGFP + renal células epiteliais (Figura 2). Por 18 dias pós-transplante, detectamos uma média de 24 EGFP + néfrons, indicando que as células dos doadores formaram o tecido novo rim (Figura 3). Para demonstrar a longo prazo enxerto, dissecamos um peixe destinatário 59 dias após o transplante (Figura 4). Este peixe tinha muitos néfrons no local da injeção (vermelho ponta de seta), além de vários néfrons em locais distantes (setas brancas), sugerindo que as células progenitoras renal são migratórias. Esta técnica é mais rápida e mais eficiente em comparação com quando as células são injetadas na circulação através do coração, que leva cerca de três meses para detectar o enxerto. <img src="/files/ftp_upload/2725/2725fig1.jpg" alt="Figura 1"/> Figura 1. Esquema geral de transplante de peixes destinatário. Non-transgênicos são irradiados para suprimir a rejeição imunológica das células transplantadas. Três dias depois, é feita uma incisão no flanco do peixe e as células progenitoras do doador são injetadas no rim cabeça exposta. A incisão é fechada com uma sutura e peixes são colocados de volta ao sistema para a recuperação. Rins de peixes destinatário são dissecados em momentos mais tarde e analisadas para enxerto e atividade de células progenitoras. <img src="/files/ftp_upload/2725/2725fig2.jpg" alt="Figura 2"/> Figura 2. Enxerto de células progenitoras após sete dias. Enxerto de células progenitoras renal é detectada no lado injetado do rim cabeça em sete dias pós-transplante, conforme determinado pela presença de grupos de células EGFP + renal epitelial (inset - visão ampliada). <img src="/files/ftp_upload/2725/2725fig3.jpg" alt="Figura 3"/> Figura 3. Enxerto de células progenitoras após 18 dias Após 18 dias pós-transplante, detectamos uma média de 24 EGFP + doador-derivado néfrons (inserir - visão ampliada de cada EGFP + néfrons).. <img src="/files/ftp_upload/2725/2725fig4.jpg" alt="Figura 4"/> Figura 4. Enxerto ea migração de células progenitoras após 59 dias. Continuamos a detectar o enxerto de células progenitoras tempo após o transplante, mesmo depois de 59 dias (cabeça de seta vermelha). Neste ponto, mais tarde, doador-derivado néfrons são detectados em locais longe do local da injeção (cabeças de seta branca), consistente com a migração 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>

Não há conflitos de interesse declarados.

Técnicas de transplante de células é crucial para estudos de regeneração e são o teste padrão ouro para medir a atividade das células-tronco. Aqui, relatamos um método de transplante de células diretamente no rim de peixe-zebra adulto para determinar a atividade das células-tronco e progenitoras. Este procedimento envolve fazer uma incisão através do músculo para expor o rim a cabeça, seguida pela injeção de células diretamente no rim. A incisão é fechada com uma sutura simples e os peixes têm operado uma taxa de sobrevivência 90-100%. Em nossos experimentos, enxertada células progenitoras renal são detectados em 80-100% dos peixes transplantado por sete dias pós-transplante. Uma limitação desta técnica é que a incisão é perto da aorta dorsal, que pode ser facilmente rompido pelo bisturi. No entanto, uma vez dominada, a taxa de sangramento pode ser reduzido a menos de um por cento. Nosso método pode ser adaptado para oferecer outros tipos de células (sangue, estromais, vasculares), bem como pequenas moléculas para o rim. O desenvolvimento desta técnica ajudará estudos antecedência regenerativa e permitir-tronco e células progenitoras potencial para ser avaliada in vivo.

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th></tr><tr><td> Seringa de agulha de insulina (28 gauge) </td><td> Becton Dickinson </td><td> 329461 </td></tr><tr><td> Célula filtro (40 uM) </td><td> Becton Dickinson </td><td> 352340 </td></tr><tr><td> Tricaina </td><td> Sigma </td><td> A5040 </td></tr><tr><td> Hamilton seringa (26 gauge) </td><td> Sigma </td><td> 20734 </td></tr><tr><td> Ethilon sutura (tamanho 9-0) </td><td> Ethicon </td><td> 2819G </td></tr></tbody></table>

transplantation of cells directly into the kidney of adult zebrafish

Medicina regenerativa baseada no transplante de células-tronco ou progenitoras para os tecidos danificados tem o potencial para tratar uma vasta gama de doenças crônicas 1. No entanto, a maioria dos órgãos não são facilmente acessíveis, exigindo a necessidade de desenvolver métodos cirúrgicos para ter acesso a essas estruturas. Neste artigo de vídeo, descrevemos um método de transplante de células diretamente no rim de adulto zebrafish, um modelo popular para estudo da regeneração e da doença 2. Peixes destinatário são pré-condicionadas por irradiação para suprimir a rejeição imunológica das células injetadas 3. Demonstramos como o rim cabeça pode ser exposto por uma incisão lateral no flanco dos peixes, seguido pela injeção de células diretamente para o órgão. Usando fluorescente etiquetado células de rim inteiro medula compreendendo uma população mista de precursores renal e hematopoiético, mostramos que progenitores néfron pode enxertar e se diferenciar em tecido renal novo - o padrão ouro de qualquer terapia baseada em células regenerativas. Esta técnica pode ser adaptada para entregar purificada-tronco progenitoras ou células e / ou pequenas moléculas para o rim, bem como outros órgãos internos e aumenta ainda mais o peixe-zebra como modelo versátil para estudar 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

Transplante de células é uma técnica essencial para o estudo da regeneração de tecidos e para o desenvolvimento de terapias baseadas em células da doença. Demonstramos aqui uma técnica de microcirurgia, que permite o transplante de células geneticamente marcado diretamente no rim de peixes paulistinhas adultos.

Transplante de células diretamente no Rim de Adulto Zebrafish

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 visualizações.  Massachusetts General Hospital. Transplante de células é uma técnica essencial para o estudo da regeneração de tecidos e para o desenvolvimento de terapias baseadas em células da doença. Demonstramos aqui uma técnica de microcirurgia, que permite o transplante de células geneticamente marcado diretamente no rim de peixes paulistinhas adultos.

Vídeo: 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.

Transplante de Medula Renal inteiro em Zebrafish Adulto

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

Biologia

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.

Transplante de ilhotas pancreáticas para a cápsula renal de ratos 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.

Transplante de lente em Zebrafish e sua Aplicação na Análise de Mutantes Eye

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.

Dissecção de Órgãos do Zebrafish Adulto

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.

Injeção intraperitoneal em Zebrafish Adulto

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.

Quantificar a freqüência de Tumor-propagação células usando Transplante de Células Limitando diluição em singênicos 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 ...

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.

Dissecção do rim Zebrafish Adulto

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.

Em Eletroporação vivo de Morpholinos na Retina Zebrafish Adulto

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.

Na eletroporação in vivo de Morpholinos no Adulto Regeneração Zebrafish Cauda Fin

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.

Gavaging Zebrafish Adulto

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