Este procedimento segue em grande medida a técnica de transplante desenvolvido para peixes caverna pelo laboratório de Bill Jeffrey.

Parte 1: Preparando os embriões Neste protocolo, vamos usar o jj símbolo xy para denotar um mutante lente hipotética zebrafish. <ol><li> Cepas de peixe-zebra são mantidos em condições de instalação padrão peixes em 28,5 C em um ciclo light/10h 14h escuro. </li><li> À noite, os machos e fêmeas lugar da cepa zebrafish jj xy: AB / TU; tg (mGFP) em um tanque com um divisor de separá-los uns dos outros. A progênie desta cruz vai expressar o transgene GFP em todos os tecidos, e serão utilizados como doadores. Em paralelo, use o mesmo procedimento para configurar o cruzamento entre não-transgênicas do tipo selvagem animais. Dependendo da finalidade desse experimento, pode-se cruzar animais de tipo selvagem no locus jj xy como doadores ou hosts. </li><li> Dentro de uma hora após a luz acende na parte da manhã, retire os divisores para permitir que os peixes para acasalar. </li><li> Depois de 15-30 minutos coletar embriões em 100 x 15 milímetros pratos Petri, limpá-los e alterar o meio (água de ovo). </li><li> Manter os embriões em uma incubadora até 28,5 C tempo desejado. </li><li> Quando estiver pronto para transplante, remover o córion manualmente com duas pinças afiadas, e incubar os embriões em EDTA 0,2% em cálcio livre ZFR por 30 minutos. </li></ol> Parte 2: Preparação de agulhas de dissecção <ol><li> O procedimento de transplante requer dois tipos de agulhas: uma aguçada, que é usada para cortar tecidos em volta da lente, remova o córion e embriões liberação de agarose, e uma agulha sem corte utilizado para mover a lente de doadores e inseri-lo no host. Aqui vamos mostrar-lhe como fazer estas agulhas. </li><li> Em primeiro lugar, a ponta de uma pipeta Pasteur deve ser cortado com uma faca de diamante. Em seguida, insira um capilar de vidro na ponta da pipeta Pasteur, para que cerca de metade do seu comprimento é dentro dela. Isto tornará mais fácil para imobilizar o fio de tungstênio pelo derretimento-lo no vidro na próxima etapa. </li><li> Em seguida, insira um fio de tungstênio fino na extremidade aberta do capilar. Derreter o vidro sobre o fio com um bico de Bunsen. Utilizando uma pinça, segure firme a fim de vidro com o fio de metal, enquanto torcendo a outra extremidade da agulha com a mão. O vidro amolecido deve espiral em torno do metal agarrando-o no lugar. </li><li> Se você está fazendo uma ponta da agulha sem corte, então este é o fim do procedimento. Para criar uma agulha afiada, segure o fio de tungstênio sobre um bico de Bunsen para 1-1,5 minutos, queimando o metal para uma ponta muito fina é criado. Verificar a qualidade da ponta de microscropy.It é uma boa idéia para esterilizar ambas as agulhas na chama por alguns segundos, imediatamente antes do transplante. </li></ol> Parte 3: Preparação dos reagentes <ol><li> Sem cálcio Zebrafish Ringer (ZFR) Solutions (116 mM NaCl, 2,9 mM KCl, 5 mM HEPES, pH 7.2) </li><li> 0,2% EDTA em cálcio livre Zebrafish Ringer Solutions (pH 7,2) </li><li> Agarose 1,2% (baixo ponto de fusão) em cálcio livre ZFR </li><li> Agarose 0,2% (baixo ponto de fusão) em cálcio livre ZFR </li><li> Médio de embriões (pH 7,0, por litro, contém: 10 ml da solução Hanks # 1, 1 ml da solução Hanks # 2, 10 ml da solução Hanks # 4, 10 ml da solução Hanks # 5, 0,35 g de bicarbonato de sódio, 300 uL de 2M de HCl, a penicilina-estreptomicina 500 mil U) Assim como em O Livro Zebrafish (University of Oregon Press, 2000). </li><li> Fio de tungstênio, ouro diâmetro, banhado a 0,1 milímetros, Alfa Aesar </li><li> Tubo capilar, 1,0 milímetros de diâmetro, instrumentos de precisão Mundial </li></ol> Parte 4: Processo de Transplante <ol><li> Em um tubo de centrífuga de plástico, prepare 1,2% baixo ponto de fusão de agarose em cálcio livre ZFR pré-aquecido a 40 C em banho-maria. </li><li> Na fase desejada (30 hpf, por exemplo), pegar os embriões em uma pipeta Pasteur, expulsá-los lentamente para dentro do tubo de centrífuga, e, lentamente, expulsá-los para dentro do tubo de centrífuga e incubar por aproximadamente 5 segundos. Os doadores e os anfitriões devem ser incubados separadamente. </li><li> Com uma pipeta, tomar-se os embriões em agarose 1,2% a partir do tubo de centrifugação e organizá-los em duas fileiras paralelas de 100 milímetros de poliestireno prato Petri, mantendo hosts e doadores distintos. Pipetando-los devagar e com cuidado, a maioria dos embriões vai se deitar de lado na agarose. Você terá que reorientar aqueles que não estão nesta posição utilizando a agulha com modo que o olho está voltado para cima antes do agarose solidifica. </li><li> Aguarde o agarose 1,2% para solidificar e depois cobri-la com 0,2% de fusão solução ponto baixo agarose. </li><li> Utilizando a agulha afiada, remova qualquer agarose ao redor dos olhos, e com muito cuidado cortou a lente para fora a partir do host e embriões de doadores usando pequenos acidentes vasculares cerebrais. Certifique-se de cortar apenas perto o suficiente para a lente para não rasgá-lo, mas também para que você não remover o tecido muito do resto do olho host. </li><li> Uma vez livre, as lentes vão flutuar no meio. Utilizando a agulha sem corte, empurre cuidadosamente o doadorlente para um pouco acima do local onde a lente de acolhimento seria normalmente, e, em seguida, empurre-o para dentro do olho com a agulha sem corte. A lente de acolhimento podem ser descartados. </li><li> Deixe embriões de doadores e de acolhimento na agarose 1,2% por 30-60 minutos, e depois libertá-los da agarose utilizando a agulha afiada, e transferi-los em uma placa de 24 poços, com meio embrião. Cada embrião deve ocupar um bem separado. Certifique-se de rotular os poços corretamente de modo que fica claro qual host que corresponde ao doador. </li><li> Incubar a 28,5 C. O doador-derivado lente pode ser distinguida da lente hospedar no lado não operados do mesmo animal com base em GFP fluorescência. Seções também podem ser feitos para medir o tamanho da lente e para determinar se diferencia corretamente. </li></ol><img src="/files/ftp_upload/1258/1258fig1.jpg" alt="figura 1" /> Fig. 1 imagem baixa ampliação de uma agulha afiada usada para lentes transplante. Um capilar de vidro (B) é inserido na pipeta Pasteur (A), e um fino fio de tungstênio (C) é inserido na extremidade aberta do capilar. O vidro sobre o fio é amenizada com um bico de Bunsen. Utilizando uma pinça, segure firme a fim de vidro com o fio de metal, enquanto torcendo a outra extremidade da pipeta Pasteur com a mão. O vidro amolecido deve espiral em torno do metal, agarrando-o no lugar. <img src="/files/ftp_upload/1258/fig2 1258.png" alt="" width="600" height="204" /> Fig. 2 As pontas dos dois tipos de agulhas usadas para a lente transplante. <img src="/files/ftp_upload/1258/1258fig3.jpg" alt="figura 3" /> Fig. 3 embriões foram submetidos a transplante de lente 30hpf. Mostrados são um embrião doador (A, B), um olho com lente de acolhimento em transplantados (C, D), eo lado controle do embrião hospedeiro (E, F). Cada olho é mostrada em ambas luz transmitida e iluminação UV conforme indicado. Fotografias foram tiradas em 48 hpf. O Q01 [9] linhagem transgênica foi utilizada neste experimento.

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

Várias etapas requerem atenção especial durante transplantes lente. <ol><li> Afiadas agulhas: É melhor preparar várias agulhas antes de realizar o transplante de lente, e na ponta das agulhas deve ser o mais fino possível. A agulha mais grossa vai rasgar mais tecido devido ao seu maior diâmetro, causando uma falha no olho para curar. </li><li> Organizando os embriões: Direita antes do transplante você deve posicionar os embriões para que eles estão mentindo em seus lados. Ao posicionar, a agarose 1,2% pod...

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<td>0.1mm diameter tungsten wire</td>
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lens transplantation in zebrafish and its application in the analysis of eye mutants

A lente tem um papel importante no desenvolvimento do cálice óptico [1,2]. Usando o zebrafish como um organismo modelo, questões relativas ao desenvolvimento da lente pode ser tratada. O peixe-zebra é útil para estudos genéticos, devido a várias características vantajosas, incluindo pequeno tamanho, alta fecundidade, ciclo de vida curto, e facilidade de cuidado. Desenvolvimento lente ocorre rapidamente em zebrafish. Em 72 hpf, a lente zebrafish é funcionalmente maduros [3]. Abundantes recursos genéticos e moleculares estão disponíveis para apoiar a investigação no zebrafish. Além disso, a semelhança do olho zebrafish aos de outros vertebrados fornece base para a sua utilização como excelente modelo animal de defeitos humanos [4-7]. Mutantes zebrafish várias lentes apresentam anormalidades, incluindo altos níveis de morte celular, que em alguns casos, leva a uma degeneração completa de tecidos lente [8]. Para determinar se as anormalidades lente são devido a causas intrínsecas ou com defeito interações com os tecidos circundantes, o transplante de uma lente de mutantes em um olho do tipo selvagem é executada. Agulhas utilizando-fogo de metal polido, lentes mutante ou tipo selvagem são cuidadosamente dissecados do animal doador, e transferido para o host. Para distinguir os tecidos do tipo selvagem e mutante, uma linha de transgênico é usado como o doador. Esta linha expressa ligada à membrana GFP em todos os tecidos, incluindo a lente. Esta técnica de transplante é uma ferramenta essencial nos estudos de mutantes lente zebrafish.

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

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

Lente de desenvolvimento envolve interações com outros tecidos. Mutantes olho várias zebrafish são caracterizados por um tamanho de lente anormalmente pequena. Aqui demonstramos um experimento de transplante de lente para determinar se este fenótipo é devido a causas intrínsecas ou interações com defeito com os tecidos que circundam a lente.

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 ...

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

Research

JoVE Journal

Biology

11.0K visualizações.  The Second Teaching Hospital of Jilin University. Lente de desenvolvimento envolve interações com outros tecidos. Mutantes olho várias zebrafish são caracterizados por um tamanho de lente anormalmente pequena. Aqui demonstramos um experimento de transplante de lente para determinar se este fenótipo é devido a causas intrínsecas ou interações com defeito com os tecidos que circundam a lente.

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

Transplantation of Whole Kidney Marrow in Adult Zebrafish

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

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

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

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

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

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

O Ladder Rung Walking Tarefa: um sistema de pontuação e sua aplicação prática.

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

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

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

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

Preparação e Cultura de explantes Rat Lens epitelial para estudar a diferenciação terminal

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

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

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

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

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

Cultura primária e Eletroporação Plasmid do Órgão de Corti murino.

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

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

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

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

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

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 ...

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 ...

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

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

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

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

Projeto e Análise do Comportamento de Preferência de Temperatura e seu Ritmo Circadiano em Drosophila

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

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

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

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

Análise de Composição Nephron e função no rim adulto Zebrafish

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

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

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

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

Aplicação sequencial de lamelas de vidro para avaliar a compressão Rigidez do Lens Mouse: tensão e morfométricas Análises

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

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

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

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

De alta resolução de imagem Time-lapse e análise automatizada de microtúbulos Dynamics em Viver umbilical humano Células Endoteliais da Veia

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

Methods Article

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