Agradecemos a Sra. Paula Roy para criação de peixe excelente, e Steve Ekker para o presente amável do plasmídeo pDB600. Estes estudos foram financiados por uma doação para SF de NHGRI.

1. Seleção e clonagem de sequências conservadas para testes <ol><li> É preciso primeiro identificar potenciais seqüências regulatórias para testes funcionais. Contamos com a conservação da sequência evolutiva como critério para selecionar as seqüências, e na maioria das vezes usar o PhastCons algoritmo, que é acessível como uma faixa no navegador Genoma UCSC (http://genome.ucsc.edu/cgi-bin/hgGateway). No entanto, existem muitos outros algoritmos disponíveis para avaliar a conservação de seqüência entre as espécies. </li><li> Uma vez selecionado o nosso seqüências, podemos projetar primers para amplificar cada seqüência de DNA genômico. Primers devem ser selecionados para amplificar a região conservada mais 30 ou mais pares de base em ambos os lados. </li><li> Para amplificação das seqüências selecionadas usamos o sistema de Takara LA TaqTM. Polimerases outro vai funcionar, mas é importante usar um mix que inclui uma enzima com capacidade de revisão, para minimizar as chances de erros introduzidos durante a amplificação. Nós verificar o tamanho amplicon por eletroforese em gel de agarose. </li><li> Usamos o pCR 8/GW/TOPO TA Cloning Kit (Invitrogen) para clonar o produto da PCR em um vetor de entrada contendo locais de recombinação Attl, para gerar o clone entrada Gateway. A reação de clonagem é mais eficiente com produtos de PCR fresco, por isso é melhor para realizar a clonagem em um dia da PCR. Transformação em linhagens DH5α é realizada seguido por espectinomicina seleção de resistência. </li><li> Nós verificamos o produto da clonagem TA por digestão de ~ 500ng de plasmídeo com EcoRI para liberar a inserir e confirmar o tamanho da pastilha por eletroforese em gel de agarose. Recomendamos seqüenciamento para verificar a seqüência de inserção e orientação; primers utilizados para amplificação também pode ser utilizado para o seqüenciamento. </li><li> Do clone de entrada, a seqüência não-codificante é conservada empurrados por recombinação LR para o nosso vector de destino gateway pronto, pGW_cfosGFP1, usando gateway LR Clonase II enzima Mix (Invitrogen). Transformação em linhagens DH5α é realizado seguido de ampicilina seleção de resistência. </li><li> Nós verificamos o produto da recombinação LR por digestão de ~ 500ng de plasmídeo com EcoRV para liberar a inserir e confirmar o tamanho da pastilha por eletroforese em gel de agarose. Uma vez que um clone exato foi identificado, nos preparamos DNA plasmídeo usando o HiSpeed ​​Qiagen ® Kit Midi Plasmid. Nós purificar ainda mais o plasmídeo utilizando o Kit de Purificação QIAquick PCR, de acordo com protocolos do fabricante, e eluir o DNA com 30 mL de água livre de RNase. O plasmídeo é diluída a uma concentração de 125 ng / mL e armazenado a 4 ° C antes de injeções. </li><li> RNA codificação funcional Tol2 enzima transposase é transcrito in vitro da pDB600 plasmid2. Purificamos o plasmídeo usando o kit Qiagen Midi-Prep, então linearizar 10-20 mg com XbaI. A síntese de mRNA é realizada após a mMACHINE mMESSAGE Kit protocolo (Ambion). </li><li> Nós purificar e precipitar o RNA de acordo com instruções do kit. Nós ressuspender RNA para uma concentração final de ~ 1μg/μl, ou 20 l para uma reação única, em água livre de RNase, e quantificar por espectrofotometria de UV. Também analisamos ~ 1μg de RNA por eletroforese em gel de agarose para verificar a transcrição de corpo inteiro. Embora uma TAE padrão ou gel TBE é adequada para esta análise, o tampão de amostra desnaturante incluído com o kit de transcrição deve ser usado de acordo com instruções do kit. </li><li> Testamos cada novo lote de transposase RNA para a eficácia através da realização de injeções com uma conhecida (Figura 1) plasmídeo. Após a verificação, o RNA é dividida em pequenas alíquotas e armazenadas a -80 ° C, para evitar o descongelamento e recongelamento repetido de alíquotas individuais. <img src="/files/ftp_upload/1722/1722fig1.jpg" alt="Figura 1" /> Figura 1. Embrião do zebrafish injetado com o rato SOX10 enhancer como controle. (Top) Brightfield imagem (Bottom) GFP imagem de fluorescência. Cada novo lote de transposase RNA é testado para sua eficácia. </li></ol> 2. Microinjeção de embriões zebrafish para análise mosaico transgênicos <ol><li> Puxamos as agulhas para a microinjeção de 1,2 milímetros de vidro de filamentos de OD capilar em um P-97 Sutter Puller Micropipeta, com um programa projetado para produzir uma dica forte com uma vela bastante acentuada para penetrar córions intacta. Nós quebramos as pontas com a mão sob um estereomicroscópio para um diâmetro exterior de cerca de 15 mm, usando uma lâmina limpa e um micrómetro. As agulhas podem ser feitas no dia anterior injeções, e armazenado em um prato coberto holding de manter limpo. </li><li> Mantemos o nosso peixe-zebra em um ciclo de luz regular escuro, com 14 horas de luz, sob conditions3 padrão. Dia anterior à realização de microinjeções, montamos peixe para acasalamentos cronometrado em tanques de criação de pequeno porte, cada uma composta por um tanque de base, uma inserção de fenda, e uma tampa de plástico. Colocamos três mulheres ou dois homens por tank em fileiras paralelas. </li><li> Na manhã do microinjeções, logo após o ciclo de luz começa, começamos a combinar machos e fêmeas no sistema de água tratada potável para produção de ovos. É importante verificar o peixe com frequência, e recolher os ovos dentro de alguns minutos de postura, para obter lotes bem sincronizados. Produção de ovos pode ser mantido por duas a três horas, continuando a combinar grupos de peixe fresco durante a manhã. </li><li> Com furo de largura, 5 1 / 4 &quot;de vidro Pasteur pipeta equipado com uma lâmpada de látex, que classificar os embriões colhidos em 60 x 15 milímetros pratos Petri, parcialmente cheio de Embrião Medium2, em grupos de 50 embriões, e marcar o tempo recolhidos em a tampa de cada prato. </li><li> Uma vez que os peixes começaram a postura, nós preparamos solução nova injecção, misturando o seguinte em um tubo de microcentrífuga no gelo: <table border="1"><tbody><tr><td> Componente </td><td></td></tr><tr><td> Transposon estoque plasmídeo (125ng/μl) </td><td> 1μl </td></tr><tr><td> Transposase RNA ações (175ng/μl) </td><td> 1μl </td></tr><tr><td> Estoque de vermelho de fenol (2% em H 2 O) </td><td> 0.5μl </td></tr><tr><td> RNase água livre </td><td> 2.5μl </td></tr></tbody></table> As agulhas de injeção são colocados em segurando pratos e preenchido por pipetagem 500 gotas nl de solução de injeção para a grande final de cada agulha. Depois o líquido é atraído para a ponta por acção capilar, a solução adicionais podem ser adicionados a um total de 1,5-2 mL. Nos preparamos, pelo menos, duas agulhas de injeção para cada solução. </li><li> A agulha cheia é carregado na mão agulha titular de um pneumático Pico bomba-injector de pressão ou similar, configurado e conectado a um tanque de N2 por instruções do fabricante. Nós calibrar volumes de injeção, medindo o diâmetro de gotículas expelidas em óleo mineral em um micrómetro. Tipicamente, um tempo de injeção de 120 ms com uma pressão de 20 psi dará uma gota de aproximadamente 1 nl, mas pequenas variações na agulha de diâmetro vai afetar esses parâmetros e recalibração pode ser requerido entre as agulhas. </li><li> As injeções são realizadas sob um estereomicroscópio com 6-10X ampliação. Nós usamos uma pinça fina para segurar o embrião no lugar, empurre a agulha de injeção com pressão constante através do córion e na gema de um embrião no estágio uma célula ou células início dois. Idealmente, nós a posição da ponta da agulha na gema, logo abaixo do blastômeros. Aproximadamente 1 nl de solução injectável é expulso ea agulha retirada. Para cada construção injetamos ≥ 200 embriões. É também muito importante, para cada ninhada de embriões colhidos, para salvar um prato de embriões controle uninjected. </li><li> Após as injeções são concluídos, podemos classificar os embriões através da remoção de ovos não fertilizados, embriões danificados, e injeções falhou (embriões sem vermelho de fenol em blastômeros). Em seguida, incubar os embriões em meio de embriões em 28.5 ° C até o momento adequado para observações. </li></ol> 3. Análise de padrões de expressão mosaico <ol><li> Após um tempo de incubação apropriado, examinamos os embriões injetados para fluorescência. O tempo depende do padrão de expressão normal do gene endógeno zebrafish, mas geralmente procuram diariamente através da primeira fertilização pós 5 dias. </li><li> Examinamos os embriões para fluorescência em um macroscópio MVX10 Olympus equipado para epifluorescência, mas qualquer microscópio semelhante capaz de imagem de baixa potência vai funcionar. Se houver um grande número de embriões que morreram ou desenvolveram de forma anormal no primeiro dia, olhar para os controles uninjected para essa embreagem para ver se eles parecem normais. Se os controles de aparência normal, o problema mais comum é a pureza insuficiente do DNA injetado. Além disso, os embriões em estágios iniciais são sensíveis à quantidade total de plasmídeo injetado, por isso, é possível que a quantificação foi impreciso e muito DNA foi injetado. </li><li> Ao examinar os embriões, tenha em mente que todos eles vão ser mosaico para a construção injetado. Será necessário analisar cuidadosamente todos os embriões, especialmente se o domínio esperado de expressão é pequena. Lembre-se também que a expressão poderia ser dinâmico; fluorescência forte no dia 1 pode desaparecer até dia 2, e os embriões negativos para a expressão dos 3 primeiros dias pode mostrar algo no dia 4. </li><li> Nós escolhemos alguns embriões com expressão mais ampla, e usá-las para documentar o padrão de fluorescência por fotomicroscópio. Após cinco dias de observações, os embriões devem ser devolvidos à facilidade e levantou como um estoque. Em vários meses, os adultos podem ser rastreados para a transmissão de células germinativas dos transgenes. </li></ol> 4. Resultados Vemos uma ampla variety de padrões e níveis de expressão, dependendo do elemento potenciador sendo analisado. Estamos muito interessados ​​em geral tecidos padrões de expressão específica, e muitas vezes são focando genes expressos no esqueleto. e são frequentemente focando genes expressos no esqueleto. A Figura 2 mostra exemplos de padrões de expressão mosaico que temos observado em nosso embriões injetados, com enhancers regulação da expressão de cartilagem e osso. Finalmente, uma parte substancial das seqüências testadas não regulam a expressão tecido específico. Para estes, que mais frequentemente ver fluorescência muito pouco, talvez apenas algumas células por embrião. Menos freqüentemente, podemos ver de fluorescência, mas apenas em dois locais comuns para a expressão ectópica, a epiderme e músculo esquelético (Figura 3). <img src="/files/ftp_upload/1722/1722fig2.jpg" alt="Figura 2" /> Figura 2. Exemplo de padrões de cartilagem e osso expressão observada em embriões injetados. <img src="/files/ftp_upload/1722/1722fig3.jpg" alt="Figura 3" /> Figura 3. Há pouca correlação entre a localização do parente enhancer do gene e regulação da expressão. Uma parte substancial das seqüências testadas não regulam a expressão tecido específico. Estes muitas vezes têm de fluorescência muito pouco, ou pode ter dois locais comuns para a expressão ectópica: a epiderme e músculo esquelético. (Top) imagem Brightfield (Bottom) GFP imagem de fluorescência.

<ol>
	<li>Fisher, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+biological+relevance+of+putative+enhancers+using+Tol2+transposon-mediated+transgenesis+in+zebrafish.">Evaluating the biological relevance of putative enhancers using Tol2 transposon-mediated transgenesis in zebrafish.</a> Nat Protoc. 1 (3), 1297-12 (2006).</li><li>Balciunas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+a+high+cargo-capacity+transposon+for+genetic+applications+in+vertebrates.">Harnessing a high cargo-capacity transposon for genetic applications in vertebrates.</a> PLoS Genet. 2 (11), e169-169 (2006).</li><li>Westerfield, 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> The Zebrafish Book. , (1995).</li></ol>

Nós demonstramos a abordagem utilizada em nosso laboratório para a análise eficiente de enhancers potencial usando transgênese zebrafish. Na maioria das vezes, usamos esta abordagem de olhar para o tecido-específicas enhancers associados com os genes humanos. Apesar da falta de homologia de seqüência mais óbvia do tempo entre humanos e peixes sequências não codificantes, descobrimos que esta abordagem é geralmente bem sucedido em identificar potenciadores para os genes de interesse para nós. Os fatores crít...

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		<th>Catalogue Number</th>
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<td>Takara LA Taq</td>
<td>&nbsp;</td>
<td>Takara Bio Inc</td>
<td>RR02M</td>
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<td>Invitrogen</td>
<td>K2500-20</td>
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<td>Gateway LR Clonase II enzyme Mix</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11791-100</td>
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<td>Library efficiency competent cells DH5&alpha;</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12535-019</td>
<td>&nbsp;</td>
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<td>Library efficiency competent cells DB3.1</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
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<td>mMESSAGE mMACHINE Kit</td>
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<td>HiSpeed Plasmid Midi Kit</td>
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mosaic zebrafish transgenesis for evaluating enhancer sequences

A conclusão da seqüência do genoma humano, juntamente com a de muitas outras espécies, destacou o desafio de atribuir função específica a não seqüências de codificação. Uma função de destaque realizadas pela fração não-codificante do genoma é regular a transcrição de genes, no entanto, não existem métodos eficazes para prever amplamente cis-regulatórias elementos de seqüência de DNA primária. Nós desenvolvemos um protocolo eficiente para avaliar o potencial funcional cis-regulatórias elementos através de transgênese zebrafish. Nossa abordagem oferece vantagens significativas sobre cultura de células-técnicas baseadas em genes developmentally importante, pois fornece informações sobre regulação do gene espacial e temporal. Por outro lado, é mais rápido e menos dispendioso do que experimentos semelhantes em camundongos transgênicos, e nós rotineiramente aplicá-la a seqüências isoladas do genoma humano. Aqui demonstramos nossa abordagem para selecionar elementos para o teste com base na conservação de seqüência e nosso protocolo para seqüências de clonagem e microinjecting-los em embriões de peixe-zebra.

Watch this Scientific Journal Video about Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences at JoVE.com

Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences

Nós demonstramos a nossa abordagem para encontrar elementos potenciais potenciador de genes regulados developmentally e avaliar a sua função através de transgênese zebrafish mosaico.

Transgénese mosaico Zebrafish de Avaliação Sequências Enhancer

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non ...

mosaic-zebrafish-transgenesis-for-evaluating-enhancer-sequences

Research

JoVE Journal

Biology

13.9K visualizações.  University of Pennsylvania. Nós demonstramos a nossa abordagem para encontrar elementos potenciais potenciador de genes regulados developmentally e avaliar a sua função através de transgênese zebrafish mosaico.

Vídeo: Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences

Zebrafish Brain Ventricle Injection

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved cavities within the brain that are filled with cerebrospinal fluid.  In zebrafish, after neural tube formation, the neuroepithelium undergoes a series of constrictions and folds while it fills with fluid resulting in brain ventricle formation.  In order to understand the process of ventricle formation, and the neuroepithelial shape changes that occur at the same time, we needed a way to visualize the ventricle space in comparison to the brain tissue.  However, the nature of transparent zebrafish embryos makes it difficult to differentiate the tissue from the ventricle space.  Therefore, we developed a brain ventricle injection technique where the ventricle space is filled with a fluorescent dye and imaged by brightfield and fluorescent microscopy.  The brightfield and the fluorescent images are then processed and superimposed in Photoshop.  This technique allows for visualization of the ventricle space with the fluorescent dye, in comparison to the shape of the neuroepithelium in the brightfield image.  Brain ventricle injection in zebrafish can be employed from 18 hours post fertilization through early larval stages.  We have used this technique extensively in our studies of brain ventricle formation and morphogenesis as well as in characterizing brain morphogenesis mutants (1-3).

After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.

Zebrafish Injection Ventrículo Cerebral

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved ...

Biologia

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression analyses by RNA interference and stable transgenesis remain limited in many species due to the particular anatomy and molecular cellular biology of the organism. For example, outside of the crown group Caenorhabditis that includes Caenorhabditis elegans3, stably transmitted transgenic lines in non-Caenorhabditis species have not been reported in this specious phylum (Nematoda), with the exception of Strongyloides stercoralis4 and Pristionchus pacificus5. To facilitate the expanding role of P. pacificus in the study of development, evolution, and behavior6-7, we describe here the current methods to use microinjection for making transgenic animals and gene knock down by RNAi. Like the gonads of C. elegans and most other nematodes, the gonads of P. pacificus is syncitial and capable of incorporating DNA and RNA into the oocytes when delivered by direct microinjection. Unlike C. elegans however, stable transgene inheritance and somatic expression in P. pacificus requires the addition of self genomic DNA digested with endonucleases complementary to the ends of target transgenes and coinjection markers5. The addition of carrier genomic DNA is similar to the requirement for transgene expression in Strongyloides stercoralis4 and in the germ cells of C. elegans. However, it is not clear if the specific requirement for the animals' own genomic DNA is because P. pacificus soma is very efficient at silencing non-complex multi-copy genes or that extrachromosomal arrays in P. pacificus require genomic sequences for proper kinetochore assembly during mitosis. The ventral migration of the two-armed (didelphic) gonads in hermaphrodites further complicates the ability to inject both gonads in individual worms8. We also demonstrate the use of microinjection to knockdown a dominant mutant (roller,tu92) by injecting double-stranded RNA (dsRNA) into the gonads to obtain non-rolling F1 progeny. Unlike C. elegans, but like most other nematodes, P. pacificus PS312 is not receptive to systemic RNAi via feeding and soaking and therefore dsRNA must be administered by microinjection into the syncitial gonads. In this current study, we hope to describe the microinjection process needed to transform a Ppa-egl-4 promoter::GFP fusion reporter and knockdown a dominant roller prl-1 (tu92) mutant in a visually informative protocol.

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

In model organisms, transgenesis can manipulate gene functions while RNAi can knockdown specific mRNA transcripts 1-2. This protocol aims to illustrate the techniques needed to introduce stably transmitted DNA and transient double stranded RNA into the necromenic nematode Pristionchus pacificus for studies in evolutionary, developmental, and behavioral biology.

RNAi Mediated Gene Knockdown e Transgénese por microinjeção no Nematóides Necromenic Pristionchus pacificus</em

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression ...

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 ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

Laser-infligido Lesão de Zebrafish muscular esquelético embrionário

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

O tomate / GFP-FLP / FRT Method for Imaging ao vivo de Mosaico Adulto<em&gt; Drosophila</em&gt; As células fotorreceptoras

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation

Mammalian spermatogenesis is a complex differentiation process that occurs in several stages in the seminiferous tubules of the testes. Currently, there is no reliable cell culture system allowing for spermatogenic differentiation in vitro, and most biological studies of spermatogenic cells require tissue harvest from animal models like the mouse and rat. Because the testis contains numerous cell types - both non-spermatogenic (Leydig, Sertoli, myeloid, and epithelial cells) and spermatogenic (spermatogonia, spermatocytes, round spermatids, condensing spermatids and spermatozoa) - studies of the biological mechanisms involved in spermatogenesis require the isolation and enrichment of these different cell types. The STA-PUT method allows for the separation of a heterogeneous population of cells - in this case, from the testes - through a linear BSA gradient. Individual cell types sediment with different sedimentation velocity according to cell size, and fractions enriched for different cell types can be collected and utilized in further analyses. While the STA-PUT method does not result in highly pure fractions of cell types, e.g. as can be obtained with certain cell sorting methods, it does provide a much higher yield of total cells in each fraction (~1 x 108 cells/spermatogenic cell type from a starting population of 7-8 x 108 cells). This high yield method requires only specialized glassware and can be performed in any cold room or large refrigerator, making it an ideal method for labs that have limited access to specialized equipment like a fluorescence activated cell sorter (FACS) or elutriator.

The STA-PUT method allows for the separation of different populations of spermatogenic cells based on size and density.

A separação de tipos de células espermatogênicas Usando STA-PUT velocidade de sedimentação

Mammalian spermatogenesis is a complex  differentiation process that occurs in several stages in the seminiferous  tubules of the testes. Currently, there ...

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca2+ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca2+ signaling.

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.

Automated Análise de Ca dinâmico<sup&gt; 2 +</sup&gt; Sinais em seqüências de imagens

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity greatly impacts cell behavior to regulate normal and pathologic processes including cell proliferation, differentiation, adhesion signaling and directional migration. In this regard, the innate ability of certain cell types to migrate towards a stiffer, or less compliant matrix substrate is referred to as durotaxis. This phenomenon plays an important role during embryonic development, wound repair and cancer cell invasion. Here, we describe a straightforward assay to study durotaxis, in vitro, using polydimethylsiloxane (PDMS) substrates. Preparation of the described durotaxis chambers creates a rigidity interface between the relatively soft PDMS gel and a rigid glass coverslip. In the example provided, we have used these durotaxis chambers to demonstrate a role for the cdc42/Rac1 GTPase activating protein, cdGAP, in mechanosensing and durotaxis regulation in human U2OS osteosarcoma cells. This assay is readily adaptable to other cell types and/or knockdown of other proteins of interests to explore their respective roles in mechanosignaling and durotaxis.

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

Many mammalian cells preferentially migrate towards a more rigid matrix or substrate through durotaxis. The goal of this protocol is to provide a simple in vitro system that can be used to study and manipulate cell durotaxis behaviors by incorporating polydimethylsiloxane (PDMS) substrates of defined rigidity, interfacing with glass coverslips.

Um sistema simplificado para a Avaliação celular Mechanosensing e Durotaxis<em&gt; In Vitro</em

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Nefrotoxina microinjeção no peixe-zebra ao modelo de lesão renal aguda

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...