We appreciate Dr. Jeong-Soo Lee for sharing the Tg(dbh:EGFP-MYCN) transgenic fish with us in our study. This work was supported by a grant 1K99CA178189-01 from the National Cancer Institute, a fellowship from the Pablove Foundation and the Friends for Life, and young investigator awards from the Alex's Lemonade Stand Foundation and the CureSearch for Children's Cancer Foundation.

NOTA: Todos os estudos de peixe-zebra e manutenção dos animais foram feitas de acordo com a Mayo Clinic Institute IACUC-aprovado protocolo # A41213. 1. As construções de ADN para Transgénese <ol><li> Amplificar uma região do promotor de 5,2 kb da dopamina-beta-hidroxilase (d βh) 8, utilizando o clone de BAC CH211-270H11 (BacPac centro de recursos (BPRC)) como um molde de ADN. Usar um adequado sistema de PCR para a amplificação por PCR de comprimento e precisos de modelos longos de ADN e os seguintes programas do ciclo de PCR: 94 ° C durante 2 min, 10 ciclos de (94 ° C, 15 seg, 50 ° C, 30 seg, 68 ° C, 8 min), seguido por 30 ciclos de (94 ° C, 15 seg, 53 ° C, 30 seg, 68 ° C, 8 min), 68 ° C, 4 min (iniciador directo 5&#39;-GGGGACAACTTTGTATAGAAAAGTTGGCGTACTCCCCCTTTTTAGG-3 &#39; e iniciador inverso 5&#39;-GGGGACTGCTTTTTTGTACAAACTTGTGTTGCTTTGTCGTCTTTTGA-3 &#39;). </li><li> Crie o d βh -pDONRP4-P1r clo entradane 8, utilizando sistema de clonagem recombinante comercial, como a porta de entrada de vários sites. Misture 1 ml de dβh purificada produto de PCR (172 ng / mL), 1 ml (150 ng / mL) pDONRP4-P1r doador vector (juntamente com outros vetores de gateway são generosas doações de Dr. Chi-Bin Chien, Univ. De Utah) , 4 ul de tampão TE (pH 8,0) com 2 ul de mistura de enzimas BP clonase, incubar durante 1 hora a 25 ° C e, em seguida, transformar a One Shot TOP10 competente E. coli de acordo com o protocolo do fabricante. </li><li> Gerar o d βh: transgênica EGFP-MYCN construir 8, utilizando sistema de clonagem recombinante mencionado na etapa 1.2. Misture 1 ml de cada clone de entrada (150 ng / L), incluindo um 5.2-kb dβh promotor (dβh -pDONR P4-P1r construção), EGFP faltando um códon de parada (PME-EGFP construção), e humano MYCN cDNA (uma oferta generosa do Dr. Hogarty no Hospital Infantil da Pennsylvania, MYCN-pDONRP2R-P3 construção), 1 ml (150 ng / mL) do vector destino modificado contendo sítios de reconhecimento I-SCEI (uma oferta generosa do Dr. C. Grabher, Karlsruhe Institute of Technology, Karlsruhe, Alemanha), 4 jul de tampão TE ( pH 8,0) com 2 ul de mistura de enzimas clonase LR, incubar durante 1 hora a 25 ° C e, em seguida, transformar a E. quimicamente competente coli, tais como Um tiro top 10 e seguir o protocolo do fabricante. </li><li> Adicione MITF: transgénico MITF construir 8 por digestão do vector de PNP-MITF com NotI e SalI enzimas de restrição durante 2 h à temperatura ambiente, e o libertado por subclonagem 2.65-kb (uma generosa oferta do Dr. D. Raible, University of Washington). fragmento de ADN que contém o promotor MITF e a sequência de codificação do gene do peixe-zebra MITF nos locais de um vector pBluescript modificado contendo flanqueiam locais de reconhecimento de I-Scel NotI e MluI (uma oferta generosa do Dr. Hui Feng, Universidade de Boston), utilizando ADN-ligase de T4 de acordo com o protocolo do fabricante. NOTA: Para aumentar a eficiência das linhas geradoras MYCN -expressing estáveis, d βh: EGFP-MYCN eo MITF:. Construções de DNA MITF são co-injectado em uma das células de embriões em estágio de nácar Nacre designa um tipo de peixe mutante sem uma crista neural derivado melanï¿½oro 36 durante o desenvolvimento. Assim, o aparecimento de células de pigmento em embriões injectados sugere a integração de MITF: ADN MITF construções no genoma. Demonstrou-se que dois ou três construções de ADN podem ser co-injectado cointegrados no genoma de peixe 31. Assim, a pigmentação causada pela expressão MITF pode servir como um marcador para a integração do dβh: transgene EGFP-MYCN e para facilitar a identificação de MYCN linhagem transgênica estável. </li><li> Misturar o d βh: EGFP-MYCN e MITF: ADN MITFconstruções na proporção de 3: 1 num volume total de 15 ul de uma reacção com 1 ml de enzima I-Scel e 0,75 ul de tampão. Certifique-se que a quantidade total de DNA na reação não exceda 750 ng. </li><li> Efectua-se a digestão I-Scel à TA durante 4 h ou O / N. No segundo dia, o I-SceI- ADNs digeridos estão prontos para microinjecção ou pode ser armazenado a -20 ° C durante a injecção no futuro. Armazenar a enzima I-Scel a -80 ° C em pequenas aliquotas para manter a eficácia da enzima. </li><li> Subclonar o ALKF1174L humana e gene ALK-tipo selvagem do vetor pCDNA3 (a oferta generosa do Dr. George no Dana-Farber Cancer Institute) 7 em sites de um vetor pENTRY1A EcoRI e NotI (a oferta generosa do Dr. C. Grabher, Instituto de Tecnologia Karlsruhe, Karlsruhe, Alemanha), utilizando ADN-ligase de T4 de acordo com o protocolo do fabricante. </li><li> Gerar o d βh: ALKF1174L &lt;/ Em&gt; ou dβh: construções transgênicas ALKWT que utilizam o sistema recombinante, como mencionado no Protocolo 1.2. Resumidamente, combinar três clones de entrada, dβh -pDONRP4-P1r, ALKF1174L- pENTRY1A (ou ALKWT -pENTRY1A) e P3e-poli, no vector de destino modificado contendo sítios de reconhecimento I-SCEI (uma oferta generosa do Dr. C. Grabher, Karlsruhe Instituto de Tecnologia de Karlsruhe, Alemanha), utilizando LR clonase enzima Mix, de acordo com o protocolo do fabricante. </li><li> Misture o d βh: ALKF1174L (ou dβh: ALKWT) e dβh: DNA mCherry constrói em uma proporção de 3: 1 e linearizar lhes com a enzima I-SCEI como descrito no Protocolo 1,5-1,6. </li></ol> 2. Microinjection <ol><li> Utilize micropipetas de vidro com 1,0 mm de diâmetro para todas as injecções 37. Quebre a ponta dos micropipetas de vidro com uma lâmina de barbear antes da injeção. Calibrar o volume de injeção por injeçãoing H 2 O em uma gota de óleo mineral. Medir o diâmetro das gotículas resultantes e ajustar o microinjector (pressão ou a duração do impulso de pressão) para garantir que o volume da injecção é inferior a 10% do volume total de células de uma célula de embriões de estágio. </li><li> Adicionar mais 0,5 ul de fresco enzima I-Scel (5 unidades / ul) em 5 mL de solução de injecção contendo construções de ADN imediatamente antes da injecção para aumentar a eficiência da transgénese 38. Injectar 50-80 pg de construções de ADN linearizado no citoplasma de embriões de estádio de uma célula. Para garantir o sucesso da injeção, misturar a amostra com 0,25% de fenol vermelho para visualização. Se as células ficam vermelhos após a injecção, isso indica a microinjecção é bem sucedido. Para garantir uma elevada taxa de sucesso para a geração de peixes transgénicos, que normalmente injetar até 500 embriões. </li><li> Para gerar peixes transgénicos que expressam estavelmente MYCN, coinject linearizado d βh: EGFP-MYCN e MITF: construções de ADN MITF (numa proporção de 3: 1) em embriões de peixe-zebra a pigmentação mutante nácar no estádio de uma célula. A expressão de MITF serve como repórter para a integração das construções transgênicas no genoma dos peixes. </li><li> Para a sobre-expressão do mosaico da ALK na de tipo selvagem ou embriões transgénicos MYCN, co-injectar o linearizado d βh: ALK F1174L (ou dβh: ALKWT) com dβh: ADN mCherry constrói (numa proporção de 3: 1) para a célula de um embriões resultantes da criação de F1 heterozigotos Tg (dβh: EGFP-MYCN) peixes transgénicos com o tipo selvagem AB peixes. Assim, metade da prole é transgênica para MCYN e metade são do tipo selvagem. </li></ol> 3. Tela de Peixe Estável ou Mosaic Transgênicos <ol><li> Para aumentar a eficiência de identificação de linhas MYCN -expressing estáveis, anestesiar os embriões injectados nácar principalmente com tricaína (0,02%) det dias 3-5 de pós-fertilização e tela para a pigmentação. Transferir os embriões com pigmentação para uma nova placa de Petri com água ovo fresco e elevá-las a maturidade sexual para posterior triagem para o peixe fundador, que carregam o d βh: transgene EGFP-MYCN nas células germinativas. </li><li> Para identificar o fundador com transmissão germinal de EGFP-MYCN, peixes F0 adulto pigmentada outcross com o tipo selvagem AB peixes e tela de embriões EGFP-positivo em 1-2 dias após a fertilização para posterior genotipagem. Coloque um único embrião F1 EGFP-positivo em um tubo de PCR e retirar todo o líquido. Adicionar 50 ul de tampão de extracção ADNg que contém 12,5 ul de tampão de lise 4x, 35 ul de H2O e 2,5 ul de proteinase K (10 mg / ml) para único embrião. Incubar a mistura reaccional durante 2-3 horas a 55 ° C, seguido de 10 min de incubação a 98 º C para inactivar a proteinase K. Para fazer 50 ml de tampão de lise 4x, adicionar 500 uL de Tris 1M (pH 8,4), 2,5 ml de 1M KCl tde o, 47 ml de H 2 O. NOTA: Depois de F0 MYCN peixes transgénicos estável são criados com o tipo selvagem AB peixe, todos os filhotes são pigmentadas. Assim, pigmentação não pode servir como marcador para a identificação de peixes MYCN- positivo. Enquanto nos peixes transgénicos MYCN, a proteína de fusão de EGFP-MYCN é expresso na PSNS, incluindo os neurónios simpáticos do gânglio cervical superior e segmentar sequencial gânglio da cadeia simpática e dos não-PSNs neurónios dopaminérgicos, tais como a medula oblonga e gânglios cranianos 8. Assim, a expressão da EGFP pode servir como um marcador para identificação dos embriões transgénicos estáveis ​​MYCN. </li><li> Utilizar 2 ul de ADNg extraídos a partir do embrião F1 pigmentada como molde, iniciadores MYCN -test F1: 5&#39;-CTG CTT AAC GAG GAG CTG TG-3 &#39;; MYCN -R3: 5&#39;-AGG CAT CGT AGG ATC TTG AG-3 &#39;eo seguinte sagacidade programah, a GC-RICH sistema de PCR: 1 ciclo de 95 ° C durante 3 min, 25 ciclos de 95 ° C durante 30 seg, 58 ° C durante 30 segundos, e 72 ° C durante 3 min. Nós normalmente genotipar 14-16 embriões pigmentados a partir de um único acasalamento para confirmar a presença do transgene integrado em MYCN os embriões pigmentadas, mais do que 6 fundador peixe superexpressando MITF e MYCN foram identificados por este método. </li><li> Levante-se o restante dos embriões F1 EGFP-positivo. Clipe Fin e do genótipo-los em 2-3 meses de idade utilizando o protocolo anterior para confirmar ainda mais a integração de MYCN transgene no peixe. Raça F1 MYCN peixes transgénicos estável com o tipo selvagem AB peixe. Co-injetar o d βh linearizado: ALK F1174L (ou dβh: ALKWT) com dβh: DNA mCherry constrói em embriões unicelulares como descrito no Protocolo 2.4. </li><li> Ordenar o MYCN -expressing embriões no experimento descrito no Protocolo 2.4 usando um fluor estereoscópicoescence microscópio e examinar os embriões injetados principalmente nos dias 1-3 de pós-fertilização para a expressão de EGFP-MYCN no PSNS. Em seguida, durante 2-5 dias de pós-fertilização, anestesiar os embriões com tricaína (0,02%) e classificar os embriões MYCN- positivas ou negativas novamente com base na expressão de mCherry nas PSNS usando um microscópio estereoscópico de fluorescência. A expressão de mCherry serve como um marcador para a co-expressão da ALK em tecidos dos animais injectados primário mosaico. NOTA: ~ 600 prole resultante da criação de peixes transgénicos MYCN estável com o tipo selvagem de peixe por grupo foram injectados com as construções de ADN linearizado, incluindo d βh: ALK F1174L e dβh: mCherry, dβh: ALKWT e dβh: mCherry, ou dβh : mCherry sozinho, respectivamente. Mosaico de expressão mCherry pode ser observado em 70-90% dos peixes primeiramente injectado. Um meio para pontualterço dos peixes injetados sobreviveram através do estágio de larva para o relógio tumor. </li><li> Levantar todo o mCherry + MYCN + e + mCherry MYCN - embriões de acordo com os protocolos padrão do livro zebrafish 39 e monitorar início tumor começando às 5 semanas pós-fertilização. </li></ol> 4. Tumor Watch em Mosaic Transgênicos Peixe <ol><li> Monitorar mCherry- positivo peixes injetados principalmente a cada 2 semanas a partir de 5 semanas pós-fertilização para a evidência de aparecimento de tumores. </li><li> Anestesiar peixe com tricaina (0,02%) e tela para a presença de mCherry - e EGFP -expressing tumores nos PSNS sob a Nikon SMZ-1500 fluorescência estereoscópica microscópio equipado com uma mira a câmera digital da DS-U1. </li><li> Para confirmar se os tumores que expressam o transgene são ALK, isolar o mCherry- e massas EGFP-ALK positivo para a genotipagem de PCR. </li><li> Usando PCR, amplificar o ADN genómicoextraiu-se a partir de tumores desenvolvidos com os seguintes iniciadores: ALK P7: 5&#39;-AGG GGT CCA CGG GTC AAT CG-3 &#39;e ALK P18: 5&#39;-TGT CTT CAG GAT GTT GCT GC-3&#39; e a sequência de reacção de PCR: 1 ciclo de 94 ° C durante 5 min, 30 ciclos de (94 ° C durante 30 seg, 55 ° C durante 30 segundos, e 72 ° C durante 60 seg). Em seguida, a sequência do produto de PCR com o iniciador P7 ALK para confirmar ainda mais a existência de mutante ou de tipo selvagem ALK em tumores mCherry-positivos. </li></ol>

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-injection+strategies+to+modify+radiation+sensitivity+and+tumor+initiation+in+transgenic+Zebrafish.">Co-injection strategies to modify radiation sensitivity and tumor initiation in transgenic Zebrafish.</a> Oncogene. 27 (30), 4242-4248 (2008).</li><li>Chen, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncogenic+mutations+of+ALK+kinase+in+neuroblastoma.">Oncogenic mutations of ALK kinase in neuroblastoma.</a> Nature. 455 (7215), 971-974 (2008).</li><li>Pugh, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetic+landscape+of+high-risk+neuroblastoma.">The genetic landscape of high-risk neuroblastoma.</a> Nature genetics. , (2013).</li><li>Berry, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ALK(F1174L)+mutation+potentiates+the+oncogenic+activity+of+MYCN+in+neuroblastoma.">The ALK(F1174L) mutation potentiates the oncogenic activity of MYCN in neuroblastoma.</a> Cancer Cell. 22 (1), 117-130 (2012).</li><li>Heukamp, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+expression+of+mutated+ALK+induces+neuroblastoma+in+transgenic+mice.">Targeted expression of mutated ALK induces neuroblastoma in transgenic mice.</a> Sci Transl Med. 4 (141), 141ra191 (2012).</li><li>Lister, J. A., Robertson, C. P., Lepage, T., Johnson, S. L., Raible, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=nacre+encodes+a+zebrafish+microphthalmia-related+protein+that+regulates+neural-crest-derived+pigment+cell+fate.">nacre encodes a zebrafish microphthalmia-related protein that regulates neural-crest-derived pigment cell fate.</a> Development. 126 (17), 3757-3767 (1999).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. 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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev Dyn. 203 (3), 253-310 (1995).</li><li>Urasaki, A., Asakawa, K., Kawakami, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+transposition+of+the+Tol2+transposable+element+from+a+single-copy+donor+in+zebrafish.">Efficient transposition of the Tol2 transposable element from a single-copy donor in zebrafish.</a> Proc Natl Acad Sci U S A. 105 (50), 19827-19832 (2008).</li><li>Caneparo, L., Pantazis, P., Dempsey, W., Fraser, S. 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R., Takahashi, K., Futreal, P. A., Chin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+patterns+of+somatic+mutations+in+cancer.">Emerging patterns of somatic mutations in cancer.</a> Nat Rev Genet. 14 (10), 703-718 (2013).</li><li>Sander, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+disruption+in+somatic+zebrafish+cells+using+engineered+TALENs.">Targeted gene disruption in somatic zebrafish cells using engineered TALENs.</a> Nat Biotechnol. 29 (8), 697-698 (2011).</li><li>Sander, J. D., Joung, J. 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The authors declare that they have no competing financial interests.

Neste estudo representativo, foi utilizado co-injecção transiente e co-expressão de LFA activado com o gene repórter mCherry em MYCN -expressing peixes transgénicos para mostrar que estes genes cooperar para acelerar significativamente o aparecimento de neuroblastoma, consistente com a nossa verificação anterior no composto estável coexpressï¿½ peixes transgénicos tanto ALK e MYCN 8 activada. Esta abordagem transgénica mosaico possui várias vantagens ...

Cancros são doenças progressivas marcados pela acumulação de mutações patológicas, exclusões e ganhos de cromossomos ao longo do tempo. Estas anormalidades genéticas pode afectar vários processos celulares que variam do ciclo celular, morte celular, no metabolismo energético e montagem do citoesqueleto de salientar as respostas, tais como a hipoxia. Assim, tumorigenesis reflete as ações coletivas de várias aberrações genéticas em toda uma gama de processos biológicos. Os recentes esforços de integração genômicas de investigação, incluindo a sequenciação do genoma inteiro, exome seqüenciamento, sequenciamento alvejado, o seqüenciamento de profundidade e estudos de associação do genoma, identificaram um número cada vez maior de alterações genéticas novos em praticamente todos os tipos de tumores 1-4. Em muitos casos, as lesões genéticas ocorrem em conjunto de uma maneira não aleatória 5-8, sugerindo a sua cooperação na patogénese da doença. Dissecando os papéis oncogênicos do grande variedade de genes expressos de maneira aberrante resultantes from essas lesões genômicas é necessário elaborar novas estratégias terapêuticas e para entender as respostas das células tumorais para esses agentes, mas isso provou ser uma tarefa difícil, que exige muito robustos sistemas de modelos animais para a realização de análise genômica funcional de alto rendimento em vivo. Apesar de mamíferos, especialmente roedores, são modelos favorecidas em biologia do câncer, o peixe-zebra começou a atrair atenção considerável. O peixe-zebra teleósteos (Dario rerio) tem sido usada como um organismo modelo para o estudo do desenvolvimento desde os anos 1960 e foi aplicado em primeiro lugar ao estudo da patogénese do tumor, em 1982, 9-11. Facilidade de manutenção, tamanho pequeno corpo, e alta fecundidade fazer o peixe-zebra um modelo robusto para telas genéticos para a frente em larga escala para identificar mutações que conferem fenótipos anormais e patológicas 10. A transparência óptica de embriões de peixes-zebra é outra característica fundamental apoiar uma maior utilização deste modelo de câncer, comoele permite que in vivo de imagens a serem realizadas para localizar o desenvolvimento do tumor em tempo real, 9, um aplicativo que é relativamente difícil em roedores 12. Recente análise genômica comparativa do genoma de referência do peixe-zebra (Zv9) revelou 26.206 genes codificadores de proteínas, com 71% tendo ortï¿½ogos humanos, dos quais 82% são correlacionados com genes associados à doença no Online mendeliana Herança em Man (OMIM) do banco de dados 13, 14. Por conseguinte, o peixe-zebra foi usado para modelar diversos tipos de cancros humanos, incluindo neuroblastoma 8, de células T aguda leucemia linfoblástica (T-ALL), 15,16, 17,18 melanoma, sarcoma de Ewing, 19, 20,21 rabdomiossarcoma, carcinoma pancreático 22, carcinoma hepatocelular e 23 mielóide malignidades 24,25, e foi selecionado como um modelo de câncer para o xenotransplante estuda 11,26. A transgênico estávelabordagem no peixe-zebra é comumente utilizada para estudar o efeito de ganho-de-função de genes no desenvolvimento normal ou patogénese da doença 27,28. Para desenvolver tal um modelo (Figura 1A), um injecta uma construção de ADN contendo o gene de interesse accionado por um promotor específico de tecido para dentro de uma células de embriões de tipo selvagem. Três a quatro meses após a injeção, quando os embriões injetados atingir a maturidade sexual, eles são outcrossed com o tipo selvagem de peixe para a tela para os que apresentaram integração do DNA construir em sua linha germinativa, o que lhes licencia como fundador peixe. Muitos factores, tais como o número de cópias e sítio de integração do transgene, afectar a expressão do transgene em linhas transgénicas estáveis. Assim, para desenvolver um modelo de tumor transgénico, várias linhas transgénicas estáveis ​​que sobre-expressam um único oncogene tem que ser gerada em primeiro lugar e rastreados para a linha de expressar o transgene a um nível que pode levar à indução de tumores. No entanto, se a sobre-expressão de um candidato oncogene é tóxico para as células germinativas, que é difícil gerar uma linha estável por transgénico que sobre-expressam o transgene directamente 29. Assim, esta abordagem pode ser demorado, com um elevado risco de falha para gerar um modelo de cancro adequado. Aqui, ilustramos uma estratégia alternativa com base na transgénese transiente mosaico (Figura 1B) que oferece vantagens únicas sobre a transgénese estável tradicional para estudo genómica funcional in vivo. Nesta abordagem, uma ou mais construções de transgenes são injectados no estádio de uma célula de embriões transgénicos ou de tipo selvagem. As construções de DNA injetado contendo transgenes são então mosaically e aleatoriamente integrado no peixes injetados primário, resultando em genótipos mistos dentro de populações de células em vários peixes individuais 30. Além disso, a co-injecção de ADN múltiplas construções de embriões unicelulares leva à co-integração na mesma célula em locais aleatórios, permitindo que um trace as células com expressão de transgenes e explorar as interações de diferentes genes durante a patogênese da doença nos animais mosaico 31. Como prova de princípio, nós transitoriamente superexpressado ALK mutacionalmente ativado (F1174L) com gene repórter mCherry no sistema nervoso simpático periférico (PSNS) sob o controle do hidroxilase dopamina beta (d βh) promotor de tipo selvagem e peixes transgênicos com superexpressão MYCN. ALK, que codifica um receptor da tirosina quinase, é o gene mais frequentemente mutado em alto risco neuroblastoma 5-7,32,33. ALK (F1174L), como uma das mutações somáticas ativadoras mais frequentes e mais potentes, é sobre-representados em MYCN- amplificado pacientes neuroblastoma de alto risco e sinergia com MYCN superexpressão para acelerar neuroblastoma tumorigenesis em ambos os ratinhos transgénicos estáveis ​​e modelos de peixes-zebra transgénicos 8,34,35. Por mosaicosobreexpressão transiente de ALK (F1174L) com mCherry nos peixes transgénicos MYCN, nós recapitulado a aceleração do aparecimento do tumor observada nos peixes transgénicos que sobre-expressam tanto estável ALK (F1174L) e MYCN, sugerindo que a estratégia de transgénese mosaico pode ser utilizado para rapidamente e eficientemente avaliar as contribuições relativas de vários oncogenes na iniciação do tumor in vivo.

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			<td height="21" style="height:21px;width:373px;">Name of Material/ Equipment</td>
			<td style="width:171px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
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			<td height="42" style="height:42px;">Expand Long Template PCR System&#160;</td>
			<td style="width:171px;">Roche Applied Science, IN</td>
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			<td height="21" style="height:21px;">pCR-TOPO vector&#160;</td>
			<td style="width:171px;">Invitrogen, CA</td>
			<td style="width:119px;">451641</td>
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		<tr height="25">
			<td height="25" style="height:25px;">T4 DNA ligase</td>
			<td style="width:171px;">New England Biolabs, MA</td>
			<td style="width:119px;">M0202M</td>
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			<td height="21" style="height:21px;">Gateway LR Clonase II enzyme 
			Mix</td>
			<td style="width:171px;">Invitrogen, CA</td>
			<td>11791-100</td>
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			<td height="21" style="height:21px;">Gateway&#174; BP Clonase&#174; II enzyme mix</td>
			<td style="width:171px;">Invitrogen, CA</td>
			<td>11789-020</td>
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			<td height="24" style="height:25px;">GC-RICH PCR System&#160;</td>
			<td style="width:171px;">Roche Applied Science, IN</td>
			<td>12 140 306 001</td>
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		<tr height="26">
			<td height="26" style="height:26px;">Meganuclease I-SceI&#160;</td>
			<td style="width:171px;">New England Biolabs, MA</td>
			<td>R0694S</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Nikon SMZ-1500 stereoscopic fluorescence microscope&#160;</td>
			<td style="width:171px;">Nikon, NY</td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nikon digital sight DS-U1 camera</td>
			<td style="width:171px;">Nikon, NY</td>
			<td style="width:119px;"></td>
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mosaic zebrafish transgenesis for functional genomic analysis of candidate cooperative genes in tumor pathogenesis

Comprehensive genomic analysis has uncovered surprisingly large numbers of genetic alterations in various types of cancers. To robustly and efficiently identify oncogenic &#8220;drivers&#8221; among these tumors and define their complex relationships with concurrent genetic alterations during tumor pathogenesis remains a daunting task. Recently, zebrafish have emerged as an important animal model for studying human diseases, largely because of their ease of maintenance, high fecundity, obvious advantages for in vivo imaging, high conservation of oncogenes and their molecular pathways, susceptibility to tumorigenesis and, most importantly, the availability of transgenic techniques suitable for use in the fish. Transgenic zebrafish models of cancer have been widely used to dissect oncogenic pathways in diverse tumor types. However, developing a stable transgenic fish model is both tedious and time-consuming, and it is even more difficult and more time-consuming to dissect the cooperation of multiple genes in disease pathogenesis using this approach, which requires the generation of multiple transgenic lines with overexpression of the individual genes of interest followed by complicated breeding of these stable transgenic lines. Hence, use of a mosaic transient transgenic approach in zebrafish offers unique advantages for functional genomic analysis in vivo. Briefly, candidate transgenes can be coinjected into one-cell-stage wild-type or transgenic zebrafish embryos and allowed to integrate together into each somatic cell in a mosaic pattern that leads to mixed genotypes in the same primarily injected animal. This permits one to investigate in a faster and less expensive manner whether and how the candidate genes can collaborate with each other to drive tumorigenesis. By transient overexpression of activated ALK in the transgenic fish overexpressing MYCN, we demonstrate here the cooperation of these two oncogenes in the pathogenesis of a pediatric cancer, neuroblastoma that has resisted most forms of contemporary treatment.

Para investigar se a sobre-expressão de F1174L ALK mutacionalmente activado ou de tipo selvagem ALK pode colaborar com MYCN na indução de neuroblastoma, que sobre-expressa ou activada humana ou ALK-ALK humano do tipo selvagem sob o controlo do promotor d βh no PSNS de peixes transgénicos que sobre-expressam MYCN. Qualquer uma das seguintes construções, dβh - ALKF1174L ou dβh - ALKWT, foram co-injectado com dβh - ...

Watch this Scientific Journal Video about Mosaic Zebrafish Transgenesis for Functional Genomic Analysis of Candidate Cooperative Genes in Tumor Pathogenesis at JoVE.com

Transgênese de peixe-zebra em mosaico para análise genômica funcional de genes candidatos cooperativos na patogênese de tumores | Biologia do Desenvolvimento | Vídeo

Mosaic Zebrafish Transgenesis for Functional Genomic Analysis of Candidate Cooperative Genes in Tumor Pathogenesis

The goal of this study is to demonstrate how the mosaic transgenesis strategy can be used in zebrafish to rapidly and efficiently assess the relative contributions of multiple oncogenes in tumor initiation and progression in vivo.

Mosaic Zebrafish Transgénese para Análise Funcional Genomic de candidatos Genes de cooperação em Tumor Patogênese

Comprehensive genomic analysis has uncovered surprisingly large numbers of genetic alterations in various types of cancers.  To robustly and efficiently ...

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11.1K visualizações.  Mayo Clinic College of Medicine, Center for Individualized Medicine. The goal of this study is to demonstrate how the mosaic transgenesis strategy can be used in zebrafish to rapidly and efficiently assess the relative contributions of multiple oncogenes in tumor initiation and progression in vivo.

Vídeo: Mosaic Zebrafish Transgenesis for Functional Genomic Analysis of Candidate Cooperative Genes in Tumor Pathogenesis

Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the cardiac transcriptome from being masked by the global embryonic transcriptome, it is necessary to collect sufficient numbers of hearts for further analyses. Furthermore, as zebrafish cardiac development proceeds rapidly, heart collection and RNA extraction methods need to be quick in order to ensure homogeneity of the samples. Here, we present a rapid manual dissection protocol for collecting functional/beating hearts from zebrafish embryos. This is an essential prerequisite for subsequent cardiac-specific RNA extraction to determine cardiac-specific gene expression levels by transcriptome analyses, such as quantitative real-time polymerase chain reaction (RT-qPCR). The method is based on differential adhesive properties of the zebrafish embryonic heart compared with other tissues; this allows for the rapid physical separation of cardiac from extracardiac tissue by a combination of fluidic shear force disruption, stepwise filtration and manual collection of transgenic fluorescently labeled hearts.

To analyse cardiac gene expression profiles during zebrafish heart development, total RNA has to be extracted from isolated hearts. Here, we present a protocol for collecting functional/beating hearts by rapid manual dissection from zebrafish embryos to obtain cardiac-specific mRNA.

Em larga escala Zebrafish Embryonic Coração de dissecação para a análise transcricional

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the ...

Biologia do Desenvolvimento

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

Análise de Zebrafish larvas Músculo Esquelético Integrity com Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

Técnica ao Target microinjeção para o desenvolvimento<em&gt; Xenopus</em&gt; Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and behavioral phenotypes. The remarkably diverse phenotypes that have evolved as marine populations adapt to countless freshwater environments, combined with the ability to cross marine and freshwater forms, provide a rare vertebrate system in which genetics can be used to map genomic regions controlling evolved traits. Excellent genomic resources are now available, facilitating molecular genetic dissection of evolved changes. While mapping experiments generate lists of interesting candidate genes, functional genetic manipulations are required to test the roles of these genes. Gene regulation can be studied with transgenic reporter plasmids and BACs integrated into the genome using the Tol2 transposase system. Functions of specific candidate genes and cis-regulatory elements can be assessed by inducing targeted mutations with TALEN and CRISPR/Cas9 genome editing reagents. All methods require introducing nucleic acids into fertilized one-cell stickleback embryos, a task made challenging by the thick chorion of stickleback embryos and the relatively small and thin blastomere. Here, a detailed protocol for microinjection of nucleic acids into stickleback embryos is described for transgenic and genome editing applications to study gene expression and function, as well as techniques to assess the success of transgenesis and recover stable lines.

Transgenic manipulations and genome editing are critical for functionally testing the roles of genes and cis-regulatory elements. Here a detailed microinjection protocol for the generation of genomic modifications (including Tol2-mediated fluorescent reporter transgene constructs, TALENs, and CRISPRs) is presented for the emergent model fish, the threespine stickleback.

Microinjeção para Transgénese e Genoma Edição na Threespine Sticklebacks

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and ...

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into the developing oocyte. Because these events rely on maternal products which typically act very soon after fertilization-that preexist inside the egg, standard approaches for expression and functional reduction involving the injection of reagents into the fertilized egg are typically ineffective. Instead, such manipulations must be performed during oogenesis, prior to or during the accumulation of maternal products. This article describes in detail a protocol for the in vitro maturation of immature zebrafish oocytes and their subsequent in vitro fertilization, yielding viable embryos that survive to adulthood. This method allows the functional manipulation of maternal products during oogenesis, such as the expression of products for phenotypic rescue and tagged construct visualization, as well as the reduction of gene function through reverse-genetics agents.

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here.

Manipulação funcional do gene materno produtos Usando<em&gt; In Vitro</em&gt; Maturação do oócito em Zebrafish

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into ...

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM). The images from these two microscopy modalities are easily merged using tissue landmarks with open source software. Here we describe the adapted method for the larval zebrafish retina. However, this method is also applicable to other types of tissues and organisms. We demonstrate that the complementary information obtained by this correlation is able to resolve the expression of mitochondrial proteins in relation with the membranes and cristae of mitochondria as well as to other compartments of the cell.

This protocol describes the necessary steps to obtain subcellular protein localization results on zebrafish retina by correlating super-resolution light microscopy and scanning electron microscopy images.

Correlativa Super-resolução e microscopia eletrônica para resolver a localização da proteína na Retina de Zebrafish

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and ...

Use of a Recombinant Mosquito Densovirus As a Gene Delivery Vector for the Functional Analysis of Genes in Mosquito Larvae

In vivo microinjection is the most commonly used gene transfer technique for analyzing the gene functions in individual mosquitoes. However, this method requires a more technically demanding operation and involves complicated procedures, especially when used in larvae due to their small size, relatively thin and fragile cuticle, and high mortality, which limit its application. In contrast, viral vectors for gene delivery have been developed to surmount extracellular and intracellular barriers. These systems have the advantages of easy manipulation, high gene transduction efficiency, long-term maintenance of gene expression, and the ability to produce persistent effects in vivo. Mosquito densoviruses (MDVs) are mosquito-specific, small single-stranded DNA viruses that can effectively deliver foreign nucleic acids into mosquito cells; however, the replacement or insertion of foreign genes to create recombinant viruses typically causes a loss of packaging and/or replication abilities, which is a barrier to the development of these viruses as delivery vectors.
Herein, we report using an artificial intronic small-RNA expression strategy to develop a non-defective recombinant Aedes aegypti densovirus (AaeDV) in vivo delivery system. Detailed procedures for the construction, packaging and quantitative analysis of the rAaeDV vectors, and for larval infection are described.
This study demonstrates, for the first time, the feasibility of developing a non-defective recombinant MDV micro RNA (miRNA) expression system, and thus providing a powerful tool for the functional analysis of genes in mosquito and establishing a basis for the application of viral paratransgenesis for controlling mosquito-borne diseases. We demonstrated that Aedes albopictus 1st instar larvae could be easily and effectively infected by introducing the virus into the water body of the larvae breeding site and that the developed rAaeDVs could be used to overexpress or knock down the expression of a specific target gene in larvae, providing a tool for the functional analysis of mosquito genes.

We report using an artificial intronic small RNA expression strategy to develop a non-defective recombinant Aedes aegypti densovirus (AaeDV) in vivo delivery system. A detailed procedure for the construction, packaging, and quantitative analysis of the rAaeDV vectors as well as for larval infection is described.

Uso de um Mosquito recombinante Densovirus como um vetor de entrega do Gene para a análise funcional de Genes em larvas do Mosquito

In vivo microinjection is the most commonly used gene transfer technique for analyzing the gene functions in individual mosquitoes. However, this method ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

Visualização da atividade elétrica celular no Zebrafish embriões adiantados e tumores

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Genotyping and Quantification of In Situ Hybridization Staining in Zebrafish

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in developmental biology for decades. Traditionally, most gene expression studies relied on visual evaluation of the ISH signal, a method that is prone to bias, particularly in cases where sample identities are known a priori. We have previously reported on a method to circumvent this bias and provide a more accurate quantification of ISH signals. Here, we present a simple guide to apply this method to quantify the expression levels of genes of interest in ISH-stained embryos and correlate that with their corresponding genotypes. The method is particularly useful to quantify spatially restricted gene expression signals in samples of mixed genotypes and it provides an unbiased and accurate alternative to the traditional visual scoring methods.

Gene editing technologies have enabled researchers to generate zebrafish mutants to investigate gene function with relative ease. Here, we provide a guide to perform parallel embryo genotyping and quantification of in situ hybridization signals in zebrafish. This unbiased approach provides greater accuracy in phenotypical analyses based on in situ hybridization.

Genotipagem e Quantificação da Coloração de Hibridização in Situ em Zebrafish

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in ...

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and molecular complexity of skeletal elements increases with development. Tissue heterogeneity, such as at the interface of cartilaginous and osseous elements with each other or with surrounding tissues, is one obstacle to the study of developing cartilage and bone. Our protocol provides a rapid method of tissue processing and isolation of cartilage and bone that yields high quality RNA for gene expression analysis. Fresh frozen tissues of mouse embryos are sectioned and brief cresyl violet staining is used to visualize cartilage and bone with colors distinct from surrounding tissues. Slides are then rapidly dehydrated, and cartilage and bone are isolated subsequently by LCM. The minimization of exposure to aqueous solutions during this process maintains RNA integrity. Mouse Meckel&rsquo;s cartilage and mandibular bone at E16.5 were successfully collected and gene expression analysis showed differential expression of marker genes for osteoblasts, osteocytes, osteoclasts, and chondrocytes. High quality RNA was also isolated from a range of tissues and embryonic ages. This protocol details sample preparation for LCM including cryoembedding, sectioning, staining and dehydrating fresh frozen tissues, and precise isolation of cartilage and bone by LCM resulting in high quality RNA for transcriptomic analysis.

This protocol describes laser capture microdissection for the isolation of cartilage and bone from fresh frozen sections of the mouse embryo. Cartilage and bone can be rapidly visualized by cresyl violet staining and collected precisely to yield high quality RNA for transcriptomic analysis.

Microdissecção de captura a laser da cartilagem embrionária do rato e osso para análise de expressão gênica

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and ...

Mosaic Zebrafish Transgénese para Análise Funcional Genomic de candidatos Genes de cooperação em Tumor Patogênese

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