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 los estudios de pez cebra y el mantenimiento de los animales se realizaron de acuerdo con el protocolo aprobado por el IACUC Clínica Mayo Institute # A41213. 1. Construcciones de DNA para la transgénesis <ol><li> Amplificar una 5,2-kb beta hidroxilasa de dopamina (d βh) región promotora 8 usando el clon BAC CH211-270H11 (desde el centro de recursos BACPAC (BPRC)) como plantilla de ADN. Utilice una los programas de ciclos siguientes para la PCR PCR sistema apropiado para larga y precisa amplificación por PCR de plantillas de ADN largos y: 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 (cebador directo 5&#39;-GGGGACAACTTTGTATAGAAAAGTTGGCGTACTCCCCCTTTTTAGG-3 &#39; y cebador inverso 5&#39;GGGGACTGCTTTTTTGTACAAACTTGTGTTGCTTTGTCGTCTTTTGA-3 &#39;). </li><li> Cree el d βh -pDONRP4-P1R entrada clone 8 utilizando el sistema de clonación recombinante comercial, como puerta de entrada para varios sitios. Mezclar 1 l de dβh purificada producto de PCR (172 ng / l), 1 l (150 ng / l) pDONRP4-P1R vector donante (junto con otros vectores de puerta de enlace son generosos donativos del doctor Chi-Bin Chien, Univ. De Utah) , 4 l de tampón TE (pH 8.0) con 2 l de BP mezcla de enzimas clonasa, se incuban durante 1 hora a 25 ° C y luego se transforman en un TOP10 Shot E. competente coli de acuerdo con el protocolo del fabricante. </li><li> Generar el d βh: transgénicos EGFP-MYCN construir 8 utilizando el sistema de clonación recombinante mencionado en el paso 1.2. Mezclar 1 l de cada clon de entrada (150 ng / l) que incluye un 5,2 kb promotor dβh (dβh -pDONR P4-P1R constructo), EGFP carece de un codón de parada (PME-EGFP construcción), y de ADNc MYCN humana (un generoso regalo del Dr. Hogarty en el Hospital de Niños de Pennsylvania, MYCN pDONRP2R-Construcción P3), 1 l (150 ng / l) del vector destino modificado que contiene sitios de reconocimiento de I-SCE (un generoso regalo del doctor C. Grabher, Karlsruhe Institute of Technology, Karlsruhe, Alemania), 4 l de tampón TE ( pH 8,0) con 2 l de mezcla de enzimas LR Clonase, incubar durante 1 hora a 25 ° C y luego transformar a E. químicamente competentes coli como One superior tiro 10 y seguir el protocolo del fabricante. </li><li> Haga MITF: transgénico MITF construir 8 digiriendo el vector PNP-MITF con enzimas de restricción NotI y SalI durante 2 horas a temperatura ambiente, y subclonando el liberado 2,65 kb (un generoso regalo del doctor D. Raible, Univ de Washington.) fragmento de ADN que contiene el promotor de MITF y la secuencia codificante del gen MITF pez cebra en los sitios NotI y MluI de un vector pBluescript modificado que contiene flanquean los sitios de reconocimiento de I-SceI (un generoso regalo del Dr. Hui Feng, Universidad de Boston), utilizando ADN ligasa de T4 de acuerdo con el protocolo del fabricante. NOTA: Para aumentar la eficiencia de la generación de líneas MYCN expresando estables, d βh: derivados de la cresta construcciones de ADN MITF se coinjected en una de las células de embriones etapa nácar Nácar designa un tipo de peces mutantes que carecen de un neural: EGFP-MYCN y la MITF. melanophore durante el desarrollo 36. Por lo tanto, la aparición de células de pigmento en los embriones inyectados sugiere la integración de MITF: DNA MITF construye en el genoma. Se ha demostrado que dos o tres constructos de ADN coinjected pueden cointegradas en el genoma de pescado 31. Por lo tanto, la pigmentación causada por la expresión de MITF puede servir como un marcador para la integración de la dβh: transgén EGFP-MYCN y para facilitar la identificación de MYCN línea transgénica estable. </li><li> Mezclar el d βh: EGFP-MYCN y MITF: DNA MITFconstrucciones en una relación 3: 1 en un volumen total de 15 l de una reacción con 1 l de enzima I-SceI y 0,75 l de tampón. Asegúrese de que la cantidad total de ADN en la reacción no exceda de 750 ng. </li><li> Llevar a cabo la digestión I-SCE a temperatura ambiente durante 4 horas o O / N. En el segundo día, el I-SceI- digirió ADN están listos para la microinyección o se pueden almacenar a -20 ° C para la inyección en el futuro. Almacenar la enzima I-SceI a -80 ° C en pequeñas alícuotas para mantener su eficiencia enzima. </li><li> Subclonar el ALKF1174L humana y el gen ALK de tipo salvaje del vector pcDNA3 (un generoso regalo del doctor George en el Dana-Farber Cancer Institute) 7 en EcoRI y NotI sitios de un vector pENTRY1A (un generoso regalo del doctor C. Grabher, Karlsruhe Institute of Technology, Karlsruhe, Alemania) utilizando ADN ligasa T4 de acuerdo con el protocolo del fabricante. </li><li> Generar el d βh: ALKF1174L &lt;/ Em&gt; o dβh: construcciones transgénicas ALKWT utilizando el sistema recombinante como se ha mencionado en el Protocolo 1.2. En pocas palabras, combinar tres clones de entrada, dβh -pDONRP4-P1R, ALKF1174L- pENTRY1A (o ALKWT -pENTRY1A) y P3E-poli, en el vector de destino modificado que contiene sitios de reconocimiento de I-SCE (un generoso regalo del doctor C. Grabher, Karlsruhe Instituto de Tecnología de Karlsruhe, Alemania), utilizando LR Clonase enzima Mix, según el protocolo del fabricante. </li><li> Mezclar la d βh: ALKF1174L (o dβh: ALKWT) y dβh: DNA mCherry construye en una proporción de 3: 1 y a alinear con la enzima I-SceI como se describe en el protocolo de 1.5 a 1.6. </li></ol> 2. La microinyección <ol><li> Utilice micropipetas de vidrio con 1,0 mm de diámetro para todas las inyecciones 37. Rompa la punta de las pipetas de vidrio con una hoja de afeitar antes de la inyección. Calibrar el volumen de inyección por inyeccióning H 2 O en una gota de aceite mineral. Medir el diámetro de las gotitas resultantes y ajustar el microinyector (presión o la duración de pulso de presión) para asegurar que el volumen de inyección es menor que 10% del volumen celular total de un embriones en fase celular. </li><li> Añadir un adicional de 0,5 l de enzima fresco I-SceI (5 unidad / l) en 5 l de solución de inyección que contiene construcciones de ADN justo antes de la inyección para aumentar la eficiencia de la transgénesis 38. Inyectar 50-80 pg de constructos de ADN linealizado en el citoplasma de embriones fase de una célula. Para asegurar el éxito de la inyección, mezclar la muestra con 0,25% de rojo fenol para la visualización. Si las células se vuelven de color rojo después de la inyección, se indica la microinyección es exitosa. Para garantizar una alta tasa de éxito para la generación de peces transgénicos, por lo general inyectamos hasta 500 embriones. </li><li> Para generar los peces transgénicos que expresan establemente MYCN, coinject linealizado d βh: EGFP-MYCN y MITF: constructos de ADN MITF (en una proporción de 3: 1) en embriones de pez cebra mutante la pigmentación de nácar en la fase de una célula. La expresión de Mitf sirve como reportero para la integración de las construcciones transgénicas en el genoma de los peces. </li><li> Para la sobreexpresión mosaico de ALK en el de tipo salvaje o MYCN embriones transgénicos, co-inyectar el linealizado d βh: ALK F1174L (o dβh: ALKWT) con dβh: DNA mCherry construye (en una proporción de 3: 1) en la célula una embriones resultantes de la cría de F1 heterocigota Tg (dβh: EGFP-MYCN) peces transgénicos con el tipo salvaje AB peces. Así, la mitad de la descendencia son transgénicos para MCYN y la mitad son de tipo salvaje. </li></ol> 3. Pantalla de pescado Estable o Mosaico transgénico <ol><li> Para aumentar la eficacia de la identificación de las líneas de MYCN expresando estables, anestesiar embriones inyectados nácar principalmente con tricaína (0,02%) at días 3-5 de postfertilización y la pantalla de la pigmentación. La transferencia de los embriones con la pigmentación a una nueva placa de Petri con agua fresca al huevo y elevarlas a la madurez sexual para una revisión adicional para los peces fundador, que lleva el d βh: transgén EGFP-MYCN en las células germinales. </li><li> Para identificar el fundador con la transmisión de la línea germinal de EGFP-MYCN, outcross pigmentada peces F0 adultos con el tipo salvaje AB pescado y pantalla para los embriones EGFP-positivas en 1-2 días después de la fertilización para su posterior genotipado. Coloque un solo embrión F1 EGFP-positivo en un tubo de PCR y eliminar todo el líquido. Añadir 50 l de tampón de extracción que contiene gDNA 12,5 l de tampón de lisis 4x, 35 l H 2 O y 2,5 l de proteinasa K (10 mg / ml) a un solo embrión. Se incuba la reacción durante 2-3 horas a 55 ° C, seguido por 10 min de incubación a 98ºC para inactivar la proteinasa K. Para hacer 50 ml de tampón de lisis 4x, añadir 500 l de Tris 1 M (pH 8,4), 2,5 ml de 1M KCl to 47 ml de H 2 O. NOTA: Después de F0 MYCN peces transgénicos estable se crían con la naturaleza tipo AB de pescado, todos los descendientes son pigmentados. Por lo tanto, la pigmentación no puede servir como marcador para la identificación de MYCN- peces positivo. Mientras que en los peces transgénicos MYCN, la proteína fusión EGFP MYCN se expresa en la PSNS, incluyendo las neuronas simpáticas del ganglio cervical superior y el ganglio segmentaria secuencial de la cadena simpática y los PSN no neuronas dopaminérgicas, tales como el bulbo raquídeo y ganglios craneales 8. Por lo tanto, la expresión de EGFP puede servir como marcador para la identificación de los embriones transgénicos estables MYCN. </li><li> Utilice 2 l de gDNA extraídos del embrión F1 pigmentados como molde, cebadores MYCN-test F1: 5&#39;-CTG CTT GAG GAG AAC CTG TG-3 &#39;; MYCN -R3: 5&#39;-AGG CAT CGT TTG AGG ATC AG-3 &#39;, y el siguiente programa de ingenioh la GC-rico sistema de PCR: 1 ciclo de 95 ° C durante 3 min, 25 ciclos de 95 ° C durante 30 segundos, 58 ° C durante 30 segundos y 72 ° C durante 3 min. Por lo general genotipo de 14-16 embriones pigmentadas de un solo apareamiento para confirmar la presencia de transgen integrado MYCN en los embriones pigmentadas, más de 6 fundador peces que sobreexpresan MYCN MITF y fueron identificados por este método. </li><li> Levanta el resto de los embriones F1 EGFP-positivas. Clip de Fin y el genotipo ellos en 2-3 meses de edad utilizando el protocolo anterior para confirmar aún más la integración de los transgenes MYCN en el pescado. Raza F1 MYCN peces transgénicos estable con el tipo salvaje AB pescado. Co-inyectar el d βh linealizado: ALK F1174L (o dβh: ALKWT) con dβh: DNA mCherry construye en los embriones de una célula como se describe en PROTOCOLO 2.4. </li><li> Ordenar la MYCN expresando embriones en el experimento descrito en el PROTOCOLO 2.4 utilizando un fluor estereoscópicamicroscopio escencia y seleccionar a los embriones inyectados principalmente en los días 1-3 de postfertilización para la expresión de EGFP-MYCN en el PSNS. Luego, durante 2-5 días después de la fecundación de, anestesiar a los embriones con tricaína (0,02%) y ordenar esos embriones MYCN- positivos o negativos de nuevo basado en la expresión de mCherry en los PSNS usando un microscopio de fluorescencia estereoscópica. La expresión de mCherry sirve como un marcador para la coexpresión de ALK en los tejidos de los animales inyectados primaria mosaico. NOTA: ~ 600 descendencia resultante de la cría de peces transgénicos MYCN estable con el tipo salvaje peces fueron inyectados por grupo con los constructos de ADN linealizado, incluyendo d βh: ALK F1174L y dβh: mCherry, dβh: ALKWT y dβh: mCherry, o dβh : mCherry solo, respectivamente. Mosaico de expresión mCherry se puede observar en el 70-90% de los peces inyectados principalmente. La mitad de unotercio de los peces inyectados sobrevivió a través de la etapa de las larvas para el reloj de tumor. </li><li> Levante todo el mCherry + MYCN + y + mCherry MYCN - embriones de acuerdo con los protocolos estándar de la libreta de pez cebra 39 y vigilar la aparición de tumores a partir de las 5 semanas después de la fecundación. </li></ol> 4. Tumor Watch en mosaico transgénico Pescado <ol><li> Monitorear peces positivo mCherry- inyectado principalmente cada 2 semanas a partir de las 5 semanas después de la fecundación para la evidencia de la aparición de tumores. </li><li> Anestesiar pescado con tricaína (0,02%) y la pantalla de la presencia de mCherry - y EGFP-expresando tumores en los PSNS bajo la Nikon SMZ-1500 microscopio de fluorescencia estereoscópica equipado con una mira la cámara digital DS-U1. </li><li> Para confirmar si los tumores están expresando el transgén ALK, aislar la mCherry- y masas EGFP-positivas para el genotipado ALK PCR. </li><li> Usando PCR, amplificar el ADN genómicoextraído de tumores desarrollados con los siguientes cebadores: ALK P7: 5&#39;-AGG CCA GGT GTC CGG AAT GC-3 &#39;y ALK P18: 5&#39;-TGT CTT CAG GCT GAT GTT GC-3&#39; y la siguiente reacción de PCR: 1 ciclo de 94 ° C durante 5 min, 30 ciclos de (94 ° C durante 30 segundos, 55 ° C durante 30 segundos y 72 ° C durante 60 segundos). Entonces, la secuencia del producto de PCR con el cebador P7 ALK Para confirmar aún más la existencia de mutante o de tipo salvaje ALK en los tumores mCherry-positivos. </li></ol>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracing+transgene+expression+in+living+zebrafish+embryos.">Tracing transgene expression in living zebrafish embryos.</a> Dev Biol. 233 (2), 329-346 (2001).</li><li>Langenau, D. 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. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+zebrafish+embryos+to+analyze+gene+function.">Microinjection of zebrafish embryos to analyze gene function.</a> J Vis Exp. (25), (2009).</li><li>Thermes, V., 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=I-SceI+meganuclease+mediates+highly+efficient+transgenesis+in+fish.">I-SceI meganuclease mediates highly efficient transgenesis in fish.</a> Mech Dev. 118 (1-2), 91-98 (2002).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. 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. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intercellular+bridges+in+vertebrate+gastrulation.">Intercellular bridges in vertebrate gastrulation.</a> PLoS One. 6 (5), e20230 (2011).</li><li>Ivics, Z., Izsvak, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+expanding+universe+of+transposon+technologies+for+gene+and+cell+engineering.">The expanding universe of transposon technologies for gene and cell engineering.</a> Mob DNA. 1 (1), 25 (2010).</li><li>Tang, Q., 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=Optimized+cell+transplantation+using+adult+rag2+mutant+zebrafish.">Optimized cell transplantation using adult rag2 mutant zebrafish.</a> Nat Methods. 11 (8), 821-824 (2014).</li><li>Watson, I. 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. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-Cas+systems+for+editing,+regulating+and+targeting+genomes.">CRISPR-Cas systems for editing, regulating and targeting genomes.</a> Nat Biotechnol. 32 (4), 347-355 (2014).</li></ol>

The authors declare that they have no competing financial interests.

En este estudio representativo, se utilizó coinyección transitoria y coexpresión de ALK activado con el gen reportero mCherry en MYCN expresando peces transgénicos para mostrar que estos genes cooperan para acelerar notablemente la aparición de neuroblastoma, coherente con nuestra conclusión anterior en el compuesto estable que coexpresan los peces transgénicos tanto ALK y MYCN 8 activado. Este enfoque transgénico mosaico posee varias ventajas sobre el mét...

Los cánceres son enfermedades progresivas marcados por la acumulación de mutaciones patológicas, deleciones y ganancias cromosómicas con el tiempo. Estas anomalías genéticas pueden afectar a múltiples procesos celulares que van desde el ciclo celular, la muerte celular, el metabolismo energético y el montaje del citoesqueleto a respuestas de estrés tales como hipoxia. Por lo tanto, la tumorigénesis refleja las acciones colectivas de múltiples aberraciones genéticas a través de un espectro de procesos biológicos. Los recientes esfuerzos de integración genómica de investigación, incluyendo la secuenciación de todo el genoma, la secuenciación del exoma, secuenciación dirigida, secuenciación profunda y estudios de asociación del genoma, han identificado un número cada vez mayor de alteraciones genéticas novedosas en prácticamente todos los tipos de tumores 1-4. En muchos casos, las lesiones genéticas ocurren juntos de una manera no aleatoria 5-8, lo que sugiere su cooperación en la patogénesis de la enfermedad. La disección de las funciones oncogénicas de la gran variedad de genes aberrantemente expresadas resultantes fesde estas lesiones genómicas es necesario diseñar nuevas estrategias terapéuticas y de entender las respuestas de las células tumorales a estos agentes, pero esto ha demostrado ser una tarea de enormes proporciones, que requiere muy robustos sistemas de modelos animales para la conducción de alto rendimiento de análisis genómico funcional en vivo. Aunque los mamíferos, especialmente roedores, son modelos favorecidos en la biología del cáncer, el pez cebra ha comenzado a atraer una atención considerable. El pez cebra teleósteos (Darío rerio) se ha utilizado como un organismo modelo para el estudio de desarrollo desde la década de 1960 y fue aplicado primero al estudio de la patogénesis del tumor en 1982 9-11. Facilidad de mantenimiento, pequeño tamaño del cuerpo, y la alta fecundidad hacen que el pez cebra un modelo sólido para pantallas adelante genéticos a gran escala para identificar las mutaciones que confieren fenotipos anormales y patológicas 10. La transparencia óptica de embriones de pez cebra es otra característica clave de apoyo a un mayor uso de este modelo de cáncer, comopermite de imágenes in vivo para ser llevado a cabo para localizar el desarrollo de tumores en tiempo real 9, una aplicación que es relativamente difícil en roedores 12. Recientes genómica comparativa análisis del genoma de referencia pez cebra (Zv9) reveló 26.206 genes codificadores de proteínas, con un 71% que tiene ortólogos humanos, de los cuales 82% están relacionados con los genes asociados a la enfermedad de la herencia mendeliana en línea en Man (OMIM) base de datos 13, 14. En consecuencia, el pez cebra se ha utilizado para modelar diversos tipos de cánceres humanos, incluyendo neuroblastoma 8, de células T de la leucemia linfoblástica aguda (LLA-T) 15,16, 17,18 melanoma, sarcoma de Ewing 19, rabdomiosarcoma 20,21, carcinoma de páncreas 22, 23 y carcinoma hepatocelular mieloides malignas 24,25, y ha sido seleccionado como un modelo de cáncer de xenotrasplantes estudia 11,26. A transgénicas establesenfoque en el pez cebra se utiliza comúnmente para estudiar el efecto de ganancia de la función de los genes en el desarrollo normal o patogénesis de la enfermedad 27,28. Para desarrollar tal un modelo (Figura 1A), se inyecta una construcción de ADN que contiene el gen de interés dirigido por un promotor específico de tejido en una de las células de tipo salvaje embriones. Tres o cuatro meses después de la inyección, cuando los embriones inyectados alcanzan la madurez sexual, se outcrossed con el tipo salvaje de pescado para detectar los que muestran la integración de la construcción de ADN en su línea germinal, lo que les otorga licencias como peces fundador. Muchos factores, tales como el número de copias y el sitio de integración del transgén, afectan a la expresión del transgén en las líneas transgénicas estables. Por lo tanto, para desarrollar un modelo de tumor transgénico, múltiples líneas transgénicas estables que sobreexpresan un único oncogén tienen que ser generada primero y proyectado para la línea que expresa el transgén en un nivel que podría conducir a la inducción de tumores. Sin embargo, si la sobreexpresión de una o candidatoncogene es tóxico para las células germinales, es difícil generar una línea transgénica estable mediante la sobreexpresión directamente el transgén 29. Por lo tanto, este enfoque puede llevar mucho tiempo, con un alto riesgo de fracaso para generar un modelo de cáncer adecuado. Aquí, se ilustra una estrategia alternativa basada en la transgénesis transitoria mosaico (Figura 1B) que proporciona ventajas únicas sobre la transgénesis estable tradicional para estudio genómico funcional in vivo. En este enfoque, una o más construcciones transgénicas se inyectan en la fase de una célula de transgénicos o de tipo salvaje embriones. Las construcciones de ADN inyectados contienen transgenes son entonces mosaically y aleatoriamente integradas en el pescado primaria inyectada, lo que resulta en una mezcla de genotipos en múltiples poblaciones celulares en peces individuales 30. Además, la coinyección de ADN múltiples constructos en embriones de una sola célula conduce a co-integración en la misma célula en sitios al azar, que permite a uno TRAce las células con expresión de los transgenes y explorar las interacciones de los diferentes genes durante la patogénesis de la enfermedad en los animales de mosaico 31. Como prueba de principio, transitoriamente overexpressed ALK mutacionalmente activado (F1174L) con el gen reportero mCherry en el sistema nervioso simpático periférico (SNPS) bajo control de la hidroxilasa beta dopamina (d βh) promotor de tipo salvaje pescado y peces transgénicos que sobreexpresan MYCN. ALK, que codifica un receptor tirosina quinasa, es el gen más frecuentemente mutado en neuroblastoma de alto riesgo 5-7,32,33. ALK (F1174L), como una de las mutaciones somáticas activación más frecuentes y potentes, es sobre-representado en MYCN- amplificado pacientes con neuroblastoma de alto riesgo y la sinergia con sobreexpresión MYCN para acelerar la tumorigénesis neuroblastoma en ratones transgénicos estables y modelos de pez cebra transgénico 8,34,35. Por mosaicola sobreexpresión transitoria de ALK (F1174L) con mCherry en el pez transgénico MYCN, que recapitula la aceleración de la aparición del tumor observado en los peces transgénicos estables que sobreexpresan tanto ALK (F1174L) y MYCN, lo que sugiere que la estrategia de la transgénesis de mosaico se puede utilizar para rápida y eficiente evaluar las contribuciones relativas de varios oncogenes en la iniciación del tumor in vivo.

<table border="0" cellpadding="0" cellspacing="0" width="663">
	<tbody>
		<tr height="21">
			<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>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Expand Long Template PCR System&#160;</td>
			<td style="width:171px;">Roche Applied Science, IN</td>
			<td style="width:119px;">11681834001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pCR-TOPO vector&#160;</td>
			<td style="width:171px;">Invitrogen, CA</td>
			<td style="width:119px;">451641</td>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gateway LR Clonase II enzyme 
			Mix</td>
			<td style="width:171px;">Invitrogen, CA</td>
			<td>11791-100</td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="24">
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
	</tbody>
</table>

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 si la sobreexpresión de F1174L ALK mutationally activado o de tipo salvaje ALK podría colaborar con MYCN en la inducción de neuroblastoma, overexpressed ya sea ALK activada humana o de tipo salvaje ALK humano bajo el control del promotor d βh en el PSNS de peces transgénicos que sobreexpresan MYCN. Cualquiera de las siguientes construcciones, dβh - ALKF1174L o dβh - ALKWT, fueron coinjected con d...

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

Mosaic Zebrafish Transgenesis for Functional Genomic Analysis of Candidate Cooperative Genes in Tumor Pathogenesis | Developmental Biology | Video

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.

Mosaico de pez cebra transgénesis de Análisis Genómica Funcional de candidatos genes cooperativos en la patogénesis del tumor

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

mosaic-zebrafish-transgenesis-for-functional-genomic-analysis

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11.0K vistas.  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.

Video: 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.

A gran escala de pez cebra embrionario del corazón Disección de Análisis transcripcional

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 ...

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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.

Análisis de pez cebra larvas esquelético Integridad del músculo con el colorante azul de Evans

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 de microinyección a Target a los países en desarrollo<em&gt; Xenopus</em&gt; riñón

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.

La microinyección de transgénesis y Genoma edición en tres espinas espinosos

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.

La manipulación de genes maternos funcional de Productos que utilizan<em&gt; In Vitro</em&gt; La maduración de ovocitos en el pez cebra

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.

Resolución súper correlativo y la microscopia electrónica para resolver la localización de la proteína en la Retina del pez cebra

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 un Mosquito recombinante Densovirus como un Vector de la entrega de genes para el análisis funcional de Genes en larvas de mosquitos

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

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.

Genotipado y cuantificación de la tinción de hibridación in situ en el pez cebra

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.

Microdisección de captura láser de cartílago embrionario de ratón y hueso para análisis de expresión génica

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

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

Generación de organoides mamarios de mosaico por trippsinización diferencial

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...