Name: mosaic zebrafish transgenesis for functional genomic analysis of candidate cooperative genes in tumor pathogenesis
Uploaded: 2015-03-31T15:00:00
Description: Watch this Scientific Journal Video about Mosaic Zebrafish Transgenesis for Functional Genomic Analysis of Candidate Cooperative Genes in Tumor Pathogenesis at JoVE.com

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: Tutti gli studi di zebrafish e manutenzione degli animali sono stati fatti in accordo con il protocollo Mayo Clinic Institute IACUC approvato # A41213. 1. DNA costrutti per Transgenesi <ol><li> Amplifica una regione del promotore 5.2-kb beta dopamina idrossilasi (d βh) 8 utilizzando il clone CH211-270H11 BAC (dal BacPac Centro risorse (BPRC)) come modello di DNA. Utilizzare un sistema adeguato per PCR lungo e accurato l&#39;amplificazione PCR di modelli di DNA lu...

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In questo studio rappresentativo, abbiamo utilizzato coiniezione transitoria e coexpression di ALK attivato con il gene reporter mCherry in MYCN esprimente il pesce transgenico per dimostrare che questi geni cooperano per accelerare notevolmente l&#39;insorgenza di neuroblastoma, in linea con la nostra scoperta precedente composto stabile transgenici coexpressing pesce sia attivato ALK e MYCN 8. Questo approccio transgenico mosaico possiede diversi vantaggi rispett...

Tumori sono malattie progressive contrassegnati da un accumulo di mutazioni patologiche, delezioni cromosomiche e guadagni nel corso del tempo. Queste anomalie genetiche possono influenzare molteplici processi cellulari che vanno dal ciclo cellulare, morte cellulare, metabolismo energetico e l&#39;assemblaggio del citoscheletro sottolineare risposte come ipossia. Quindi, tumorigenesi riflette le azioni collettive di più aberrazioni genetiche attraverso una gamma di processi biologici. I recenti sforzi integrative genomiche di ricerca, tra cui intero sequenziamento del genoma, exome sequencing, sequenziamento mirato, sequenziamento profondo e gli studi di associazione sull&#39;intero genoma, hanno identificato un numero crescente di nuove alterazioni genetiche in essenzialmente tutti i tipi di tumori 1-4. In molti casi, le lesioni genetiche presentano insieme in maniera non casuale 5-8, suggerendo loro cooperazione nella patogenesi della malattia. Analisi dei ruoli oncogeniche della vasta gamma di geni espressi aberrante derivanti fROM queste lesioni genomiche È necessario elaborare nuove strategie terapeutiche e di comprendere le risposte delle cellule tumorali a questi agenti, ma questo ha dimostrato di essere un compito arduo, che richiede molto robusti sistemi modello animale per lo svolgimento di high-throughput analisi genomica funzionale vivo. Anche se i mammiferi, in particolare roditori, sono modelli favorite in biologia del cancro, il pesce zebra ha iniziato ad attirare una notevole attenzione. Il pesce zebra teleosteo (Dario rerio) è stata utilizzata come organismo modello per lo studio dello sviluppo dal 1960 ed è stata applicata la prima allo studio della patogenesi del tumore nel 1982 9-11. Facilità di manutenzione, di piccola dimensione del corpo, e l&#39;alta fecondità rendono il zebrafish un modello robusto per schermi genetici a termine su larga scala per identificare le mutazioni che conferiscono fenotipi anormali e patologiche 10. La trasparenza ottica di embrioni di zebrafish è un altro elemento chiave a sostegno più ampio uso di questo modello di cancro, comepermette imaging in vivo ad essere condotti per individuare lo sviluppo del tumore in tempo reale 9, un&#39;applicazione che è relativamente difficile nei roditori 12. Genomica comparativa recente analisi del genoma di riferimento zebrafish (Zv9) ha rivelato 26.206 geni codificanti proteine, il 71% ha ortologhi umani, di cui l&#39;82% sono correlati con i geni associati alla malattia della linea mendeliana Inheritance in Man (OMIM) del database 13, 14. Di conseguenza, il pesce zebra è stato utilizzato per modellare diversi tipi di tumori umani, tra cui 8 neuroblastoma, cellule T leucemia linfoblastica acuta (T-ALL) 15,16, il melanoma 17,18, sarcoma di Ewing 19, rabdomiosarcoma 20,21, carcinoma pancreatico 22, 23 e carcinoma epatocellulare mieloide neoplasie 24,25, ed è stato selezionato come un modello di cancro per lo xenotrapianto studi 11,26. A transgenico stabileapproccio in zebrafish è comunemente usato per studiare l&#39;effetto di guadagno-di-funzione dei geni nello sviluppo normale o malattia patogenesi 27,28. Per sviluppare un tale modello (Figura 1A), si inietta un costrutto di DNA contenente il gene di interesse azionato da un promotore tessuto-specifico in una cellule wild-type embrioni. Tre o quattro mesi dopo l&#39;iniezione, quando gli embrioni iniettati raggiungono la maturità sessuale, sono outcrossed con wild-type di pesce per lo screening per i più mostrano integrazione del DNA costruire nella loro linea germinale, che concede in licenza il pesce fondatore. Molti fattori, come il numero di copie e sito di integrazione del transgene, influenzano l&#39;espressione del transgene in linee transgeniche stabili. Così, per sviluppare un modello di tumore transgenico, più linee transgeniche stabili sovraesprimono un singolo oncogene devono essere generati prima e screening per la linea esprimere il transgene ad un livello che potrebbe condurre a tumori indotti. Tuttavia, se la sovraespressione di un candidato oncogene è tossico per le cellule germinali, è difficile generare una linea transgenica stabile sovraespressione direttamente il transgene 29. Pertanto, questo approccio può richiedere molto tempo, con un elevato rischio di fallimento per generare un modello di cancro adatto. Qui illustriamo una strategia alternativa basata su mosaico transgenesi transitoria (Figura 1B), che fornisce vantaggi unici rispetto transgenesi tradizionale stabile per lo studio di genomica funzionale in vivo. In questo approccio, una o più costrutti transgenici sono iniettati nello stadio unicellulare di transgenici o wild-type embrioni. I costrutti di DNA contenenti iniettate transgeni sono poi mosaically e casualmente integrati nel pesce iniettato primario, con conseguente genotipi misti in popolazioni di cellule più in singoli pesci 30. Inoltre, co-iniezione di DNA multiple costruisce in embrioni di una cellule porta alla co-integrazione nella stessa cella in siti casuali, permettendo di TRACe le cellule con l&#39;espressione dei transgeni ed esplorare le interazioni dei diversi geni durante patogenesi della malattia negli animali mosaico 31. Come prova di principio, siamo transitoriamente sovraespresso ALK mutationally attivato (F1174L) con mCherry gene reporter nel sistema nervoso simpatico periferico (PSNS) sotto il controllo del idrossilasi beta dopamina (d βh) promotore wild-type di pesce e pesce transgenico sovraespressione MYCN. ALK, che codifica un recettore tirosina chinasi, è il gene più frequentemente mutato in neuroblastoma ad alto rischio 5-7,32,33. ALK (F1174L), come una delle mutazioni somatiche attivazione più frequenti e potenti, è sovra-rappresentata in MYCN- amplificato pazienti neuroblastoma ad alto rischio e con synergizes MYCN sovraespressione di accelerare neuroblastoma tumorigenesi sia nei topi transgenici stabili e transgenici modelli zebrafish 8,34,35. By mosaicosovraespressione transiente di ALK (F1174L) con mCherry nel pesce transgenico MYCN, abbiamo ricapitolato l&#39;accelerazione di insorgenza del tumore osservata nei pesci transgenici che sovraesprimono stabile sia ALK (F1174L) e MYCN, suggerendo che la strategia transgenesi mosaico può essere utilizzato per rapidamente ed efficacemente valutare i contributi relativi di più oncogeni nel tumore iniziazione in vivo.

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			<td height="21" style="height:21px;">Gateway&#174; BP Clonase&#174; II enzyme mix</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.

Per verificare se la sovraespressione di mutationally attivato F1174L ALK o wild-type ALK potrebbe collaborare con MYCN in induzione neuroblastoma, abbiamo sovraespresso o ALK umano attivo o wild-type ALK umana sotto il controllo del promotore d βh nel PSNS di pesci transgenici iperespressione MYCN. Uno dei seguenti costrutti, dβh - ALKF1174L o dβh - ALKWT, sono stati coinjected con dβh - mCherry in u...

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

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 Transgenesi per analisi genomica funzionale di geni candidati cooperative in Tumor Patogenesi

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

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