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 lunghe e seguenti programmi ciclo di PCR: 94 ° C per 2 min, 10 cicli di (94 ° C, 15 sec, 50 ° C, 30 sec, 68 ° C, 8 min), seguita da 30 cicli di (94 ° C, 15 sec, 53 ° C, 30 sec, 68 ° C, 8 min), 68 ° C, 4 min (forward primer 5&#39;-GGGGACAACTTTGTATAGAAAAGTTGGCGTACTCCCCCTTTTTAGG-3 &#39; e primer reverse 5&#39;GGGGACTGCTTTTTTGTACAAACTTGTGTTGCTTTGTCGTCTTTTGA-3 &#39;). </li><li> Creare il d βh -pDONRP4-P1R entry clone 8 utilizzando commercial Sistema clonazione ricombinante come gateway di multisito. Mescolare 1 ml di dβh purificato prodotto della PCR (172 ng / ml), 1 ml (150 ng / ml) pDONRP4-P1R donatore vettore (insieme ad altri vettori di gateway sono doni generosi del dottor Chi-Bin Chien, Univ. Di Utah) , 4 microlitri di tampone TE (pH 8.0) con 2 ml di BP mix clonase enzima, incubare per 1 ora a 25 ° C e poi a trasformare One Shot TOP10 competente E. coli secondo il protocollo del produttore. </li><li> Generare il d βh: transgenica EGFP-MYCN costruire 8 utilizzando il sistema di clonazione ricombinante di cui al punto 1.2. Mescolare 1 ml di ogni clone di entrata (150 ng / mL) tra cui un-kb 5.2 promoter dβh (dβh -pDONR P4-P1R costrutto), EGFP manca un codone di stop (PME-EGFP costrutto), e umano MYCN cDNA (un dono generoso Dr. Hogarty all&#39;ospedale dei Bambini di Pennsylvania, MYCN-pDONRP2R-P3 costrutto), 1 ml (150 ng / ml) del vettore di destinazione modificato contenente siti di riconoscimento I-SCEI (un dono generoso del Dr. C. Grabher, Karlsruhe Institute of Technology, Karlsruhe, Germania), 4 pl di tampone TE ( pH 8.0) con 2 ml di LR Clonase miscela enzimatica, incubare per 1 ora a 25 ° C e poi a trasformare chimicamente competente E. coli quali One top sparato 10 e seguire il protocollo del produttore. </li><li> Fai MITF: transgenica MITF costruire 8 digerendo il vettore PNP-MITF con NotI e Sali enzimi di restrizione per 2 ore a RT, e la subcloning rilasciato 2.65-kb (un dono generoso da Dr. D. Raible, Univ of Washington.) frammento di DNA che contiene il promotore MITF e la sequenza codificante del gene MITF zebrafish nei siti NotI e MluI di un vettore pBluescript modificato contenente accompagnamento siti di riconoscimento I-SCEI (un dono generoso da Dr. Hui Feng, Boston University), utilizzando DNA ligasi T4 secondo il protocollo del produttore. NOTA: per aumentare l&#39;efficienza di generare linee MYCN esprimente stabili, d βh: EGFP-MYCN e MITF:. Costrutti DNA MITF sono coinjected in una delle cellule di embrioni fase madreperla Nacre designa un tipo di pesce mutante manca un neurale derivati ​​dalla cresta melanophore durante lo sviluppo 36. Così, la comparsa di cellule del pigmento negli embrioni iniettati suggerisce l&#39;integrazione di MITF: DNA MITF costruisce nel genoma. È stato dimostrato che due o tre costrutti di DNA possono essere coinjected cointegrate nel genoma pesci 31. Così, pigmentazione causata da espressione MITF può servire come marker per l&#39;integrazione della dβh: transgene EGFP-MYCN e per una più facile individuazione di MYCN linea transgenica stabile. </li><li> Mescolare il d βh: EGFP-MYCN e MITF: DNA MITFcostrutti in un rapporto 3: 1 in un volume totale di una reazione di 15 microlitri di 1 ml di I-SCEI enzima e 0,75 ml di tampone. Assicurarsi che la quantità totale di DNA nella reazione non superi 750 ng. </li><li> Effettuare la digestione I-SCEI a temperatura ambiente per 4 ore o O / N. Il secondo giorno, la I-SceI- digerito DNA sono pronti per microiniezione o possono essere conservati a -20 ° C per l&#39;iniezione in futuro. Conservare l&#39;enzima I-SCEI a -80 ° C in piccole aliquote per mantenere la sua efficacia enzima. </li><li> Subclone la ALKF1174L umano e wild-type del gene ALK dal pcDNA3 vettore (un dono generoso del Dr. George al Dana-Farber Cancer Institute) 7 in EcoRI e NotI siti di un vettore pENTRY1A (un dono generoso del Dr. C. Grabher, Karlsruhe Institute of Technology, Karlsruhe, Germania) utilizzando DNA ligasi T4 secondo il protocollo del produttore. </li><li> Generare il d βh: ALKF1174L &lt;/ Em&gt; o dβh: costrutti transgenici ALKWT utilizzando il sistema ricombinante di cui al protocollo 1.2. In breve, unire tre cloni di ingresso, dβh -pDONRP4-P1R, ALKF1174L- pENTRY1A (o ALKWT -pENTRY1A) e P3E-polyA, nel vettore di destinazione modificato contenente siti di riconoscimento I-SCEI (un dono generoso del Dr. C. Grabher, Karlsruhe Institute of Technology, Karlsruhe, Germania), con LR Clonase enzima Mix, secondo il protocollo del produttore. </li><li> Mescolare il d βh: ALKF1174L (o dβh: ALKWT) e dβh: DNA mCherry costruisce in un rapporto di 3: 1 e linearizzare con I-SCEI enzima, come descritto nel protocollo 1,5-1,6. </li></ol> 2. Microiniezione <ol><li> Utilizzare micropipette di vetro con diametro di 1,0 mm per tutte le iniezioni di 37. Spezzare la punta delle micropipette di vetro con una lametta prima dell&#39;iniezione. Calibrare il volume di iniezione per iniezioneing H 2 O in una goccia di olio minerale. Misurare il diametro delle goccioline risultanti e regolare la microinjector (pressione o durata di impulso di pressione) per assicurare che il volume di iniezione è inferiore al 10% del volume cellulare totale di uno embrioni cellule stadio. </li><li> Aggiungere un ulteriore 0,5 l di fresco I-SCEI enzima (5 unità / ml) in 5 ml di soluzione iniettabile contenente DNA costruisce destra prima dell&#39;iniezione per aumentare l&#39;efficienza della transgenesi 38. Iniettare 50-80 pg di costrutti di DNA linearizzate nel citoplasma di una cellula-embrioni stadio. Per assicurare il successo di iniezione, miscelare il campione con 0,25% di rosso fenolo per la visualizzazione. Se le cellule diventano rossi dopo l&#39;iniezione, indica la microiniezione è riuscita. Per garantire un alto tasso di successo per la generazione di pesci transgenici, di solito iniettare fino a 500 embrioni. </li><li> Per generare pesci transgenici che esprimono stabilmente MYCN, coinject il linearizzato d βh: EGFP-MYCN e MITF: costrutti DNA MITF (in un rapporto di 3: 1) in embrioni di pigmentazione mutante madreperla zebrafish allo stadio unicellulare. L&#39;espressione di Mitf serve come reporter per l&#39;integrazione di costrutti transgenici nel genoma pesci. </li><li> Per sovraespressione mosaico di ALK nel wild-type o MYCN embrioni transgenici, co-iniettare il linearizzato d βh: ALK F1174L (o dβh: ALKWT) con dβh: DNA mCherry costruisce (in un rapporto di 3: 1) nella cellula uno embrioni risultanti da allevamento di F1 eterozigoti Tg (dβh: EGFP-MYCN) pesci transgenici con wild-type AB pesce. Così, la metà dei figli sono transgenici per MCYN e mezzo sono wild-type. </li></ol> 3. Schermo per Pesce stabile o Mosaic transgenici <ol><li> Per aumentare l&#39;efficienza di identificazione delle linee MYCN esprimente stabili, anestetizzare gli embrioni madreperla principalmente iniettati con tricaine (0,02%) at giorni 3-5 di postfertilization e schermo per la pigmentazione. Trasferire gli embrioni con pigmentazione ad una nuova capsula di Petri con acqua fresca all&#39;uovo ed aumentare loro di maturità sessuale per ulteriori controlli per il pesce fondatore, che portano il d βh: transgene EGFP-MYCN nelle cellule germinali. </li><li> Per identificare il fondatore con la trasmissione germinale di EGFP-MYCN, outcross pigmentato pesce F0 adulto con wild-type AB pesci e dello schermo per embrioni EGFP-positivi a 1-2 giorni dopo la fecondazione per ulteriori genotipizzazione. Posizionare un singolo embrione F1 EGFP-positivi in ​​un tubo di PCR e rimuovere tutto il liquido. Aggiungere 50 ml di tampone di estrazione gDNA che contiene 12,5 ml di tampone di lisi 4x, 35 ml di H 2 O e 2,5 ml proteinasi K (10 mg / ml) per singolo embrione. Incubare la reazione per 2-3 ore a 55 ° C, seguita da 10 minuti di incubazione a 98 º C per inattivare proteinasi K. Per 50 ml di tampone di lisi 4x, aggiungere 500 ml di 1M Tris (pH 8,4), 2,5 ml di 1M KCl to 47 ml di H 2 O. NOTA: Dopo F0 MYCN stabile pesci transgenici sono allevati con wild-type AB pesce, tutti i figli sono pigmentate. Così, la pigmentazione non può servire come marcatore per l&#39;identificazione di MYCN- pesci positive. Mentre nel pesce transgenico MYCN, la proteina di fusione EGFP-MYCN è espresso nella PSNS, compresi i neuroni simpatici del ganglio cervicale superiore e sequenziale segmentale gangli della catena simpatica ei PSNs non neuroni dopaminergici, come il midollo allungato e gangli cranica 8. Così, l&#39;espressione di EGFP può servire come marker per l&#39;identificazione degli embrioni transgenici stabili MYCN. </li><li> Utilizzare 2 ml di gDNA estratte dall&#39;embrione F1 pigmentata come modello, primer MYCN-test F1: 5&#39;-CTG CTT GAG AAC GAG CTG TG-3 &#39;; MYCN R3: 5&#39;-AGG CAT CGT TTG AGG ATC AG-3 &#39;, e la seguente ingegno programmah GC-RICH PCR: 1 ciclo di 95 ° C per 3 minuti, 25 cicli di 95 ° C per 30 sec, 58 ° C per 30 sec e 72 ° C per 3 min. Noi di solito genotipo 14-16 embrioni pigmentati da un unico accoppiamento per confermare la presenza di transgene MYCN integrati negli embrioni pigmentati, più di 6 fondatore pesce iperespressione MITF e MYCN sono stati individuati con questo metodo. </li><li> Sollevare il resto degli embrioni F1 EGFP-positivi. Clip Fin e genotipo a 2-3 mesi di età utilizzando il protocollo di cui sopra per confermare ulteriormente l&#39;integrazione di MYCN transgene nel pesce. Razza F1 MYCN pesci transgenici stabile con wild-type AB pesce. Co-iniettare il linearizzato d βh: ALK F1174L (o dβh: ALKWT) con dβh: DNA mCherry costruisce in embrioni uno cellule, come descritto nel protocollo 2.4. </li><li> Ordina la MYCN esprimente embrioni nell&#39;esperimento descritto nel protocollo 2.4 utilizzando un fluoro stereoscopicaMicroscopio escence e schermare gli embrioni principalmente iniettati a giorni 1-3 del postfertilization per l&#39;espressione di EGFP-MYCN nella PSNS. Poi, nei giorni 2-5 di postfertilization, anestetizzare gli embrioni con tricaine (0,02%) e ordinare quegli embrioni MYCN- positivi o negativi di nuovo in base all&#39;espressione dei mCherry nelle PSNS utilizzando un microscopio a fluorescenza stereoscopico. L&#39;espressione di mCherry serve come un marcatore per la coespressione di ALK nei tessuti degli animali primaria mosaico iniettati. NOTA: ~ 600 prole derivante dall&#39;allevamento di MYCN stabile pesci transgenici con wild-type di pesce sono stati iniettati per gruppo con i costrutti di DNA linearizzate, tra cui d βh: ALK F1174L e dβh: mCherry, dβh: ALKWT e dβh: mCherry, o dβh : mCherry solo, rispettivamente. Espressione Mosaico di mCherry può essere osservato nel 70-90% dei pesci principalmente iniettato. La metà di One-terzo del pesce iniettato sopravvissuto attraverso la fase di larve per la vigilanza del tumore. </li><li> Sollevare tutto il mCherry + MYCN + e mCherry + MYCN - embrioni secondo i protocolli standard del libro zebrafish 39 e monitorare tumore insorgenza inizio alle cinque settimane postfertilization. </li></ol> 4. Tumore Orologio in Mosaic Transgenic Pesce <ol><li> Monitorare mCherry- positivo pesci principalmente iniettata ogni 2 settimane a partire da 5 settimane postfertilization per l&#39;evidenza di tumore insorgenza. </li><li> Anestetizzare pesce con tricaine (0,02%) e lo schermo per la presenza di mCherry - e EGFP esprimente tumori nei PSNS sotto la Nikon SMZ-1500 microscopio a fluorescenza stereoscopico dotato di una vista fotocamera digitale DS-U1. </li><li> Per verificare se i tumori esprimono il transgene ALK, isolare il mCherry- e masse EGFP-positivi per ALK genotipizzazione PCR. </li><li> Usando PCR, amplificare DNA genomicoestratto da tumori sviluppati con i seguenti primer: ALK P7: 5&#39;-AGG CCA GGT GTC CGG AAT GC-3 &#39;e ALK P18: 5&#39;-TGT CTT CAG GCT GAT GTT GC-3&#39; e la seguente reazione PCR: 1 ciclo di 94 ° C per 5 minuti, 30 cicli di (94 ° C per 30 sec, 55 ° C per 30 sec e 72 ° C per 60 sec). Quindi, la sequenza del prodotto PCR con ALK P7 fondo per confermare ulteriormente l&#39;esistenza di mutante o wild-type ALK nei tumori mCherry-positivi. </li></ol>

<ol>
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J., Dellaire, G., Berman, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hooking+the+big+one:+the+potential+of+zebrafish+xenotransplantation+to+reform+cancer+drug+screening+in+the+genomic+era.">Hooking the big one: the potential of zebrafish xenotransplantation to reform cancer drug screening in the genomic era.</a> Dis Model Mech. 7 (7), 745-754 (2014).</li><li>Patton, E. E., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+art+and+design+of+genetic+screens:+zebrafish.">The art and design of genetic screens: zebrafish.</a> Nat Rev Genet. 2 (12), 956-966 (2001).</li><li>Lieschke, G. J., Currie, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+human+disease:+zebrafish+swim+into+view.">Animal models of human disease: zebrafish swim into view.</a> Nat Rev Genet. 8 (5), 353-367 (2007).</li><li>Igoucheva, O., Alexeev, V., Yoon, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+cellular+responses+to+exogenous+DNA+in+mammalian+cells+and+its+effect+on+oligonucleotide-directed+gene+modification.">Differential cellular responses to exogenous DNA in mammalian cells and its effect on oligonucleotide-directed gene modification.</a> Gene Ther. 13 (3), 266-275 (2006).</li><li>Koster, R. 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=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.

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.

<table><tbody><tr><td>Expand Long Template PCR System&amp;nbsp;</td><td>Roche Applied Science, IN</td><td>11681834001</td><td></td></tr><tr><td>pCR-TOPO vector&amp;nbsp;</td><td>Invitrogen, CA</td><td>451641</td><td></td></tr><tr><td>T4 DNA ligase</td><td>New England Biolabs, MA</td><td>M0202M</td><td></td></tr><tr><td>Gateway LR Clonase II enzyme&lt;br/&gt; Mix</td><td>Invitrogen, CA</td><td>11791-100</td><td></td></tr><tr><td>Gateway&amp;reg; BP Clonase&amp;reg; II enzyme mix</td><td>Invitrogen, CA</td><td>11789-020</td><td></td></tr><tr><td>GC-RICH PCR System&amp;nbsp;</td><td>Roche Applied Science, IN</td><td>12 140 306 001</td><td></td></tr><tr><td>Meganuclease I-SceI&amp;nbsp;</td><td>New England Biolabs, MA</td><td>R0694S</td><td></td></tr><tr><td>Nikon SMZ-1500 stereoscopic fluorescence microscope&amp;nbsp;</td><td>Nikon, NY</td><td></td><td></td></tr><tr><td>Nikon digital sight DS-U1 camera</td><td>Nikon, NY</td><td></td><td></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.

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

Transgenesi del mosaico del pesce zebra per l'analisi genomica funzionale dei geni cooperativi candidati nella patogenesi dei tumori | Biologia dello sviluppo | 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.

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 ...

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

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11.1K Views.  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

Transplantation of Zebrafish Pediatric Brain Tumors into Immune-competent Hosts for Long-term Study of Tumor Cell Behavior and Drug Response

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to assess potential patient response to therapy. Current methods largely depend on using syngeneic or immune-compromised animals to avoid rejection of the tumor graft. Such methods require the use of specific genetic strains that often prevent the analysis of immune-tumor cell interactions and/or are limited to specific genetic backgrounds. An alternative method in zebrafish takes advantage of an incompletely developed immune system in the embryonic brain before 3 days, where tumor cells are transplanted for use in short-term assays (i.e., 3 to 10 days). However, these methods cause host lethality, which prevents the long-term study of tumor cell behavior and drug response. This protocol describes a simple and efficient method for the long-term orthotopic transplantation of zebrafish brain tumor tissue into the fourth ventricle of a 2-day-old immune-competent zebrafish. This method allows: 1) long-term study of tumor cell behaviors, such as invasion and dissemination; 2) durable tumor response to drugs; and 3) re-transplantation of tumors for the study of tumor evolution and/or the impact of different host genetic backgrounds. In summary, this technique allows cancer researchers to assess engraftment, invasion, and growth at distant sites, as well as to perform chemical screens and cell competition assays over many months. This protocol can be extended to studies of other tumor types and can be used to elucidate mechanisms of chemoresistance and metastasis.

The transplantation of cancer cells is an important tool for the identification of cancer mechanisms and therapeutic responses. Current techniques depend on immune-incompetent animals. Here, we describe a method to transplant zebrafish tumor cells into immune-competent embryos for the long-term analysis of tumor cell behavior and in vivo drug responses.

Il trapianto di tumori del cervello pediatrico di Zebrafish in ospedali immunitario-competenti per uno studio a lungo termine del comportamento della cellula tumorale e della risposta ai farmaci

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to ...

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Ricerca sul cancro

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

Screening per Modificatori melanoma utilizzando un modello Zebrafish Tumore autoctone

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

Medicina

Generation of Zebrafish Larval Xenografts and Tumor Behavior Analysis

Zebrafish larval xenografts are being widely used for cancer research to perform in vivo and real-time studies of human cancer. The possibility of rapidly visualizing the response to anti-cancer therapies (chemo, radiotherapy, and biologicals), angiogenesis and metastasis with single cell resolution, places the zebrafish xenograft model as a top choice to develop preclinical studies.

The zebrafish larval xenograft assay presents several experimental advantages compared to other models, but probably the most striking is the reduction of size scale and consequently time. This reduction of scale allows single cell imaging, the use of a relatively low number of human cells (compatible with biopsies), medium-high-throughput drug screenings, but most importantly enables a significant reduction of the time of the assay. All these advantages make the zebrafish xenograft assay extremely attractive for future personalized medicine applications.

Many zebrafish xenograft protocols have been developed with a wide diversity of human tumors; however, a general and standardized protocol to efficiently generate zebrafish larval xenografts is still lacking. Here we provide a step-by-step protocol, with tips to generate xenografts and guidelines for tumor behavior analysis, whole-mount immunofluorescence, and confocal imaging quantification.

Here, we provide a step-by-step protocol, with tips to generate xenografts and guidelines for tumor behavior analysis, whole-mount immunofluorescence, and confocal imaging quantification.

Generazione di xenografti larvali zebrafish e analisi del comportamento tumorale

Zebrafish larval xenografts are being widely used for cancer research to perform in vivo and real-time studies of human cancer. The possibility of rapidly ...

Zebrafish Model of Neuroblastoma Metastasis

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing technologies applied in zebrafish modeling, the ease of maintenance, high-yield productivity, and powerful live imaging altogether make the zebrafish a valuable model system to study metastasis and cellular and molecular bases underlying this process in vivo. The first zebrafish neuroblastoma (NB) model of metastasis was developed by overexpressing two oncogenes, MYCN and LMO1, under control of the dopamine-beta-hydroxylase (d&#946;h) promoter. Co-overexpressed MYCN and LMO1 led to the reduced latency and increased penetrance of neuroblastomagenesis, as well as accelerated distant metastasis of tumor cells. This new model reliably reiterates many key features of human metastatic NB, including involvement of clinically relevant and metastasis-associated genetic alterations; natural and spontaneous development of metastasis in vivo; and conserved sites of metastases. Therefore, the zebrafish model possesses unique advantages to dissect the complex process of tumor metastasis in vivo.

This paper introduces the method of developing, characterizing, and tracking in real-time the tumor metastasis in zebrafish model of neuroblastoma, specifically in the transgenic zebrafish line with overexpression of MYCN and LMO1, which develops metastasis spontaneously.

Zebrafish Modello di Metastasi del Neuroblastoma

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing ...

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.

Visualizzazione dell'attività elettrica cellulare in Zebrafish embrioni in anticipo e tumori

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

Biologia dello sviluppo

Global Gene Expression Analysis Using a Zebrafish Oligonucleotide Microarray Platform

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray technology is used in numerous biological disciplines in a variety of applications including global gene expression analysis in relation to developmental stage, to a disease state, and in toxic responses. Herein, we include a demonstration of global gene expression analysis using a comprehensive zebrafish-specific oligonucleotide microarray platform. The zebrafish expression microarray platform contains 385,000 probes, 60 base pairs in length, interrogating 37,157 targets with up to 12 probes per target. For this platform, all cDNA and genomic information available for the zebrafish was collected from various genomic databases including Ensembl (http://www.ensembl.org), VEGA (http://vega.sanger.ac.uk), UCSC (http://genome.ucsc.edu), and ZFIN (http://www.zfin.org). As a result this expression array provides complete coverage of the current zebrafish transcriptome. The zebrafish expression microarray was printed by Roche NimbleGen (Madison, WI). This technical demonstration includes the fluorescent labeling of a cDNA product, hybridization of the labeled cDNA product to the microarray platform, and array scanning for signal acquisition using the one color analysis strategy.  

Gene microarrays are powerful tools in gene expression profiling at a genome-wide level. This technology has application in a variety of biological disciplines including developmental biology and toxicology. In this video, we detail a protocol for global gene expression analysis using a comprehensive oligonucleotide microarray platform for the zebrafish.

Analisi di espressione genica globale Utilizzando un Zebrafish piattaforma Microarray oligonucleotidi

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray ...

Biologia

Normal and Malignant Muscle Cell Transplantation into Immune Compromised Adult Zebrafish

Zebrafish have become a powerful tool for assessing development, regeneration, and cancer. More recently, allograft cell transplantation protocols have been developed that permit engraftment of normal and malignant cells into irradiated, syngeneic, and immune compromised adult zebrafish. These models when coupled with optimized cell transplantation protocols allow for the rapid assessment of stem cell function, regeneration following injury, and cancer. Here, we present a method for cell transplantation of zebrafish adult skeletal muscle and embryonal rhabdomyosarcoma (ERMS), a pediatric sarcoma that shares features with embryonic muscle, into immune compromised adult rag2E450fs homozygous mutant zebrafish. Importantly, these animals lack T cells and have reduced B cell function, facilitating engraftment of a wide range of tissues from unrelated donor animals. Our optimized protocols show that fluorescently labeled muscle cell preparations from &#945;-actin-RFP transgenic zebrafish engraft robustly when implanted into the dorsal musculature of rag2 homozygous mutant fish. We also demonstrate engraftment of fluorescent-transgenic ERMS where fluorescence is confined to cells based on differentiation status. Specifically, ERMS were created in AB-strain myf5-GFP; mylpfa-mCherry double transgenic animals and tumors injected into the peritoneum of adult immune compromised fish. The utility of these protocols extends to engraftment of a wide range of normal and malignant donor cells that can be implanted into dorsal musculature or peritoneum of adult zebrafish.

Here, we present a protocol for cell transplantation of zebrafish skeletal muscle and embryonal rhabdomyosarcoma (ERMS) into adult immune compromised rag2E450fs homozygous mutant zebrafish. This protocol allows for the efficient analysis of regeneration and malignant transformation of muscle cells.

Normali e maligne trapianto muscolare cellulare in immunitario compromesso Zebrafish adulti

Zebrafish have become a powerful tool for assessing development, regeneration, and cancer. More recently, allograft cell transplantation protocols have ...

Immunologia e infezioni

Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non coding sequences. One prominent function carried out by the non coding fraction of the genome is to regulate gene transcription; however, there are no effective methods to broadly predict cis-regulatory elements from primary DNA sequence. We have developed an efficient protocol to functionally evaluate potential cis-regulatory elements through zebrafish transgenesis. Our approach offers significant advantages over cell-culture based techniques for developmentally important genes, since it provides information on spatial and temporal gene regulation. Conversely, it is faster and less expensive than similar experiments in transgenic mice, and we routinely apply it to sequences isolated from the human genome. Here we demonstrate our approach to selecting elements for testing based on sequence conservation and our protocol for cloning sequences and microinjecting them into zebrafish embryos.

We demonstrate our approach to finding potential enhancer elements from developmentally regulated genes and evaluating their function through mosaic zebrafish transgenesis.

Mosaico Transgenesi Zebrafish per la valutazione sequenze Enhancer

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

Utilizzo delle larve di pesce zebra per studi efficienti sui tumori in vivo

A Simple, Rapid, and Effective Method for Tumor Xenotransplantation Analysis in Transparent Zebrafish Embryos

In vivo studies of tumor behavior are a staple of cancer research; however, the use of mice presents significant challenges in cost and time. Here, we present larval zebrafish as a transplant model that has numerous advantages over murine models, including ease of handling, low expense, and short experimental duration. Moreover, the absence of an adaptive immune system during larval stages obviates the need to generate and use immunodeficient strains. While established protocols for xenotransplantation in zebrafish embryos exist, we present here an improved method involving embryo staging for faster transfer, survival analysis, and the use of flow cytometry to assess disease burden. Embryos are staged to facilitate rapid cell injection into the yolk of the larvae and cell marking to monitor the consistency of the injected cell bolus. After injection, embryo survival analysis is assessed up to 7 days post injection (dpi). Finally, disease burden is also assessed by marking transferred cells with a fluorescent protein and analysis by flow cytometry. Flow cytometry is enabled by a standardized method of preparing cell suspensions from zebrafish embryos, which could also be used in establishing the primary culture of zebrafish cells. In summary, the procedure described here allows a more rapid assessment of the behavior of tumor cells in vivo with larger numbers of animals per study arm and in a more cost-effective manner.

Autore in primo piano: Avanzamento delle terapie antitumorali utilizzando lo xenotrapianto tumorale nel pesce zebra

We describe a protocol for xenotransplantation into the yolk of transparent zebrafish embryos that is optimized by a simple, rapid staging method. Post-injection analyses include survival and assessing the disease burden of xenotransplanted cells by flow cytometry.

Un metodo semplice, rapido ed efficace per l'analisi dello xenotrapianto tumorale in embrioni trasparenti di zebrafish

In vivo studies of tumor behavior are a staple of cancer research; however, the use of mice presents significant challenges in cost and time. Here, we ...

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

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Il trapianto di tumori del cervello pediatrico di Zebrafish in ospedali immunitario-competenti per uno studio a lungo termine del comportamento della cellula tumorale e della risposta ai farmaci

Screening per Modificatori melanoma utilizzando un modello Zebrafish Tumore autoctone

Generazione di xenografti larvali zebrafish e analisi del comportamento tumorale

Zebrafish Modello di Metastasi del Neuroblastoma

Visualizzazione dell'attività elettrica cellulare in Zebrafish embrioni in anticipo e tumori

Analisi di espressione genica globale Utilizzando un Zebrafish piattaforma Microarray oligonucleotidi

Normali e maligne trapianto muscolare cellulare in immunitario compromesso Zebrafish adulti

Mosaico Transgenesi Zebrafish per la valutazione sequenze Enhancer

Un metodo semplice, rapido ed efficace per l'analisi dello xenotrapianto tumorale in embrioni trasparenti di zebrafish

Un metodo semplice, rapido ed efficace per l'analisi dello xenotrapianto tumorale in embrioni trasparenti di zebrafish