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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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. 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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 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.

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 | 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.

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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11.0K 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

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.

Su larga scala Zebrafish embrionali Cuore dissezione per l&#39;analisi trascrizionale

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.

Analisi delle larve Zebrafish muscolo scheletrico Integrità con Blu di Evans Dye

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

Technique to Target Microinjection to the Developing Xenopus Kidney

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

Technique to Target Microinjection to the Developing Xenopus Kidney

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

Tecnica a Target microiniezione per lo sviluppo<em&gt; Xenopus</em&gt; Kidney

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

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

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

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

Microiniezione per Transgenesi e Genome Editing in Threespine spinarelli

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.

Manipolazione funzionale delle materne genica Utilizzando<em&gt; In Vitro</em&gt; La maturazione degli ovociti in Zebrafish

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

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

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

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

Microscopia elettronica e correlativo super-risoluzione per risolvere la localizzazione della proteina nella Retina di Zebrafish

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

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

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

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

Utilizzo di una zanzara ricombinante Densovirus come un vettore di consegna del Gene per l'analisi funzionale di geni in larve di zanzara

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.

Genotipizzazione e quantificazione della colorazione dell'ibridazione in situ nel pesce zebra

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.

Microdissezione laser di cattura della cartilagine embrionale del topo e dell'osso per l'analisi dell'espressione genica

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.

Generazione di organoidi mammari mosaici per provepsinizzazione differenziale

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