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.

NOTE: All zebrafish studies and maintenance of the animals were done in accord with Mayo Clinic Institute IACUC-approved protocol # A41213.
1. DNA Constructs for Transgenesis
<ol>
	<li>Amplify a 5.2-kb dopamine beta hydroxylase (d&#946;h) promoter region8 using the CH211-270H11 BAC clone (from BACPAC resources center (BPRC)) as a DNA template. Use a PCR system appropriate for long and accurate PCR amplification of long DNA templates and the following cycle progra...

<ol>
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The authors declare that they have no competing financial interests.

In this representative study, we used transient coinjection and coexpression of activated ALK with the mCherry reporter gene in MYCN-expressing transgenic fish to show that these genes cooperate to markedly accelerate the onset of neuroblastoma, consistent with our previous finding in compound stable transgenic fish coexpressing both activated ALK and MYCN 8. This mosaic transgenic approach possesses several distinct advantages over the conventional method. Most imp...

Cancers are progressive diseases marked by the accumulation of pathologic mutations, deletions and chromosome gains over time. These genetic abnormalities can affect multiple cellular processes ranging from the cell cycle, cell death, energetic metabolism and assembly of the cytoskeleton to stress responses such as hypoxia. Hence, tumorigenesis reflects the collective actions of multiple genetic aberrations across a spectrum of biological processes. Recent integrative genomic research efforts, including whole genome sequencing, exome sequencing, targeted sequencing, deep sequencing and genome-wide association studies, have identified a growing number of novel genetic alterations in essentially all types of tumors 1-4. In many instances, the genetic lesions occur together in a nonrandom manner 5-8, suggesting their cooperation in disease pathogenesis. Dissecting the oncogenic roles of the large array of aberrantly expressed genes resulting from these genomic lesions is necessary to devise new therapeutic strategies and to understand the responses of tumor cells to these agents, but this has proved to be a daunting task, requiring very robust animal model systems for the conduct of high-throughput functional genomic analysis in vivo.
Although mammals, especially rodents, are favored models in cancer biology, the zebrafish has begun to attract considerable attention. The teleost zebrafish (Dario rerio) has been used as a model organism for development study since the 1960s and was first applied to the study of tumor pathogenesis in 1982 9-11. Ease of maintenance, small body size, and high fecundity make the zebrafish a robust model for large-scale forward genetic screens to identify mutations that confer abnormal and pathological phenotypes 10. The optical transparency of zebrafish embryos is another key feature supporting wider use of this cancer model, as it allows in vivo imaging to be conducted to locate tumor development in real time 9, an application that is relatively difficult in rodents 12. Recent comparative genomics analysis of the zebrafish reference genome (Zv9) revealed 26,206 protein-coding genes, with 71% having human orthologues, of which 82% are correlated with disease-associated genes in the Online Mendelian Inheritance in Man (OMIM) database 13,14. Consequently, the zebrafish has been used to model diverse types of human cancers, including neuroblastoma 8, T-cell acute lymphoblastic leukemia (T-ALL) 15,16, melanoma 17,18, Ewing&#8217;s sarcoma 19, rhabdomyosarcoma 20,21, pancreatic carcinoma 22, hepatocellular carcinoma 23 and myeloid malignancies 24,25, and has been selected as a cancer model for xenotransplantation studies 11,26.
A stable transgenic approach in zebrafish is commonly used to study the effect of gain-of-function of genes in normal development or disease pathogenesis 27,28. To develop such a model (Figure 1A), one injects a DNA construct containing the gene of interest driven by a tissue-specific promoter into one-cell wild-type embryos. Three to four months after injection, when the injected embryos reach sexual maturity, they are outcrossed with wild-type fish to screen for the ones showing integration of the DNA construct in their germline, which licenses them as founder fish. Many factors, such as the copy number and integration site of the transgene, affect expression of the transgene in stable transgenic lines. Thus, to develop a transgenic tumor model, multiple stable transgenic lines overexpressing a single oncogene have to be generated first and screened for the line expressing the transgene at a level that might lead to tumor induction. However, if overexpression of a candidate oncogene is toxic to germ cells, it is difficult to generate a stable transgenic line by directly overexpressing the transgene 29. Hence, this approach can be time-consuming, with a high risk of failure to generate a suitable cancer model.
Here, we illustrate an alternative strategy based on mosaic transient transgenesis (Figure 1B) that provides unique advantages over traditional stable transgenesis for functional genomic study in vivo. In this approach, one or more transgene constructs are injected into the one-cell stage of transgenic or wild-type embryos. The injected DNA constructs containing transgenes are then mosaically and randomly integrated into the primary injected fish, resulting in mixed genotypes within multiple cell populations in individual fish 30. Moreover, coinjection of multiple DNA constructs in one-cell embryos leads to co-integration into the same cell at random sites, allowing one to trace the cells with expression of transgenes and explore the interactions of different genes during disease pathogenesis in the mosaic animals 31. As proof of principle, we transiently overexpressed mutationally activated ALK (F1174L) with mCherry reporter gene in the peripheral sympathetic nervous system (PSNS) under control of the dopamine beta hydroxylase (d&#946;h) promoter in wild-type fish and transgenic fish overexpressing MYCN. ALK, which encodes a receptor tyrosine kinase, is the most frequently mutated gene in high-risk neuroblastoma 5-7,32,33. ALK (F1174L), as one of the most frequent and potent somatic activating mutations, is over-represented in MYCN-amplified high-risk neuroblastoma patients and synergizes with MYCN overexpression to accelerate neuroblastoma tumorigenesis in both stable transgenic mice and transgenic zebrafish models 8,34,35. By mosaic transient overexpression of ALK (F1174L) with mCherry in the MYCN transgenic fish, we recapitulated the acceleration of tumor onset observed in the stable transgenic fish overexpressing both ALK (F1174L) and MYCN, suggesting that the mosaic transgenesis strategy can be used to rapidly and efficiently assess the relative contributions of multiple oncogenes in tumor initiation 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>
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			<td height="21" style="height:21px;">Gateway&#174; BP Clonase&#174; II enzyme mix</td>
			<td style="width:171px;">Invitrogen, CA</td>
			<td>11789-020</td>
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			<td height="24" style="height:25px;">GC-RICH PCR System&#160;</td>
			<td style="width:171px;">Roche Applied Science, IN</td>
			<td>12 140 306 001</td>
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			<td height="26" style="height:26px;">Meganuclease I-SceI&#160;</td>
			<td style="width:171px;">New England Biolabs, MA</td>
			<td>R0694S</td>
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			<td height="21" style="height:21px;">Nikon SMZ-1500 stereoscopic fluorescence microscope&#160;</td>
			<td style="width:171px;">Nikon, NY</td>
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			<td height="21" style="height:21px;">Nikon digital sight DS-U1 camera</td>
			<td style="width:171px;">Nikon, NY</td>
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mosaic zebrafish transgenesis for functional genomic analysis of candidate cooperative genes in tumor pathogenesis

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

To investigate whether overexpression of mutationally activated ALKF1174L or wild-type ALK could collaborate with MYCN in neuroblastoma induction, we overexpressed either activated human ALK or wild-type human ALK under control of the d&#946;h promoter in the PSNS of transgenic fish overexpressing MYCN. Either of the following constructs, d&#946;h-ALKF1174L or d&#946;h-ALKWT, were coinjected with d&#94...

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

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

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

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 Görüntüleme.  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.