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 programs for PCR: 94 &#176;C for 2 min, 10 cycles of (94 &#176;C, 15 sec, 50 &#176;C, 30 sec, 68 &#176;C, 8 min), followed by 30 cycles of (94 &#176;C, 15 sec, 53 &#176;C, 30 sec, 68 &#176;C, 8 min), 68 &#176;C, 4 min (forward primer 5&#8217;-GGGGACAACTTTGTATAGAAAAGTTGGCGTACTCCCCCTTTTTAGG-3&#8217; and reverse primer 5&#8217;- GGGGACTGCTTTTTTGTACAAACTTGTGTTGCTTTGTCGTCTTTTGA-3&#8217;).</li>
	<li>Create the d&#946;h-pDONRP4-P1R entry clone 8 using commercial recombinant cloning system such as multisite gateway. Mix 1 &#956;l of purified d&#946;h PCR product (172 ng/&#956;l), 1 &#956;l (150 ng/&#956;l) pDONRP4-P1R donor vector (together with other gateway vectors are generous gifts from Dr. Chi-Bin Chien, Univ. of Utah), 4 &#956;l of TE buffer (pH 8.0) with 2 &#181;l of BP clonase enzyme mix, incubate for 1 hr at 25 &#176;C and then transform to One Shot TOP10 competent E. coli according to the manufacturer&#39;s protocol.</li>
	<li>Generate the d&#946;h:EGFP-MYCN transgenic construct 8 using recombinant cloning system mentioned in step 1.2. Mix 1 &#956;l of each entry clone (150 ng/&#956;l ) including a 5.2-kb d&#946;h promoter (d&#946;h-pDONR P4-P1R construct), EGFP lacking a stop codon (pME-EGFP construct), and human MYCN cDNA (a generous gift from Dr. Hogarty at the Children&#8217;s Hospital of Pennsylvania, MYCN-pDONRP2R-P3 construct), 1 &#956;l (150 ng/&#956;l ) of the modified destination vector containing I-SceI recognition sites (a generous gift from Dr. C. Grabher, Karlsruhe Institute of Technology, Karlsruhe, Germany), 4 &#956;l of TE buffer (pH 8.0) with 2 &#181;l of LR Clonase enzyme mix, incubate for 1 hr at 25 &#176;C and then transform to chemically competent E. coli such as One shot top 10 and follow the manufacturer&#39;s protocol.</li>
	<li>Make mitf:mitf transgenic construct 8 by digesting the PNP-mitf vector (a generous gift from Dr. D. Raible, Univ. of Washington) with NotI and SalI restriction enzymes for 2 h at RT, and by subcloning the released 2.65-kb DNA fragment that contains the mitf promoter and the coding sequence of the zebrafish mitf gene into the NotI and MluI sites of a modified pBluescript vector containing flanking I-SceI recognition sites (a generous gift from Dr. Hui Feng, Boston University), using T4 DNA ligase according to the manufacturer&#8217;s protocol. 
	NOTE: To increase the efficiency of generating stable MYCN-expressing lines, d&#946;h:EGFP-MYCN and the mitf:mitf DNA constructs are coinjected into one-cell stage nacre embryos. Nacre designates a type of mutant fish lacking a neural crest-derived melanophore during development 36. Thus, the appearance of pigment cells in the injected embryos suggests the integration of mitf:mitf DNA constructs into the genome. It has been demonstrated that two or three coinjected DNA constructs can be cointegrated into the fish genome 31. Thus, pigmentation caused by mitf expression can serve as a marker for the integration of the d&#946;h:EGFP-MYCN transgene and for easier identification of MYCN stable transgenic line.</li>
	<li>Mix the d&#946;h:EGFP-MYCN and mitf:mitf DNA constructs at a 3:1 ratio in a total volume of a 15 &#956;l reaction with 1 &#956;l of I-SceI enzyme and 0.75 &#956;l of buffer. Ensure that the total amount of DNA in the reaction does not exceed 750 ng.</li>
	<li>Carry out the I-SceI digestion at RT&#160;for 4 hr or O/N. On the second day, the I-SceI-digested DNAs are ready for microinjection or can be stored at -20 &#176;C for injection in the future. Store the I-SceI enzyme at -80 &#176;C in small aliquots to maintain its enzyme efficiency.</li>
	<li>Subclone the human ALKF1174L and wild-type ALK gene from the PCDNA3 vector (a generous gift from Dr. George at Dana-Farber Cancer Institute) 7 into EcoRI and NotI sites of a pENTRY1A vector (a generous gift from Dr. C. Grabher, Karlsruhe Institute of Technology, Karlsruhe, Germany) using T4 DNA ligase according to the manufacturer&#8217;s protocol.</li>
	<li>Generate the d&#946;h:ALKF1174L or d&#946;h:ALKWT transgenic constructs using the recombinant system as mentioned in Protocol 1.2. Briefly, combine three entry clones, d&#946;h-pDONRP4-P1R, ALKF1174L-pENTRY1A (or ALKWT-pENTRY1A) and p3E-polyA, into the modified destination vector containing I-SceI recognition sites (a generous gift from Dr. C. Grabher, Karlsruhe Institute of Technology, Karlsruhe, Germany), using LR Clonase enzyme Mix, according to manufacturer&#39;s protocol.</li>
	<li>Mix the d&#946;h: ALKF1174L (or d&#946;h:ALKWT) and d&#946;h:mCherry DNA constructs at a 3:1 ratio and linearize them with I-SceI enzyme as described in PROTOCOL 1.5-1.6.</li>
</ol>
2. Microinjection
<ol>
	<li>Use glass micropipettes with 1.0 mm diameter for all injections 37. Break off the tip of the glass micropipettes with a razor blade before injection. Calibrate the injection volume by injecting H2O into a drop of mineral oil. Measure the diameter of the resulting droplets and adjust the microinjector (pressure or duration of pressure pulse) to ensure that the injection volume is less than 10% of the total cell volume of one cell-stage embryos.</li>
	<li>Add an additional 0.5 &#956;l of fresh I-SceI enzyme (5 unit/&#956;l) in 5 &#956;l of injection solution containing DNA constructs right before the injection to increase the efficiency of transgenesis 38. Inject 50-80 pg of linearized DNA constructs into the cytoplasm of one-cell stage embryos. To ensure the success of injection, mix the sample with 0.25% phenol red for visualization. If cells turn red after injection, it indicates the microinjection is successful. To ensure a high success rate for generating transgenic fish, we typically inject as many as 500 embryos.</li>
	<li>To generate transgenic fish stably expressing MYCN, coinject the linearized d&#946;h:EGFP-MYCN and mitf:mitf DNA constructs (at a 3:1 ratio) into embryos of the pigmentation mutant nacre zebrafish at the one-cell stage. The expression of Mitf serves as a reporter for the integration of transgenic constructs in the fish genome.</li>
	<li>For mosaic overexpression of ALK in the wild-type or MYCN transgenic embryos, co-inject the linearized d&#946;h:ALK F1174L (or d&#946;h:ALKWT) with d&#946;h:mCherry DNA constructs (at a 3:1 ratio) into the one-cell embryos resulting from breeding of F1 heterozygous Tg(d&#946;h:EGFP-MYCN) transgenic fish with wild-type AB fish. Thus, half of offspring are transgenic for MCYN and half are wild-type.</li>
</ol>
3. Screen for Stable or Mosaic Transgenic Fish
<ol>
	<li>To increase the efficiency of identification of stable MYCN-expressing lines, anesthetize primarily injected nacre embryos with tricaine (0.02%) at days 3-5 of postfertilization and screen for pigmentation. Transfer the embryos with pigmentation to a new petri dish with fresh egg water and raise them to sexual maturity for further screening for the founder fish, which carry the d&#946;h:EGFP-MYCN transgene in the germ cells.</li>
	<li>To identify the founder with germline transmission of EGFP-MYCN, outcross pigmented F0 adult fish with wild-type AB fish and screen for EGFP-positive embryos at 1-2 days post fertilization for further genotyping.&#160;Place a single EGFP-positive F1 embryo into a PCR tube and remove all liquid. Add 50 &#956;l of gDNA extraction buffer which contains 12.5 &#956;l of 4x lysis buffer, 35 &#956;l H2O and 2.5 &#956;l proteinase K (10 mg/ml) to single embryo. Incubate the reaction for 2-3 hr at 55 &#176;C, followed by 10 min of incubation at 98&#186;C to inactivate proteinase K. To make 50 ml of 4x lysis buffer, add 500 &#956;l of 1M Tris (pH 8.4), 2.5 ml of 1M KCl to 47 ml of H2O.&#160;NOTE: After F0&#160;MYCN stable transgenic fish are bred with wild-type AB fish, all of the offspring are pigmented. Thus, pigmentation cannot serve as marker for the identification of&#160;MYCN-positive fish. While in the&#160;MYCN&#160;transgenic fish, the EGFP-MYCN fusion protein is expressed in the PSNS, including the sympathetic neurons of the superior cervical ganglia and sequential segmental ganglion of the sympathetic chain and the non-PSNS dopaminergic neurons, such as the medulla oblongata and cranial ganglia&#160;8. Thus, the expression of&#160;EGFP&#160;can serve as a marker for identification of the&#160;MYCN&#160;stable transgenic embryos.</li>
	<li>Use 2 &#956;l of gDNA extracted from the pigmented F1 embryo as template, primers MYCN-test F1: 5&#8217;-CTG CTT GAG AAC GAG CTG TG-3&#8217;; MYCN-R3: 5&#8217;-AGG CAT CGT TTG AGG ATC AG-3&#8217;, and the following program with the GC-RICH PCR System: 1 cycle of 95 &#176;C for 3 min, 25 cycles of 95 &#176;C for 30 sec, 58 &#176;C for 30 sec, and 72 &#176;C for 3 min. We typically genotype 14-16 pigmented embryos from a single mating to confirm the presence of integrated MYCN transgene in the pigmented embryos, more than 6 founder fish overexpressing mitf and MYCN were identified by this method.</li>
	<li>Raise up the remainder of the EGFP-positive F1 embryos. Fin clip and genotype them at 2-3 month of age using the above protocol to further confirm the integration of MYCN transgene into the fish. Breed F1 MYCN stable transgenic fish with wild-type AB fish. Co-inject the linearized d&#946;h:ALK F1174L (or d&#946;h:ALKWT) with&#160;d&#946;h:mCherry DNA constructs into the one-cell embryos as described in PROTOCOL 2.4.</li>
	<li>Sort the MYCN-expressing embryos in the experiment described in PROTOCOL 2.4 using a stereoscopic fluorescence microscope and screen the primarily injected embryos at days 1-3 of postfertilization for the expression of EGFP-MYCN in the PSNS. Then, during days 2-5 of postfertilization, anesthetize the embryos with tricaine (0.02%) and sort those MYCN-positive or negative embryos again based on the expression of mCherry in the PSNS using a stereoscopic fluorescence microscope. The expression of mCherry serves as a marker for the coexpression of ALK in tissues of the mosaic primary injected animals. 
	NOTE: ~600 offspring resulting from the breeding of MYCN stable transgenic fish with wild-type fish were injected per group with the linearized DNA constructs, including d&#946;h:ALK F1174L and d&#946;h:mCherry, d&#946;h:ALKWT and d&#946;h:mCherry, or d&#946;h:mCherry alone, respectively. Mosaic expression of mCherry can be observed in 70-90% of the primarily injected fish. One-half to one-third of the injected fish survived through the larvae stage for tumor watch.</li>
	<li>Raise all of the mCherry+MYCN+ and mCherry+MYCN- embryos according to the standard protocols from the zebrafish book 39 and monitor tumor onset beginning at 5 weeks postfertilization.</li>
</ol>
4. Tumor Watch in Mosaic Transgenic Fish
<ol>
	<li>Monitor mCherry-positive primarily injected fish every 2 weeks starting at 5 weeks postfertilization for evidence of tumor onset.</li>
	<li>Anesthetize fish with tricaine (0.02%) and screen for the presence of mCherry- and EGFP-expressing tumors in the PSNS under the Nikon SMZ-1500 stereoscopic fluorescence microscope equipped with a digital sight DS-U1 camera.</li>
	<li>To confirm whether the tumors are expressing the ALK transgene, isolate the mCherry- and EGFP-positive masses for ALK genotyping PCR.</li>
	<li>Using PCR, amplify genomic DNA extracted from developed tumors with the following primers: ALK P7: 5&#8217;-AGG CCA GGT GTC CGG AAT GC-3&#8217; and ALK P18: 5&#8217;-TGT CTT CAG GCT GAT GTT GC-3&#8217; and the following PCR reaction: 1 cycle of 94 &#176;C for 5 min, 30 cycles of (94 &#176;C for 30 sec, 55 &#176;C for 30 sec, and 72 &#176;C for 60 sec). Then, sequence the PCR product with ALK P7 primer to further confirm the existence of mutant or wild-type ALK in the mCherry-positive tumors.</li>
</ol>

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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 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.

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			<td height="21" style="height:21px;width:373px;">Name of Material/ Equipment</td>
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			<td style="width:119px;">Catalog Number</td>
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			<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>
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			<td height="21" style="height:21px;">pCR-TOPO vector&#160;</td>
			<td style="width:171px;">Invitrogen, CA</td>
			<td style="width:119px;">451641</td>
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			<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>
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			<td height="21" style="height:21px;">Gateway LR Clonase II enzyme 
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			<td style="width:171px;">Invitrogen, CA</td>
			<td>11791-100</td>
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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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		<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>
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		<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>
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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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11.1K 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

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.

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 ...

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.

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.

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.

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.

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.

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.

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.

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.

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.

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