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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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetic+landscape+of+high-risk+neuroblastoma.">The genetic landscape of high-risk neuroblastoma.</a> Nature genetics. , (2013).</li><li>Berry, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ALK(F1174L)+mutation+potentiates+the+oncogenic+activity+of+MYCN+in+neuroblastoma.">The ALK(F1174L) mutation potentiates the oncogenic activity of MYCN in neuroblastoma.</a> Cancer Cell. 22 (1), 117-130 (2012).</li><li>Heukamp, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+expression+of+mutated+ALK+induces+neuroblastoma+in+transgenic+mice.">Targeted expression of mutated ALK induces neuroblastoma in transgenic mice.</a> Sci Transl Med. 4 (141), 141ra191 (2012).</li><li>Lister, J. A., Robertson, C. P., Lepage, T., Johnson, S. L., Raible, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=nacre+encodes+a+zebrafish+microphthalmia-related+protein+that+regulates+neural-crest-derived+pigment+cell+fate.">nacre encodes a zebrafish microphthalmia-related protein that regulates neural-crest-derived pigment cell fate.</a> Development. 126 (17), 3757-3767 (1999).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+zebrafish+embryos+to+analyze+gene+function.">Microinjection of zebrafish embryos to analyze gene function.</a> J Vis Exp. (25), (2009).</li><li>Thermes, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=I-SceI+meganuclease+mediates+highly+efficient+transgenesis+in+fish.">I-SceI meganuclease mediates highly efficient transgenesis in fish.</a> Mech Dev. 118 (1-2), 91-98 (2002).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev Dyn. 203 (3), 253-310 (1995).</li><li>Urasaki, A., Asakawa, K., Kawakami, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+transposition+of+the+Tol2+transposable+element+from+a+single-copy+donor+in+zebrafish.">Efficient transposition of the Tol2 transposable element from a single-copy donor in zebrafish.</a> Proc Natl Acad Sci U S A. 105 (50), 19827-19832 (2008).</li><li>Caneparo, L., Pantazis, P., Dempsey, W., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intercellular+bridges+in+vertebrate+gastrulation.">Intercellular bridges in vertebrate gastrulation.</a> PLoS One. 6 (5), e20230 (2011).</li><li>Ivics, Z., Izsvak, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+expanding+universe+of+transposon+technologies+for+gene+and+cell+engineering.">The expanding universe of transposon technologies for gene and cell engineering.</a> Mob DNA. 1 (1), 25 (2010).</li><li>Tang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+cell+transplantation+using+adult+rag2+mutant+zebrafish.">Optimized cell transplantation using adult rag2 mutant zebrafish.</a> Nat Methods. 11 (8), 821-824 (2014).</li><li>Watson, I. R., Takahashi, K., Futreal, P. A., Chin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+patterns+of+somatic+mutations+in+cancer.">Emerging patterns of somatic mutations in cancer.</a> Nat Rev Genet. 14 (10), 703-718 (2013).</li><li>Sander, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+disruption+in+somatic+zebrafish+cells+using+engineered+TALENs.">Targeted gene disruption in somatic zebrafish cells using engineered TALENs.</a> Nat Biotechnol. 29 (8), 697-698 (2011).</li><li>Sander, J. D., Joung, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-Cas+systems+for+editing,+regulating+and+targeting+genomes.">CRISPR-Cas systems for editing, regulating and targeting genomes.</a> Nat Biotechnol. 32 (4), 347-355 (2014).</li></ol>

The authors declare that they have no competing financial interests.

In 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 important, it enables rapid examination of the effects of coexpressing candidate oncogenes in primarily injected animals (F0 generation) without the requirement for stable transgenic animals, such as eliminating the excessive time and labor typically involved in the breeding and identification of ALK-expressing stable transgenic fish. We also examined the effect of overexpression of wild-type ALK, with or without MYCN overexpression, on tumor initiation. The results show that transient overexpression of wild-type ALK in our fish model is not sufficient to initiate neuroblastoma tumorigenesis, regardless of the status of the MYCN oncogene. It would be interesting to test in the future whether transient coexpression of both activated ALK and MYCN in wild-type fish could induce earlier onset of tumorigenesis in the primarily injected fish. This study would more faithfully mimic the actual disease context, in which somatic alterations of both genes co-occur in a subset of high-risk neuroblastoma patients 5.
An important design element of the mosaic transient transgenic strategy is the inclusion of a reporter gene for coinjection with any candidate oncogenes of interest, which aids in screening for animals with successful integration of transgenes among the primarily injected zebrafish embryos and to monitor the positive fish for tumor onset and progression. To increase the efficiency of transgenesis, we have applied the I-SceI meganuclease-mediated transgenic approach, which significantly increases the uniform promoter-dependent expression of transgenes from 26% of the primarily injected embryos without meganuclease to 76% of injected embryos with meganuclease 38. Although use of the I-SceI meganuclease-mediated transgenic approach has improved the efficiency of transgenesis remarkably, the percentage of injected embryos positively for the transgenes still remains well below 100%. Thus, the expression of a coinjected reporter gene can serve as a marker to identify injected embryos with successful integration of candidate transgenes before further monitoring the fish for the onset of tumors. In contrast, a Tol2 transposon-mediated transgenic approach has become a popular genetic tool in model vertebrates and has been successfully used for transgenesis, insertional mutagenesis, gene trapping, and enhancer trapping 40,41. Coinjection of Tol2 transposons with transposase mRNA into fertilized embryos can facilitate early integration events that increases the efficiency of chromosomal integration and potentiates successful germline transmission of the transgene42. However, due to the &#8220;cut-and-paste&#8221; mechanism of DNA transposition, a single copy of transgene is integrated into the chromosome per insertion locus42. Thus, coinjected candidate genes are most likely integrated individually into the fish genome at different sites, which could lead to the expression of candidate genes in different cells and a more complicated transient and mosaic expression pattern of coinjected genes in the primarily injected fish.
Overall, the current study supports the future use of mosaic transgenesis as a rapid means to evaluate the collaborative, perhaps synergistic, relationships among multiple oncogenes in a high-throughput manner, a challenge that is difficult to meet with other animal models, including rodents. Thus far, this strategy has also been successfully applied in transgenic zebrafish to identify genes that suppress the activated RAS-induced initiation of rhabdomyosarcoma 31 and modify the radiation sensitivity of MYC-induced T-cell acute lymphoblastic leukemia 31. Furthermore, Langenau et al, 15 have demonstrated that combining the coinjection approach with a heat-shock-inducible transgenic approach can induce transgene expression by heat shock in the primarily injected fish, which would be very useful in exploring the cooperative roles of oncogenes in tumor progression, as it permits gene expression to be turned on after the primary tumor is established. Very recently, Langenau and colleagues developed the first immunocompromised zebrafish model by targeting rag2 gene with engineered zinc-finger nucleases 43. Loss of function of rag2 permitted robust and long-term engraftment of multiple tissues and cancer cells 43, an advantage that could be useful in transplanting the heterogeneous cancer cells that overexpress candidate transgenes and assessing the effect of mosaic transgene expression on self-renewal capacity, therapeutic responses and the growth rate of these cancer cells.
Finally, mosaic transgenesis offers another unique advantage over stable transgenesis. In stable transgenic animals, the transgenes are integrated into the genome of every single cell in the body and are heritable from generation to generation 27. However, many cancers arise from genetic alterations in somatic cells instead of inherited mutations in germ cells 44. Thus, the mosaic pattern of transgene integration in the primary injected fish and the mixed genotypes within cell populations of individual mosaic transgenic fish would better recapitulate the somatic defects found in patients and would avoid the artificial positional effect caused by a single insertion site in a stable transgenic line. On the other hand, the mosaicism and small population of cells overexpressing the transgenes in the primarily injected fish makes mechanistic study more difficult.
In summary, the mosaic transgenic strategy provides a robust tool for rapid and effective assessment of the cooperative contribution of oncogenes to tumor initiation and spread. This advance is expected to generate seminal information on the interplay among candidate oncogenes, which is urgently needed for further productive investigation of oncogenic mechanisms and pathways and for developing effective targeted therapy. Increasing the efficiency of cointegration of more than three coinjected DNA constructs would open the opportunity for examination of the complex relationships among concurrently expressed multigene combinations in various types of cancers. Challenges for the future will be to modify the mosaic transgenic strategy to accommodate any genomic or epigenetic alteration that could play a role in the neoplastic process, rather than limit its use to mutations that change only protein-coding or to genes are overexpressed, gained or amplified. Recently, improved genome editing techniques, such as transcription activator-like effector nucleases (TALENS) 45 and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems 46, have become widely used to more rapidly and efficiently modify endogenous genes in various types of cells and organisms 46. Such techniques would allow us to perform targeted, highly efficient modifications of genome sequence and gene expression in the zebrafish model system and to better understand the mechanism(s) underlying the role of genomic or epigenetic alterations in tumor pathogenesis. These, in turn, are&#160;likely to spur the development of novel molecular therapeutics for a range of cancers.

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

mosaic zebrafish transgenesis for functional genomic analysis of candidate cooperative genes in tumor pathogenesis

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

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&#946;h-mCherry into one-cell wild-type or MYCN transgenic embryos (Figure 2A) 8. The expression of mCherry served as a marker for the coexpression of ALK in tissues of the mosaic primary injected animals. All of the expected genotypes were represented in the injected fish: (1) MYCN-expressing fish with mosaic coexpression of activated ALKF1174L and mCherry, designated MYCN;mosaic ALKmut; (2) MYCN-expressing fish with mosaic coexpression of wild-type ALK and mCherry, designated MYCN;mosaic ALKWT; (3) MYCN-expressing fish with mosaic expression of mCherry, designated MYCN;mosaic mCherry; (4) wild-type fish with mosaic coexpression of activated ALKF1174L and mCherry, designated WT;mosaic ALKmut; and (5) wild-type fish with mosaic coexpression of wild-type ALK and mCherry, designated WT;mosaic ALKWT. A tumor watch was then performed every 2 weeks from 5 weeks postfertilization (wpf) on a total of 492 injected animals.
Eight GFP+/mCherry+ tumors arose in the interrenal gland, the human adrenal gland equivalent, by 9 wpf in the MYCN-expressing fish coinjected with d&#946;h-ALKF1174L and d&#946;h-mCherry (Figures 2B and 3). These tumors were histologically, immunohistochemically, and ultrastructurally comparable to human neuroblastoma (data can be found in reference 8). By contrast, no tumors were observed by 9 weeks of age in the MYCN-expressing fish coinjected with d&#946;h-ALKWT and d&#946;h-mCherry (Figures 2C and 3, p = 0.002) or injected with d&#946;h-mCherry alone (Figures 2D and 3, p = 0.007). Tumors arose in both the MYCN;mosaic ALKWT and MYCN;mosaic mCherry fish with the same rate of induction after 12 weeks of age. In addition, neuroblastomas were not detected in any wild-type fish coinjected with either the wild-type ALK and mCherry or ALKF1174L and mCherry transgenes during the 15 weeks of monitoring. Taken together, these findings show that mosaic overexpression of mutationally activated ALK accelerates MYCN-induced tumor onset, regardless of the integration site in individual mosaic animals, and that overexpression of wild-type ALK at the levels driven by the d&#946;h promoter does not appear to collaborate with MYCN overexpression to induce neuroblastoma in this model system.
<img alt="Figure 1" src="/files/ftp_upload/52567/52567fig1.jpg" /> 
Figure 1: Schematic outline of conventional stable and mosaic transient zebrafish transgenic strategies in the study of the Cooperative Contributions of Candidate Oncogenes to Tumorigenesis. (A) Stable transgenic strategy. Linearized transgenic DNA construct containing candidate oncogene is injected into one-cell stage wild-type or nacre mutant zebrafish embryos where the transgene can be integrated into the fish genome at random genomic loci. Integration of transgene into the germ cells is required for the generation of stable transgenic line. Primarily injected embryos take 3 to 4 months to reach sexual maturity (F0 generation of fish), which will be outcrossed with wild-type or nacre mutant fish to screen for the founder fish with the transgene germline transmission. The resultant offspring of founder fish, termed the F1 generation, carry the heterozygous alleles of the transgene in their genome. To assess the collaborative effects of two candidate oncogenes, such as gene &#8220;a&#8221; and gene &#8220;b&#8221;, in tumorigenesis, we bred the F1 progeny of two stable transgenic lines overexpressing these transgenes, with only a quarter of offspring carrying the compound transgenes. Because of the relatively low efficiency of generating stable transgenic lines, this approach is not feasible for high-throughput functional genomic analyses. (B) Mosaic transient transgenic strategy. Similar to the stable transgenesis, linearized transgenic DNA constructs containing candidate genes can be injected into one-cell stage wild-type embryos. Because the transgene does not have to be integrated into the genome of germ cells, much time and effort can be saved in the process of identifying founder fish. Alternatively, if the transgenic fish line overexpressing one of the candidate oncogenes is available (in our case, MYCN transgenic fish were developed), linearized transgenic DNA constructs containing other candidate genes can be injected into one-cell stage transgenic embryos to study their cooperation in tumorigenesis. Up to three transgene constructs can be coinjected and coexpressed in the primarily injected fish 31; thus, the interaction of candidate oncogenes in tumorigenesis can be assessed in the primarily injected fish. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52567/52567fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52567/52567fig2.jpg" /> 
Figure 2: Mosaic Expression of Activated ALK Accelerates the Onset of MYCN-induced Neuroblastoma 8. Modified figure with permission from Elsevier (reference 3466510609819). (A) Approach for constructing mosaic transgenesis. (B-F) 2-day-old larvae of primarily injected embryos. Lateral views of stable EGFP expression in the dopaminergic neurons, including cranial ganglia (CG, arrowheads) in merged fluorescence-brightfield images (upper panels). Lateral views of mosaic mCherry expression (lower panels) in the cranial ganglia (CG, arrowheads), medulla oblongata (MO, arrows). Some ectopic mCherry expression is observed in the non-PSNS and non-dopaminergic neurons. (G-K) 7-week-old primarily injected fish. (B, G) MYCN-expressing fish coinjected with d&#946;h-ALKF1174L and d&#946;h-mCherry constructs (MYCN;mosaic ALKmut). EGFP- and mCherry-positive tumors arose at 7 weeks (white empty arrows in G). (C, H) MYCN-expressing fish coinjected with d&#946;h-ALKWT and d&#946;h-mCherry constructs (MYCN;mosaic ALKWT). (D, I) MYCN-expressing fish injected with d&#946;h-mCherry construct alone (MYCN; mosaic&#160;mCherry). (E, J) Wild-type (WT) fish coinjected with d&#946;h-ALKF1174L and d&#946;h-mCherry constructs (WT; mosaic ALKmut). (F, K) Wild-type (WT) fish coinjected with d&#946;h-ALKWT and d&#946;h-mCherry constructs (WT;mosaic ALKWT). Neuroblastomas were not observed in the MYCN-expressing fish coinjected with d&#946;h-ALKWT and d&#946;h-mCherry or d&#946;h-mCherry alone at 7 wpf, or in any of the siblings that did not inherit the MYCN transgene and were injected with either the ALKWT gene or the ALKF1174L gene. Scale bars, 100 &#956;m in B-F and 1 mm in G-K. A Nikon SMZ-1500 stereoscopic fluorescence microscope equipped with a digital sight DS-U1 camera and a Leica MZ10F stereoscopic fluorescence microscope equipped with a Leica DFC 345FX digital camera were used to capture the fluorescent images.The acquired images were processed and compiled with Adobe Photoshop and Illustrator CS3 (Adobe) software. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52567/52567fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52567/52567fig3.jpg" /> 
Figure 3: Onset of Neuroblastoma in MYCN Transgenic Fish or Wild-type (WT) Fish as Mosaics Coinjected with the DNA Constructs. Reprinted with permission from Elsevier (reference 3466510609819). d&#946;h-ALKF1174L and d&#946;h-mCherry (mosaic ALKmut); d&#946;h-ALKWT and d&#946;h-mCherry (mosaic ALKWT); or d&#946;h-mCherry (mosaic&#160;mCherry) alone 8. The difference between tumor onset by 9 wpf in the MYCN-expressing fish coinjected with d&#946;h-ALKF1174L and d&#946;h-mCherry (MYCN;mosaic ALKmut) and that in the MYCN line coinjected with d&#946;h-ALKWT and d&#946;h-mCherry (MYCN;mosaic ALKWT) or d&#946;h-mCherry alone (MYCN; mosaic&#160;mCherry) is significant at p = 0.002 and p = 0.007, respectively, by two-tailed Fisher&#8217;s exact test.

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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

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

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

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

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

Cancer Research

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

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

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

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

Medicine

Generation of Zebrafish Larval Xenografts and Tumor Behavior Analysis

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

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

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

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

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

Zebrafish Model of Neuroblastoma Metastasis

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

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

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

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

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

Global Gene Expression Analysis Using a Zebrafish Oligonucleotide Microarray Platform

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

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

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

Normal and Malignant Muscle Cell Transplantation into Immune Compromised Adult Zebrafish

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

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

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

Immunology and Infection

Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences

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

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

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

Utilizing Larval Zebrafish for Efficient In Vivo Tumor Studies

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

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

Author Spotlight: Advancing Cancer Therapeutics Using Tumor Xenotransplantation in Zebrafish

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

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

The Zebrafish Tol2 System: A Modular and Flexible Gateway-Based Transgenesis Approach

Fetal alcohol spectrum disorders (FASD) are characterized by a highly variable set of structural defects and cognitive impairments that arise due to prenatal ethanol exposure. Due to the complex pathology of FASD, animal models have proven critical to our current understanding of ethanol-induced developmental defects. Zebrafish have proven to be a powerful model to examine ethanol-induced developmental defects due to the high degree of conservation of both genetics and development between zebrafish and humans. As a model system, zebrafish possess many attributes that make them ideal for developmental studies, including large numbers of externally fertilized embryos that are genetically tractable and translucent. This allows researchers to precisely control the timing and dosage of ethanol exposure in multiple genetic contexts. One important genetic tool available in zebrafish is transgenesis. However, generating transgenic constructs and establishing transgenic lines can be complex and difficult. To address this issue, zebrafish researchers have established the transposon-based Tol2 transgenesis system. This modular system uses a multisite Gateway cloning approach for the quick assembly of complete Tol2 transposon-based transgenic constructs. Here, we describe the flexible Tol2 system toolbox and a protocol for generating transgenic constructs ready for zebrafish transgenesis and their use in ethanol studies.

This work describes a protocol for the modular Tol2 transgenesis system, a gateway-based cloning method to create and inject transgenic constructs into zebrafish embryos.

Fetal alcohol spectrum disorders (FASD) are characterized by a highly variable set of structural defects and cognitive impairments that arise due to ...