Co-injection strategies to modify radiation sensitivity and tumor initiation in transgenic Zebrafish

Langenau, Dm M.; Keefe, Matthew D.; Storer, Ny Y.; Jette, Ca A.; Smith, Ach C. H.; Ceol, Cj J.; Bourque, Caitlin; Look, A. Thomas; Zon, Li I.

doi:10.1038/onc.2008.56

Cited by 68 publications

(81 citation statements)

References 20 publications

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“…These tumors were fluorescently labeled by co-injection of embryos with both an oncogene and fluorescent marker; because the DNA co-segregates, any cell expressing the oncogene will also express the fluorescent protein 17 . While fluorescence is recommended because it facilitates tracking of tumor growth without the need to sacrifice the animal and provides a straight-forward way to isolate tumor cells by FACS, it is possible to label tumor cells with antibodies to tumorspecific makers to facilitate their purification, though antibody staining in zebrafish may require optimization.…”

Section: Discussionmentioning

confidence: 99%

Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish

Blackburn

Liu

Langenau

2011

JoVE

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Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as tumor-propagating cells, or tumor-initiating cells. It is thought that targeting these self-renewing cells for destruction will block tumor progression and stop relapse, greatly improving patient prognosis 1 . The most common way to determine the frequency of self-renewing cells within a tumor is a limiting dilution cell transplantation assay, in which tumor cells are transplanted into recipient animals at increasing doses; the proportion of animals that develop tumors is used the calculate the number of self-renewing cells within the original tumor sample 2, 3 . Ideally, a large number of animals would be used in each limiting dilution experiment to accurately determine the frequency of tumor-propagating cells. However, large scale experiments involving mice are costly, and most limiting dilution assays use only 10-15 mice per experiment.Zebrafish have gained prominence as a cancer model, in large part due to their ease of genetic manipulation and the economy by which large scale experiments can be performed. Additionally, the cancer types modeled in zebrafish have been found to closely mimic their counterpart human disease 4 . While it is possible to transplant tumor cells from one fish to another by sub-lethal irradiation of recipient animals, the regeneration of the immune system after 21 days often causes tumor regression 5 . The recent creation of syngeneic zebrafish has greatly facilitated tumor transplantation studies [6][7][8] . Because these animals are genetically identical, transplanted tumor cells engraft robustly into recipient fish, and tumor growth can be monitored over long periods of time. Syngeneic zebrafish are ideal for limiting dilution transplantation assays in that tumor cells do not have to adapt to growth in a foreign microenvironment, which may underestimate self-renewing cell frequency 9,10 . Additionally, one-cell transplants have been successfully completed using syngeneic zebrafish 8 and several hundred animals can be easily and economically transplanted at one time, both of which serve to provide a more accurate estimate of self-renewing cell frequency.Here, a method is presented for creating primary, fluorescently-labeled T-cell acute lymphoblastic leukemia (T-ALL) in syngeneic zebrafish, and transplanting these tumors at limiting dilution into adult fish to determine self-renewing cell frequency. While leukemia is provided as an example, this protocol is suitable to determine the frequency of tumor-propagating cells using any cancer model in the zebrafish. Video LinkThe video component of this article can be found at https://www.jove.com/video/2790/ Protocol 1. DNA microinjection of zebrafish embryos 1. Linearize rag2::cMyc and rag2::GFP 11 DNA constructs by digesting 10μg of each plasmid with NotI at 37°C overnight.2. Phenol:chloroform extract and precipitate the plasmids, and resuspend each in 20μL H 2 O. 3. Determine the...

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Section: Discussionmentioning

confidence: 99%

Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish

Blackburn

Liu

Langenau

2011

JoVE

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“…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. Please click here to view a larger version of this figure.…”

Section: Representative Resultsmentioning

confidence: 99%

“…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.…”

Section: Discussionmentioning

confidence: 99%

“…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 dβh:EGFP-MYCN transgene and for easier identification of MYCN stable transgenic line.…”

Section: Protocolmentioning

confidence: 99%

“…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 (dβh) promoter in wild-type fish and transgenic fish overexpressing MYCN.…”

Section: Introductionmentioning

confidence: 99%

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

Ung

Guo

Zhu

et al. 2015

JoVE

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Comprehensive genomic analysis has uncovered surprisingly large numbers of genetic alterations in various types of cancers. To robustly and efficiently identify oncogenic "drivers" 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. Video LinkThe video component of this article can be found at

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Cancer Modeling by Transgene Electroporation in Adult Zebrafish (TEAZ)

Montal,

Suresh,

et al. 2023

Methods in Molecular Biology

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Co-injection strategies to modify radiation sensitivity and tumor initiation in transgenic Zebrafish

Cited by 68 publications

References 20 publications

Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish

Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish

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

Cancer Modeling by Transgene Electroporation in Adult Zebrafish (TEAZ)

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