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Introduction

Protocol

Representative Results

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Acknowledgements

Materials

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Cancer Research

Zebrafish Model of Neuroblastoma Metastasis

Published: March 14th, 2021

DOI:

10.3791/62416

1Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Mayo Clinic Cancer Center

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 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 (dβh) 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.

Zebrafish has been widely used and applied to several areas of research, especially in cancer. This model provides many advantages-such as its robust reproduction, cost-effective maintenance, and versatile visualization of tumor growth and metastasis-all of which make zebrafish a powerful tool to study and investigate the cellular and molecular bases of tumorigenesis and metastasis. New techniques for large-scale genome mapping, transgenesis, genes overexpression or knockout, cell transplantation, and chemical screens have immensely augmented the power of the zebrafish model1. During the past few years, many zebrafish lines have been developed ....

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All research methods using zebrafish and animal care/maintenance were performed in compliance with the institutional guidelines at Mayo Clinic.

1. Preparation and microinjection of transgene constructs for the development of LMO1 transgenic zebrafish line with overexpression in PSNS

  1. To develop the LMO1-pDONR221 entry clone, amplify the coding region of human LMO1 from cDNA obtained from human cell line using PCR.
    1. Make a 25 µL reaction as .......

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To determine whether LMO1 synergizes with MYCN to affect NB pathogenesis, transgenic constructs that drive expression of either LMO1 (dβh:LMO1 and dβh:mCherry) or MYCN (dβh:EGFP-MYCN) in the PSNS cells under control of the dβh promoter were injected into zebrafish embryos13. As illustrated in Figure 1A, after the development of stable transgenic lines and validation of their ge.......

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Zebrafish has been commonly used in research for the past few decades, especially in cancer research, for obvious reasons, such as its ease of maintenance, robust reproduction, and clear advantages for in vivo imaging1,28. The zebrafish model can be easily manipulated embryonically due to their external fertilization and development, which complements well to mammalian model organisms, such as rats and mice, for large-scale genetic studies

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This work was supported by a grant R01 CA240323 (S.Z.) from the National Cancer Institute; a grant W81XWH-17-1-0498 (S.Z.) from the United States Department of Defense (DoD); a V Scholar award from the V Foundation for Cancer Research (S.Z.) and a Platform Grant from the Mayo Center for Biomedical Discovery (S.Z.); and supports from the Mayo Clinic Cancer Center and Center for Individualized Medicine (S.Z.).

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Name Company Catalog Number Comments
3,3’-Diaminobenzidine (DAB) Vector Kit Vector SK-4100
Acetic Acid Fisher Scientific / Acros Organic 64-19-7
Agarose GP2 Midwest Scientific 009012-36-6
Anti-Tyrosine Hydroxylase (TH) Antibody Pel-Freez P40101
Avidin/Biotin Blocking Kit Vector SP-2001
BOND Intense R Detection Leica Biosystems DS9263
BOND primary antibody diluent Leica Biosystems Newcastle, Ltd. AR9352
BOND-MAX IHC instrument Leica Biosystems Newcastle, Ltd. N/A fully automated IHC staining system
CH211-270H11 BAC clone BACPAC resources center (BRFC) N/A
Compound microscope equipped with DP71 camera Olympus AX70
Cytoseal XYL (xylene based mounting medium) Richard-Allan Scientific 8312-4
Eosin Leica 3801601 ready-to-use (no preparation needed)
Ethanol Carolina 86-1263
Expand Long Template PCR System Roche Applied Science, IN 11681834001
Gateway BP Clonase II enzyme mix Invitrogen, CA 11789-020
Gateway LR Clonase II enzyme mix Invitrogen, CA 11791-100
Goat anti-Rb secondary antibody (Biotinylated) Dako E0432
Hematoxylin Solution, Harris Modified Sigma Aldrich Chemical Company Inc. / SAFC HHS-32-1L
HRP Avidin D Vector A-2004
Hydrochloric Acid Aqua Solutions 4360-1L
Hydrogen Peroxide, 3% Fisher Scientific H324-500
I-SceI enzyme New England Biolabs, MA R0694L
Kanamycin sulfate Teknova, Inc. K2150
Kimberly-Clark Professional Kimtech Science Kimwipes Fisher Scientific 34133
Lithium Carbonate Sigma Aldrich Chemical Company Inc. / SAFC 554-13-2
Microtome for sectioning Leica Biosystems RM2255
One Shot TOP10 Chemically Competent E. coli Invitrogen C404006
p3E-polyA  Dr. Chi-Bin Chien, Univ. of Utah N/A a generous gift
(Please refer to webpage http://tol2kit.genetics.utah.edu/index.php/Main_Page to obtain material, which is freely distrubted as described.)
Parafin wax Surgipath Paraplast 39603002 Parrafin to parafin
Paraformaldehyde Alfa Aesar A11313
pDEST vector (modified destination vector containing I-SceI recognition sites) Dr. C. Grabher, Karlsruhe Institute of Technology, Karlsruhe, Germany N/A a generous gift
pDONR 221 gateway donor vector Thermo Fisher Scientific 12536-017
pDONRP4-P1R donor vector  Dr. Chi-Bin Chien, Univ. of Utah N/A a generous gift
Phenol red, 0.5% Sigma Aldrich  P0290
Phosphate Buffered Saline (PBS), 10X BioRad 1610780
Picrosirrius red stain kit Polysciences 24901-250
pME-mCherry Addgene 26028 (DBH construct)
Proteinase K, recombinant, PCR Grade Roche 21712520
QIAprep Spin MiniPrep Kit Qiagen 27104
RDO Rapid Decalcifier Apex Enginerring RDO04
Sodium Azide (NaN3) Sigma Aldrich 26628-22-8
Stereo fluorescence microscope Leica MZ10F
Stereoscopic fluorescence microscope equipped with a digital sight DS-U1 camera for imaging Nikon SMZ-1500
Taq DNA Polymerase New England Biolabs, MA M0273L
Tissue-Tek VIP® 6 AI Vacuum Infiltration Processor Sakura N/A Model #: VIP-6-A1
Tricaine-S Western Chemical Incorporated 20513
Xylene Thermo Fisher Scientific X3P1GAL

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