GSBS Student Publications

Student Author(s)

Sharanya Iyengar

GSBS Program

Cancer Biology

UMMS Affiliation

Program in Molecular Medicine; Department of Cancer Biology

Date

11-13-2012

Document Type

Article

Disciplines

Cancer Biology | Cell Biology | Molecular Biology | Research Methods in Life Sciences

Abstract

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors. 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 model 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 recombined. 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:BRAF(V600E));p53(lf);mitfa(lf) zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis 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.

Rights and Permissions

Copyright © 2012 Journal of Visualized Experiments. Publisher PDF posted as allowed by the publisher's author rights policy at https://www.jove.com/files/Author_License_Agreement.pdf.

DOI of Published Version

10.3791/50086

Source

J Vis Exp. 2012 Nov 13;(69):e50086. doi: 10.3791/50086. Link to article on publisher's site

Related Resources

Link to Article in PubMed

Keywords

Cancer Biology, Issue 69, Medicine, Genetics, Molecular Biology, Melanoma, zebrafish, Danio rerio, mitfa, melanocytes, tumor model, miniCoopR

Journal Title

Journal of visualized experiments : JoVE

PubMed ID

23183931

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