miniCoopR Vector-based Transgenesis: A Technique to Screen Melanoma-inducing Genes in Zebrafish Tumor Model
Published: April 30, 2023
Abstract
Source: Iyengar, S. et al. Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model. J. Vis. Exp. (2012)
This video describes a method to screen for the melanoma modifying genes using a transgenic zebrafish tumor model. When injected with a melanoma-inducing miniCoopR vector, the zebrafish develops melanomas and can therefore be used to study the genetic factors affecting tumor development in zebrafish.
Protocol
All procedures involving animals have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Screening for Melanoma Onset Modifiers Create Gateway middle entry clones by PCR, amplifying the full-length open reading frame of genes of interest (GOI) and recombining into pDONR 221 using BP clonase II (Invitrogen). Use Multisite Gateway technology (Invitrogen) to recombine p5E_mitfa, pME_GOI, Tol2kit #302 p3E_SV40pol…
Representative Results
Figure 1. Screening for melanoma onset modifiers using the miniCoopR assay. A) Schematic of the miniCoopR assay. Embryo with rescued melanocytes (arrowhead) containing the miniCoopR vector and the gene of interest. Scale bar = 250 μM. Adult with greater than 4 mm2 melanocyte rescue (arrowhead). Scale bar = 500 μM B) MiniCoopR-EGFP re…
Divulgaciones
The authors have nothing to disclose.
Materials
| Gateway recombination reagents | Invitrogen | ||
| miniCoopR | |||
| Mitfa antibody | |||
| FITC goat anti-rabbit IgG antibody | Invitrogen | ||
| Vectashield | Vector Labs | H-1000 | |
| casper Zebrafish | |||
| 701N 10 μl Syringe | Hamilton/Fisher | 14-824 | |
| 40 μM filter | BD Falcon/Fisher | 352340 | |
| FBS | Invitrogen | 26140079 |
Citar este artículo
miniCoopR Vector-based Transgenesis: A Technique to Screen Melanoma-inducing Genes in Zebrafish Tumor Model. J. Vis. Exp. (Pending Publication), e20363, doi: (2023).