Tol2 Transposon-Mediated Zebrafish Transgenesis: A Procedure to Generate Transgenic Zebrafish Following the Coinjection of Tol2 System Into Fertilized Zebrafish Embryos

Published: April 30, 2023

Abstract

Source: Silic, M. R., et al. Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors. J. Vis. Exp. (2018)

In this video, we demonstrate a procedure to generate a stable zebrafish transgenic line constitutively expressing a fluorescent voltage indicator protein following the co-injection of the Tol2 transposon system comprising the transposase mRNA and Tol2 transposon construct encoding the fluorescent voltage indicator.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Microinjection Set up 4-6 breeding tanks with at least 2 males and 2 females the afternoon before injection. These fish must not be fed in the afternoon. This step will reduce the amount of fish waste and save time to clean them out the next morning, while also helping to induce a breeding response. The follow…

Divulgazioni

The authors have nothing to disclose.

Materials

Agarose RA  Amresco  N605-500G  For making the injection gels
Bright field dissection scope   Nikon SMZ 745  Dechorionation, microinjection, mounting
Disposable transfer pipette 3.4 ml  Thermo Scientific  13-711-9AM  Fish embryos and water transfer
Forceps #5  WPI  500342 Dechorionation and needle breaking
Loading tip  Eppendorf  930001007 For loading injection solution into capilary needles
Microinjection mold Adaptive Science Tools  TU-1  To prepare agaorse mold tray for holding fish embryos during injection
Microinjector  WPI  Pneumatic Picopump PV820  Microinjection injector
Micro-manipulator  WPI  Microinjector MM3301R  Microinjection operation
Micropipette puller  Sutter instrument  P-1000  For preparing capillary needle
Mineral oil  Amresco  J217-500ml  For calibrating injection volume
Plastic Petri dishes VWR  25384-088  For holding fish or fish embryos during imaging process
Stage Micrometer  Am Scope  MR100  Microinjection volume calibration
Thin wall glass capillaries  WPI  TW100F-4  Raw glass for making cappilary needle

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Citazione di questo articolo
Tol2 Transposon-Mediated Zebrafish Transgenesis: A Procedure to Generate Transgenic Zebrafish Following the Coinjection of Tol2 System Into Fertilized Zebrafish Embryos. J. Vis. Exp. (Pending Publication), e20982, doi: (2023).

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