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 |
Tags
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).