Contents
The Tol2kit
Tol2kit v1.0, released January 2007
Generating stable transgenic zebrafish lines has historically been laborious, for three reasons:
- Building transgenesis constructs by conventional subcloning can be difficult, especially when using long coding or regulatory sequences
- Injecting plasmid DNA yields mosaic transient expression and low germline transgenesis rates
- When screening for stable integrations, transgenes that express fluorescent proteins (e.g. GFP) are easy to detect, but other transgenes are not.
The Tol2kit uses site-specific recombination-based cloning (multisite Gateway technology) to allow quick, modular assembly of promoter-coding sequence-3' tag constructs in a Tol2 transposon backbone.
It includes a variety of widely-useful entry clones, including hsp70 and beta-actin promoters; cytoplasmic, nuclear, and membrane-localized fluorescent proteins; and IRES-driven GFP cassettes for bicistronic expression. It includes two Tol2-based destination vector, one with a cmlc2:egfp transgenesis marker.
About this site
This wiki provides the documentation for the Tol2kit; it is maintained by the Chien lab.
- Basic Gateway principles
- List of entry and destination vectors
- Protocols
- Components used for the Tol2kit
- Sample results with the Tol2kit
- References
We are currently preparing a methods paper describing the Tol2kit.
Links
- Tol2kit blog for discussion and questions about the system
- Lawson lab Gateway site
- Invitrogen multisite Gateway manual 11/29/04
Wiki help
Consult the User's Guide for information on using the wiki software.