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Revision as of 17:23, 30 January 2007

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.

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.

• Configuration settings list
• MediaWiki FAQ