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''Tol2kit v1.0, released January 2007'' | ''Tol2kit v1.0, released January 2007'' | ||
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| + | Generating stable transgenic zebrafish lines has historically been laborious, for three reasons. (1) Building transgenesis constructs by conventional subcloning can be difficult, especially when using long coding sequences, or using large genomic fragments to drive tissue-specific expression. (2) Injecting plasmid DNA yields mosaic transient expression and low germline transgenesis rates. (3) When screening for stable integrations, transgenes that express fluorescent proteins (e.g. GFP) are easy to detect, but other transgenes are not. | ||
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| + | 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. | ||
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| + | 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 == | == Links == | ||
Revision as of 17:21, 30 January 2007
Welcome to the Tol2kit documentation site
Tol2kit v1.0, released January 2007
Generating stable transgenic zebrafish lines has historically been laborious, for three reasons. (1) Building transgenesis constructs by conventional subcloning can be difficult, especially when using long coding sequences, or using large genomic fragments to drive tissue-specific expression. (2) Injecting plasmid DNA yields mosaic transient expression and low germline transgenesis rates. (3) 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.