| Line 8: | Line 8: | ||
* When screening for stable integrations, transgenes that express fluorescent proteins (e.g. GFP) are easy to detect, but other transgenes are not. | * 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. | + | The Tol2kit uses site-specific recombination-based cloning ([[Basic Gateway principles|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 | + | It includes a variety of widely-useful [[List of entry and destination vectors|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 [[List of entry and destination vectors|destination vectors]], one with a ''cmlc2:egfp'' transgenesis marker. |
== About this site == | == About this site == | ||
| Line 24: | Line 24: | ||
We are currently preparing a methods paper describing the Tol2kit. If you have questions or comments, please post them on the | We are currently preparing a methods paper describing the Tol2kit. If you have questions or comments, please post them on the | ||
| − | [http://tol2kit.blogspot.com Tol2kit blog]. | + | [http://tol2kit.blogspot.com Tol2kit blog]. If you construct other entry or destination clones, we would love to post information about them here. |
== Links == | == Links == | ||
Revision as of 13:11, 2 February 2007
Contents
The Tol2kit
Tol2kit v1.0, released February 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 vectors, 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. Please bear with us; it went live on 28 January and there are still many sections that are under construction.
- 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. If you have questions or comments, please post them on the Tol2kit blog. If you construct other entry or destination clones, we would love to post information about them here.
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.