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The Tol2kit

Tol2kit v1.2, released November 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.

• 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 Kwan lab.

• List of entry and destination clones
• Protocols

If you have questions or comments about the Tol2kit, please contact Kristen Kwan. If you construct other entry or destination clones, we would love to post information about them here.

Clone Distribution

We are freely distributing all of the plasmids described here. For reasons of convenience, we usually distribute them as a complete set, spotted as DNA onto filter paper. If you have an older version (e.g. v1.0), we can send you an "upgrade kit" to the latest version. Since the destination vectors and transposase plasmid are all derived from Dr. Koichi Kawakami's original constructs, we ask that you please first contact Dr. Kawakami and execute an MTA for use of the Tol2 transposase construct and plasmids based on Tol2.

We are also happy for labs to share these plasmids or pass them on to others, as long as you pass on the address of this wiki, and the receiving lab has executed a Tol2 MTA with Dr. Kawakami. We especially encourage different labs at the same institution to share plasmids. This reduces our workload (generating maxipreps and spotting plasmid) as well as yours (growing up clones).

To request the Tol2kit, please send an email to Kristen Kwan.

Citing the Tol2kit

A methods paper describing the Tol2kit has been published in Developmental Dynamics.

Here is a link to the paper and the PDF. Please cite this paper in manuscripts using the Tol2kit.

Links

• Tol2kit blog for discussion and questions about the system

• Lawson lab Gateway site

• Kawakami lab site

• Invitrogen multisite Gateway manual Version D 3/7/07