Discussing zebrafish genetic methods

This blog was originally set up to help plan a workshop held at the 2007 SCZI. During this workshop, it was clear that while there are some powerful new advances in genetic methods for the zebrafish, there are important questions about some existing methods, and several methods (notably homologous recombination and RNAi) which would be great for the field, but do not yet exist.

We've left this blog up as a forum in which people can discuss some of these methods. Below are several blog entries describing particular methods. Please add questions and comments under the appropriate heading.

--Chi-Bin Chien, Koichi Kawakami, Todd Evans, Hazel Sive

Monday, January 22, 2007

Gateway-based systems for building expression vectors

Building complex expression vectors with components such as large regulatory elements, fluorescent protein fusions, transgenesis markers, or bicistronic (IRES) markers can be extremely time-consuming. The labs of Nathan Lawson and Chi-Bin Chien have both built systems using multisite Gateway technology, which allows quick construction of expression vectors in a Tol2 backbone. Given Gateway-based "ORFeome" libraries, it should also become possible to do large-scale misexpression screens.

We have already built many entry vectors that allow introduction of ubiquitous promoters (beta-actin, hsp70, CMV/SP6), fluorescent protein markers (EGFP, mCherry, and nuclear and membrane-tagged versions of both), as well as N- and C-terminal fusions, and several IRES tags. Nathan and Chi-Bin made a short presentation on these constructs, and distributed clones at the meeting.

For documentation and more information, see:

http://chien.neuro.utah.edu/tol2kitwiki

or
http://lawsonlab.umassmed.edu

We also know that Jochen Wittbrodt's lab has built a similar system using I-SceI vectors, while Shannon Fisher and Michael Nonet have built systems using "classic" [not multisite] Gateway.
Posted by at

No comments:

Post a Comment

Subscribe to: Post Comments (Atom)