J.A. Villefranc and Dr. Amigo contributed equally to this work.
Gateway compatible vectors for analysis of gene function in the zebrafish
Article first published online: 18 OCT 2007
Copyright © 2007 Wiley-Liss, Inc.
Volume 236, Issue 11, pages 3077–3087, November 2007
How to Cite
Villefranc, J. A., Amigo, J. and Lawson, N. D. (2007), Gateway compatible vectors for analysis of gene function in the zebrafish. Dev. Dyn., 236: 3077–3087. doi: 10.1002/dvdy.21354
- Issue published online: 18 OCT 2007
- Article first published online: 18 OCT 2007
- Manuscript Accepted: 24 AUG 2007
- National Heart, Blood, and Lung Institute. Grant Numbers: R01HL079266, F31 HL081927
- Ruth Kirchstein Minority Predoctoral Fellowship award
- Gateway cloning
The recent establishment of recombination-based cloning systems has greatly facilitated the analysis of gene function by allowing rapid and high-efficiency generation of plasmid constructs. However, the use of such an approach in zebrafish requires the availability of recombination-compatible plasmids that are appropriate for functional studies in zebrafish embryos. In this work, we describe the construction and validation of Gateway compatible vectors based on commonly used zebrafish plasmids. We have generated pCS-based plasmids that allow rapid generation of both N-terminal and C-terminal fusion proteins, and we demonstrate that mRNA synthesized from these plasmids encodes functional native or fusion proteins in injected zebrafish embryos. In parallel, we have established similar Gateway plasmids containing Tol2 cis elements that promote efficient integration into the zebrafish genome and allow expression of native or fusion proteins in a tissue-specific manner in the zebrafish embryo. Finally, we demonstrate the use of this system to rapidly identify tissue-specific cis elements to aid the establishment of blood vessel-specific transgenic constructs. Taken together, this work provides an important platform for the rapid functional analyses of open reading frames in zebrafish embryos. Developmental Dynamics 236:3077–3087, 2007. © 2007 Wiley-Liss, Inc.