The Tol2kit: A multisite gateway-based construction kit for Tol2 transposon transgenesis constructs
Article first published online: 15 OCT 2007
Copyright © 2007 Wiley-Liss, Inc.
Volume 236, Issue 11, pages 3088–3099, November 2007
How to Cite
Kwan, K. M., Fujimoto, E., Grabher, C., Mangum, B. D., Hardy, M. E., Campbell, D. S., Parant, J. M., Yost, H. J., Kanki, J. P. and Chien, C.-B. (2007), The Tol2kit: A multisite gateway-based construction kit for Tol2 transposon transgenesis constructs. Dev. Dyn., 236: 3088–3099. doi: 10.1002/dvdy.21343
- Issue published online: 18 OCT 2007
- Article first published online: 15 OCT 2007
- Manuscript Accepted: 28 AUG 2007
- National Eye Institute. Grant Number: RO1 EY12873
- Dana Foundation
- NIH. Grant Numbers: 5T32 HD07491, 2P01 CA68484-11A1, RO1 HL66292
- American Cancer Society. Grant Number: PF-06-146-01-DDC
- EMCV IRES;
- nuclear localization signal
Transgenesis is an important tool for assessing gene function. In zebrafish, transgenesis has suffered from three problems: the labor of building complex expression constructs using conventional subcloning; low transgenesis efficiency, leading to mosaicism in transient transgenics and infrequent germline incorporation; and difficulty in identifying germline integrations unless using a fluorescent marker transgene. The Tol2kit system 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 destination vector with a cmlc2:EGFP (enhanced green fluorescent protein) transgenesis marker and a variety of widely useful entry clones, including hsp70 and beta-actin promoters; cytoplasmic, nuclear, and membrane-localized fluorescent proteins; and internal ribosome entry sequence–driven EGFP cassettes for bicistronic expression. The Tol2kit greatly facilitates zebrafish transgenesis, simplifies the sharing of clones, and enables large-scale projects testing the functions of libraries of regulatory or coding sequences. Developmental Dynamics 236:3088–3099, 2007. © 2007 Wiley-Liss, Inc.