The Tol2kit: A multisite gateway-based construction Kit for Tol2 transposon transgenesis constructs

Kristen M. Kwan, Esther Fujimoto, Clemens Grabher, Benjamin D. Mangum, Melissa E. Hardy, Douglas Simon Campbell, John M. Parant, H. Joseph Yost, John P. Kanki, Chi Bin Chien

研究成果: Article

882 引用 (Scopus)

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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.

元の言語English
ページ(範囲)3088-3099
ページ数12
ジャーナルDevelopmental Dynamics
236
発行部数11
DOI
出版物ステータスPublished - 2007 11 1
外部発表Yes

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ASJC Scopus subject areas

  • Developmental Biology

これを引用

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., & Chien, C. B. (2007). The Tol2kit: A multisite gateway-based construction Kit for Tol2 transposon transgenesis constructs. Developmental Dynamics, 236(11), 3088-3099. https://doi.org/10.1002/dvdy.21343