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The Tol2kit: A multisite gateway‐based construction kit for Tol2 transposon transgenesis constructs
Author(s) -
Kwan Kristen M.,
Fujimoto Esther,
Grabher Clemens,
Mangum Benjamin D.,
Hardy Melissa E.,
Campbell Douglas S.,
Parant John M.,
Yost H. Joseph,
Kanki John P.,
Chien ChiBin
Publication year - 2007
Publication title -
developmental dynamics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.634
H-Index - 141
eISSN - 1097-0177
pISSN - 1058-8388
DOI - 10.1002/dvdy.21343
Subject(s) - transgenesis , biology , mcherry , subcloning , green fluorescent protein , genetics , transposable element , transgene , computational biology , microbiology and biotechnology , gene , plasmid , genome , reproductive technology , embryogenesis
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.