The Tol2kit: A multisite gateway‐based construction kit for <i>Tol2</i> transposon transgenesis constructs

TLDR

Transgenesis is a key method for studying gene function, but in zebrafish it has been hampered by complex construct assembly, low efficiency, mosaicism, and difficulty detecting germline integration. The Tol2kit employs multisite Gateway recombination to rapidly assemble promoter–coding sequence–3′ tag constructs into a Tol2 transposon backbone, providing a destination vector with a cmlc2:EGFP marker and a suite of entry clones for common promoters, fluorescent proteins, and bicistronic expression. Using the Tol2kit, zebrafish transgenesis is greatly facilitated, clone sharing is simplified, and large‑scale projects testing libraries of regulatory or coding sequences become feasible. © 2007 Wiley‑Liss, Inc.; Developmental Dynamics 236:3088–3099.

Abstract

Abstract 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.