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

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

1094 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)3088-3099
Number of pages12
JournalDevelopmental Dynamics
Volume236
Issue number11
DOIs
Publication statusPublished - 2007 Nov 1
Externally publishedYes

Keywords

  • Bicistronic
  • EGFP
  • EMCV IRES
  • mCherry
  • Nuclear localization signal
  • Transposon

ASJC Scopus subject areas

  • Developmental Biology

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