The Tol2kit: A Multisite Gateway-Based Construction Kit for Tol2 Transposon Transgenesis Constructs

Dev Dyn. 2007 Nov;236(11):3088-99. doi: 10.1002/dvdy.21343.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Genetically Modified*
  • Cloning, Molecular / methods*
  • DNA Transposable Elements*
  • DNA, Recombinant / genetics*
  • Gene Transfer Techniques*
  • Genetic Techniques
  • Genetic Vectors
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Plasmids / genetics
  • Recombination, Genetic
  • Transposases / metabolism
  • Zebrafish / genetics*
  • Zebrafish / metabolism

Substances

  • DNA Transposable Elements
  • DNA, Recombinant
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • Transposases