Advanced zebrafish transgenesis with Tol2 and application for Cre/lox recombination experiments

Methods Cell Biol. 2011;104:173-94. doi: 10.1016/B978-0-12-374814-0.00010-0.

Abstract

Spatio-temporal transgene regulation by transgenic DNA recombinases is a central tool for genetic research in multicellular organisms, with excellent applications for misexpression and lineage tracing experiments. Cre recombinase-controlled lox site recombination is a cornerstone of contemporary mouse genetics, and Cre/lox techniques therefore attract increasing interest in the zebrafish field. Tol2-mediated zebrafish transgenesis now provides a stable platform for lox cassette transgenes, while the ease of drug treatments in zebrafish makes the model an ideal candidate for Tamoxifen/4-hydroxytamoxifen-inducible CreER(T2) experiments. In this chapter, we will first introduce the basics of Cre/lox methodology, CreER(T2) regulation by Tamoxifen/4-hydroxytamoxifen, as well as the benefits of Tol2 transgenesis for Cre/lox experiments. We will then in detail outline practical experimental steps for Tol2 transgenesis toward the creation of single-insertion transgenes. Lastly, we will introduce protocols for 4-hydroxytamoxifen-mediated CreER(T2) induction to perform spatio-temporal lox transgene regulation experiments in zebrafish embryos.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Genetically Modified*
  • Cloning, Molecular / methods
  • DNA Transposable Elements*
  • Female
  • Genetic Markers
  • Genetic Testing / methods
  • Genetic Vectors / isolation & purification
  • Integrases / genetics
  • Integrases / metabolism
  • Male
  • Microinjections / methods
  • Mutagenesis, Insertional / methods*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Recombination, Genetic*
  • Viral Proteins / genetics
  • Viral Proteins / metabolism
  • Zebrafish / genetics*

Substances

  • DNA Transposable Elements
  • Genetic Markers
  • Recombinant Proteins
  • Viral Proteins
  • Cre recombinase
  • Integrases