ZFIN ID: ZDB-PUB-110921-12
Advanced Zebrafish Transgenesis with Tol2 and Application for Cre/lox Recombination Experiments
Mosimann, C., and Zon, L.I.
Date: 2011
Source: Methods in cell biology   104: 173-194 (Chapter)
Registered Authors: Mosimann, Christian, Zon, Leonard I.
Keywords: Tol2, trangenesis, transgenes, Cre, CreERT2, IoxP, lineage tracing, tamoxifen, 4-OHT
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*
PubMed: 21924163 Full text @ Meth. Cell. Biol.
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.