PUBLICATION

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

Authors
Felker, A., Mosimann, C.
ID
ZDB-PUB-160725-25
Date
2016
Source
Methods in cell biology   135: 219-44 (Chapter)
Registered Authors
Felker, Anastasia, Mosimann, Christian
Keywords
Cre/lox, Genetics, Genome engineering, Lineage tracing, Recombination, Tamoxifen, Tol2, Transgenesis, Zebrafish
MeSH Terms
  • Animals
  • Animals, Genetically Modified/genetics*
  • Embryo, Nonmammalian/drug effects
  • Embryonic Development/drug effects
  • Embryonic Development/genetics*
  • Gene Transfer Techniques*
  • Integrases/genetics*
  • Recombination, Genetic
  • Tamoxifen/pharmacology
  • Transgenes/genetics
  • Zebrafish/genetics
  • Zebrafish/growth & development
PubMed
27443928 Full text @ Meth. Cell. Biol.
Abstract
Spatiotemporal transgene regulation by transgenic DNA recombinases is a central tool for reverse genetics in multicellular organisms, with excellent applications for misexpression and lineage tracing experiments. One of the most widespread technologies for this purpose is Cre recombinase-controlled lox site recombination that is attracting increasing interest in the zebrafish field. Tol2-mediated zebrafish transgenesis provides a stable platform to integrate lox cassette transgenes, while the amenability of the zebrafish embryo to drug treatments makes the model an ideal candidate for tamoxifen-inducible CreERT2 experiments. In addition, advanced transgenesis technologies such as phiC31 or CRISPR-Cas9-based knock-ins are even further promoting zebrafish transgenesis for Cre/lox applications. In this chapter, we will first introduce the basics of Cre/lox methodology, CreERT2 regulation by tamoxifen, as well as the utility of Tol2 and other contemporary transgenesis techniques for Cre/lox experiments. We will then outline in detail practical experimental steps for efficient transgenesis toward the creation of single-insertion transgenes and will introduce protocols for 4-hydroxytamoxifen-mediated CreERT2 induction to perform spatiotemporal lox transgene regulation experiments in zebrafish embryos. Last, we will discuss advanced experimental applications of Cre/lox beyond traditional lineage tracing approaches.
Genes / Markers
Figures
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping