title: Contemporary zebrafish transgenesis with Tol2 and application for Cre/lox recombination experiments.
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.
authors.affiliation: University of Zürich, Zürich, Switzerland.
authors.forename: A
authors.initials: A
authors.lastname: Felker
authors.affiliation: University of Zürich, Zürich, Switzerland.
authors.forename: C
authors.initials: C
authors.lastname: Mosimann
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics*; D004625:Embryo, Nonmammalian / Q000187:drug effects; D047108:Embryonic Development / Q000187:drug effects / Q000235:genetics*; D018014:Gene Transfer Techniques; D019426:Integrases / Q000235:genetics*; D011995:Recombination, Genetic; D013629:Tamoxifen / Q000494:pharmacology; D019076:Transgenes / Q000235:genetics; D015027:Zebrafish / Q000235:genetics / Q000254:growth & development
chemical_list: D013629:Tamoxifen; C045073:Cre recombinase; D019426:Integrases
keywords: Cre/lox; Genetics; Genome engineering; Lineage tracing; Recombination; Tamoxifen; Tol2; Transgenesis; Zebrafish