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

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.