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

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

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1
Howard Hughes Medical Institute, Boston, Massachusetts, USA.

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.

[Indexed for MEDLINE]

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