title: Advanced zebrafish transgenesis with Tol2 and application for Cre/lox recombination experiments.
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.
authors.affiliation: Howard Hughes Medical Institute, Boston, Massachusetts, USA.
authors.forename: Christian
authors.initials: C
authors.lastname: Mosimann
authors.forename: Leonard I
authors.initials: LI
authors.lastname: Zon
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D003001:Cloning, Molecular / Q000379:methods; D004251:DNA Transposable Elements; D005260:Female; D005819:Genetic Markers; D005820:Genetic Testing / Q000379:methods; D005822:Genetic Vectors / Q000302:isolation & purification; D019426:Integrases / Q000235:genetics / Q000378:metabolism; D008297:Male; D008845:Microinjections / Q000379:methods; D016254:Mutagenesis, Insertional / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D015027:Zebrafish / Q000235:genetics*
chemical_list: D004251:DNA Transposable Elements; D005819:Genetic Markers; D011994:Recombinant Proteins; D014764:Viral Proteins; C045073:Cre recombinase; D019426:Integrases