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Standard zebrafish transgenesis involves random transgene integration with resource-intensive screening. While phiC31 integrase–basedattP/attBrecombination has streamlined transgenesis in mice andDrosophila, validatedattP-based landing sites for universal applications are lacking in zebrafish. Here, we developedphiC31 Integrase Genomic Loci Engineered for Transgenesis(pIGLET) as transgenesis approach, with twoattPlanding sitespIGLET14aandpIGLET24bfrom well-validated Tol2 transgenes. Both sites facilitate diverse transgenesis applications including reporters and Cre/loxPtransgenes. ThepIGLET14aandpIGLET24blanding sites consistently yield 25 to 50% germline transmission, substantially reducing the resources needed for transgenic line generation. Transgenesis into these sites enables reproducible expression patterns in F0 zebrafish embryos for enhancer discovery and testing of gene regulatory variants. Together, our new landing sites streamline targeted, reproducible zebrafish transgenesis as a robust platform for various applications while minimizing the workload for generating transgenic lines. 

ABSTRACT Transgenesis is an essential technique for any genetic model. Tol2-based transgenesis paired with Gateway-compatible vector collections has transformed zebrafish transgenesis with an accessible modular system. Here, we establish several next-generation transgenesis tools for zebrafish and other species to expand and enhance transgenic applications. To facilitate gene regulatory element testing, we generated Gateway middle entry vectors harboring the small mouse beta-globin minimal promoter coupled to several fluorophores, CreERT2 and Gal4. To extend the color spectrum for transgenic applications, we established middle entry vectors encoding the bright, blue-fluorescent protein mCerulean and mApple as an alternative red fluorophore. We present a series of p2A peptide-based 3′ vectors with different fluorophores and subcellular localizations to co-label cells expressing proteins of interest. Finally, we established Tol2 destination vectors carrying the zebrafish exorh promoter driving different fluorophores as a pineal gland-specific transgenesis marker that is active before hatching and through adulthood. exorh-based reporters and transgenesis markers also drive specific pineal gland expression in the eye-less cavefish (Astyanax). Together, our vectors provide versatile reagents for transgenesis applications in zebrafish, cavefish and other models.