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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\u2032 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.<\/jats:p>","DOI":"10.1242\/dev.201531","type":"journal-article","created":{"date-parts":[[2023,3,28]],"date-time":"2023-03-28T09:01:01Z","timestamp":1679994061000},"source":"Crossref","is-referenced-by-count":38,"title":["Next-generation plasmids for transgenesis in zebrafish and beyond"],"prefix":"10.1242","volume":"150","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4516-029X","authenticated-orcid":false,"given":"Cassie L.","family":"Kemmler","sequence":"first","affiliation":[{"name":"University of Colorado, School of Medicine, Anschutz Medical Campus 1 , Department of Pediatrics, Section of Developmental Biology, 12801 E 17th Avenue, Aurora, CO 80045 , USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8056-5762","authenticated-orcid":false,"given":"Hannah R.","family":"Moran","sequence":"additional","affiliation":[{"name":"University of Colorado, School of Medicine, Anschutz Medical Campus 1 , Department of Pediatrics, Section of Developmental Biology, 12801 E 17th Avenue, Aurora, CO 80045 , USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1351-3572","authenticated-orcid":false,"given":"Brooke F.","family":"Murray","sequence":"additional","affiliation":[{"name":"University of Utah 2 Department of Human Genetics , , Salt Lake City, UT 84112 , USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aaron","family":"Scoresby","sequence":"additional","affiliation":[{"name":"University of Utah 2 Department of Human Genetics , , Salt Lake City, UT 84112 , USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John R.","family":"Klem","sequence":"additional","affiliation":[{"name":"University of Louisville School of Medicine 3 Department of Biochemistry and Molecular 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Campus 1 , Department of Pediatrics, Section of Developmental Biology, 12801 E 17th Avenue, Aurora, CO 80045 , USA"},{"name":"University of Zurich 5 Department of Molecular Life Sciences , , 8057 Z\u00fcrich , Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sibylle","family":"Burger","sequence":"additional","affiliation":[{"name":"University of Zurich 5 Department of Molecular Life Sciences , , 8057 Z\u00fcrich , Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1021-0015","authenticated-orcid":false,"given":"Gianluca","family":"D'Agati","sequence":"additional","affiliation":[{"name":"University of Zurich 5 Department of Molecular Life Sciences , , 8057 Z\u00fcrich , Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9837-6916","authenticated-orcid":false,"given":"Charles","family":"Betz","sequence":"additional","affiliation":[{"name":"University of Basel 6 Growth & Development, Biozentrum, Spitalstrasse 41 , , 4056 Basel , Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ann-Christin","family":"Puller","sequence":"additional","affiliation":[{"name":"University of Zurich 5 Department of Molecular Life Sciences , , 8057 Z\u00fcrich , Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3734-2026","authenticated-orcid":false,"given":"Anastasia","family":"Felker","sequence":"additional","affiliation":[{"name":"University of Zurich 5 Department of Molecular Life Sciences , , 8057 Z\u00fcrich , Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2209-2941","authenticated-orcid":false,"given":"Karolina","family":"Ditrychova","sequence":"additional","affiliation":[{"name":"University of Zurich 5 Department of Molecular Life Sciences , , 8057 Z\u00fcrich , Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Seraina","family":"B\u00f6tschi","sequence":"additional","affiliation":[{"name":"University of Zurich 5 Department of Molecular Life Sciences , , 8057 Z\u00fcrich , Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5171-0016","authenticated-orcid":false,"given":"Markus","family":"Affolter","sequence":"additional","affiliation":[{"name":"University of Basel 6 Growth & Development, Biozentrum, Spitalstrasse 41 , , 4056 Basel , Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3248-2772","authenticated-orcid":false,"given":"Nicolas","family":"Rohner","sequence":"additional","affiliation":[{"name":"Stowers Institute for Medical Research 4 , Kansas City, MO 64110 , USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7838-5823","authenticated-orcid":false,"given":"C. Ben","family":"Lovely","sequence":"additional","affiliation":[{"name":"University of Louisville School of Medicine 3 Department of Biochemistry and Molecular Genetics , , Louisville, KY 40202 , USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0052-275X","authenticated-orcid":false,"given":"Kristen M.","family":"Kwan","sequence":"additional","affiliation":[{"name":"University of Utah 2 Department of Human Genetics , , Salt Lake City, UT 84112 , USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7137-3910","authenticated-orcid":false,"given":"Alexa","family":"Burger","sequence":"additional","affiliation":[{"name":"University of Colorado, School of Medicine, Anschutz Medical Campus 1 , Department of Pediatrics, Section of Developmental Biology, 12801 E 17th Avenue, Aurora, CO 80045 , USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0749-2576","authenticated-orcid":false,"given":"Christian","family":"Mosimann","sequence":"additional","affiliation":[{"name":"University of Colorado, School of Medicine, Anschutz Medical Campus 1 , Department of Pediatrics, Section of Developmental Biology, 12801 E 17th Avenue, Aurora, CO 80045 , USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"237","published-online":{"date-parts":[[2023,4,19]]},"reference":[{"key":"2023041912194295600_DEV201531C1","doi-asserted-by":"publisher","first-page":"191","DOI":"10.1016\/j.ydbio.2011.01.002","article-title":"Transgenerational analysis of transcriptional silencing in zebrafish","volume":"352","author":"Akitake","year":"2011","journal-title":"Dev. 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