We used the Tc1/mariner family transposable element Sleeping Beauty (SB) for transgenesis and long-term expression studies in the zebrafish (Danio rerio), a popular organism for clinical disease, vertebrate patterning, and cell biology applications. SB transposase enhanced the transgenesis and expression rate sixfold (from 5 to 31%) and more than doubled the total number of tagged chromosomes over standard, plasmid injection-based transgenesis methods. Molecular analysis of these loci demonstrated a precise integration of these elements into recipient chromosomes with genetic footprints diagnostic of transposition. GFP expression from transposase-mediated integrants was Mendelian through the eighth generation. A blue-shifted GFP variant (BFP) and a red fluorescent protein (DsRed) were also useful transgenesis markers, indicating that multiple reporters are practical for use with SB in zebrafish. We showed that SB is suitable for tissue-specific transgene applications using an abbreviated gamma-crystallin GFP cassette. Finally, we describe a general utility transposon vector for chromosomal engineering and molecular genetics experiments in zebrafish. Together, these data indicate that SB is an efficient tool for transgenesis and expression in zebrafish, and that the transposon will be useful for gene expression in cell biology applications as well as an insertional mutagen for gene discovery during development.
Bibliographical noteFunding Information:
We thank David Largaespada, Scott McIvor, Adam Dupuy, and Karl Clark for helpful discussions throughout the course of this work. For technical assistance, we give special thanks to Erin Walsh, Cheryl Saunders, Ken Finley, Daiva Urneziene, and Rachel Dries. We also thank Eleanor Chen and David Largaespada for comments on the manuscript. This work has been supported by the Arnold and Mabel Beckman Foundation and NIH Grants DA14546 and RRO6625.
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- Insertional mutagenesis
- Sleeping Beauty
- Transgene expression