Highly efficient transgenesis mediated by Tip100 transposon system in medaka.

Abstract

Transgenesis mediated by transposon is an effective approach for introducing exogenous DNA into the nuclear genome and establishing stable transgenic strains that efficiently express genetic tools. Although the DNA transposon Tol2 is widely used for transgenesis in zebrafish, its endogenous transpositional activity can lead to unintended transgene mobilization, making it unsuitable for transgenesis in medaka (Oryzias latipes). Here, we demonstrated that the DNA transposon Tip100, originally identified in the common morning glory (Ipomoea purpurea), an ornamental plant, can serve as a useful tool for transgenesis in Japanese medaka. The GFP transgene cassette, when co-injected with Tip100 transposase mRNA, was expressed in significantly higher number of somatic cells in the injected fish. Furthermore, a transgene flanked by truncated recognition sequences (100 bp each) exhibited expression levels comparable to those of the original vector containing the full 2.2 kb recognition sequence. Injection of a transgene driven by a germline-specific promoter revealed that fish injected with Tip100 mRNA exhibited a significantly higher germline transmission rate (42/68; 62.7%) compared to those injected without the mRNA (13/62; 21.0%). We successfully established transgenic strains by outcrossing injected founders with GFP-positive germ cells (7/7; 100%) and demonstrated that the transgenes were randomly integrated into the medaka genome, generating 8-bp duplications at the insertional sites-an insertional signature of the hAT superfamily of transposons. Our findings indicate that the Tip100 system is a promising tool for generating stable transgenic strains that express various genetic tools in medaka and potentially other fish species.