Identification of a functional transposase of the Tol2 element, an Ac-like element from the Japanese medaka fish, and its transposition in the zebrafish germ lineage

Key findings

Kawakami et al. identified the first functional transposase from a vertebrate genome by demonstrating Tol2 transposition in zebrafish germline. Zebrafish eggs were co-injected with in vitro transcribed Tol2 transposase mRNA and a nonautonomous Tol2-tyr plasmid carrying a 1.5 kb transposase deletion. Eight founder fish were raised to adulthood and mated with non-injected fish to generate F1 progeny for genomic analysis.

Germline transposition was confirmed in F1 offspring from one of eight injected founders, with 50% of F1 fish containing integrated Tol2 DNA without vector sequences. PCR and Southern hybridization identified four distinct genomic integration loci among F1 progeny. Individual F1 fish carried two or three different Tol2 insertions, demonstrating highly mosaic germline incorporation during embryogenesis in the founder fish.

Sequencing analysis revealed that all integrated Tol2 elements were flanked by zebrafish genomic sequences with characteristic 8-bp target site duplications at integration sites. This duplication signature confirmed authentic cut-and-paste transposition rather than illegitimate recombination. The study established Tol2 as the first autonomous DNA transposon identified from vertebrates.