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

Parts used

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.

Abstract

The Tol2 element of the medaka fish Oryzias latipes belongs to the hAT family of transposons (hobo/Ac/Tam3). We report here identification of a functional transposase of Tol2 that is capable of catalyzing its transposition in the germ line of zebrafish Danio rerio. A transcript produced from Tol2 encodes a putative transposase. Zebrafish fertilized eggs were coinjected with mRNA transcribed in vitro, using cDNA of the Tol2 transcript as a template and a plasmid DNA harboring a mutant Tol2, which had a deletion in the putative transposase gene but retained necessary cis sequences. The injected fish were raised to adulthood and mated to noninjected fish, and genomic DNA of the progeny fish were analyzed by PCR and Southern hybridization. Half of F(1) fish obtained from one of eight injected fish contained the Tol2 DNA in their genomes but not the vector portion. Among these F(1) fish, Tol2 insertions at four different loci were identified, and some F(1) fish carried two or three different Tol2 insertions, indicating that the germ line of the founder fish is highly mosaic. Sequencing analyses revealed that, in all cases, Tol2 was surrounded by zebrafish genomic sequences, and an 8-bp duplication was created at the target site, indicating that Tol2 was integrated in the zebrafish genome through transposition. This study identifies an autonomous member of a DNA-based transposable element from a vertebrate genome. The Tol2 transposon system should thus be used to develop novel transgenesis and insertional mutagenesis methods in zebrafish and possibly in other fishes.