Transposition of the Tol2 element, an Ac-like element from the Japanese medaka fish Oryzias latipes, in mouse embryonic stem cells

Parts used

Key findings

• Kawakami and Noda developed a two-component Tol2 transposon system for mouse embryonic stem cells comprising the T2KPKneo donor plasmid with a nonautonomous Tol2 element (2754 bp: 2228 bp left end + 526 bp right end flanking PGK-neo cassette) and pCAGGS-T2TP helper plasmid expressing full-length transposase cDNA under the CAG promoter. This constituted the first demonstration that a natural vertebrate transposon encoding a fully functional transposase could catalyze transposition in mammalian cells.
• Mouse ES cells co-transfected with 50 μg donor plasmid and increasing amounts of transposase helper plasmid yielded G418-resistant colonies in a dose-dependent manner: 1,300 colonies (0 μg control), 2,400 (37.5 μg), 3,900 (75 μg), 6,400 (150 μg), and 14,000 colonies (300 μg). The transposase activity elevated chromosomal integration efficiency approximately 10-fold compared to nonhomologous recombination, demonstrating that transposition significantly enhanced stable genomic integration in mammalian cells without requiring endogenous repair factors.
• Inverse PCR and Southern blot analysis of 8 independent ES clones confirmed precise transposition events with characteristic 8-bp target site duplications flanking all integrated Tol2 elements. Sequencing revealed T2KPKneo insertions surrounded by mouse genomic DNA without deletions or rearrangements at integration sites, unlike nonhomologous recombination events. Single-copy integrations mapped to diverse genomic loci including gene exons and introns, establishing the Tol2 system's potential for insertional mutagenesis in mammals.

Abstract

Abstract The Tol2 transposable element of the Japanese medaka fish belongs to the hAT family of transposons including hobo of Drosophila, Ac of maize, and Tam3 of snapdragon. To date, Tol2 is the only natural transposon in vertebrates that has ever been shown to encode a fully functional transposase. It has not been known, however, whether Tol2 can transpose in vertebrates other than fish. We report here transposition of Tol2 in mouse embryonic stem (ES) cells. We constructed a transposon donor plasmid containing a nonautonomous Tol2 element with the neomycin resistance gene and a helper plasmid capable of expressing the transposase and introduced the donor plasmid with various amounts of the helper plasmid by electroporation into mouse ES cells. The number of G418-resistant ES colonies increased as the amount of helper plasmid was increased, in a dose-dependent manner, indicating that the transposase activity elevated the integration efficiency. These G418-resistant ES colonies were cloned and the structure of the junction of the integrated Tol2 element and the genomic DNA was analyzed by inverse PCR. In those clones, Tol2 was surrounded by mouse genomic sequences and an 8-bp direct repeat was created adjacent to both ends of Tol2, indicating that Tol2 was integrated in the genome through transposition. The Tol2 transposon system is thus active in mouse as well as in fish. We propose that it should be used as a genetic tool to develop novel gene transfer, transgenesis, and mutagenesis methods in mammals.