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

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.