Tol2: a versatile gene transfer vector in vertebrates

Key findings

Kawakami characterized the minimal Tol2 cis-sequences required for transposition, demonstrating that constructs containing 200 bp and 150 bp from left and right ends respectively were sufficient for activity. These sequences include 12 bp terminal inverted repeats and subterminal regions essential for transposase recognition. The Tol2 vector maintained full transpositional activity with DNA inserts as large as 11 kb, substantially exceeding Tc1/mariner-type transposon size limitations.

The Tol2 transposon system achieved germline transmission in 50-70% of injected zebrafish at frequencies ranging from 3% to 100% per founder, representing 10-14× improvement over plasmid microinjection (5% transmission). Microinjection of transposon-donor plasmid with synthetic transposase mRNA enabled stable genomic integration during early embryogenesis. Transgenic F1 offspring demonstrated persistent multi-generational transgene expression without gene silencing, validated across multiple regulatory elements including EF1α, Fugu tyrp1, askopos, and heat-inducible Gal4/UAS systems.

Tol2 transposition was demonstrated to be active in all tested vertebrate cell types, including zebrafish, Xenopus, chicken, mouse, and human cells. The autonomous Tol2 element (4.7 kb) encodes a fully functional 649 amino acid transposase that catalyzes cut-and-paste transposition with 8 bp target site duplications. This broad vertebrate compatibility established Tol2 as a versatile gene transfer vector across diverse model organisms and therapeutic applications.