Tol2: a versatile gene transfer vector in vertebrates
Vertebrate DNA transposons were mostly non-autonomous, with no natural transposase usable as a vector. This review describes Tol2, an autonomously active medaka transposon validated for transgenesis in fish, chicken, mouse, and human cells. It carries 11-kb inserts.
Parts used
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.
Abstract
The medaka fish Tol2 element is an autonomous transposon that encodes a fully functional transposase. The transposase protein can catalyze transposition of a transposon construct that has 200 and 150 base pairs of DNA from the left and right ends of the Tol2 sequence, respectively. These sequences contain essential terminal inverted repeats and subterminal sequences. DNA inserts of fairly large sizes (as large as 11 kilobases) can be cloned between these sequences without reducing transpositional activity. The Tol2 transposon system has been shown to be active in all vertebrate cells tested thus far, including zebrafish, Xenopus, chicken, mouse, and human. In this review I describe and discuss how the Tol2 transposon is being applied to transgenic studies in these vertebrates, and possible future applications.