Harnessing a High Cargo-Capacity Transposon for Genetic Applications in Vertebrates

Balciunas et al. (2006). PLoS Genetics DOI: 10.1371/journal.pgen.0020169

Key findings

The Tol2 transposon was characterized with minimal sequences (miniTol2: 261 bp 5' + 202 bp 3' sequences) that showed activity statistically indistinguishable from full-length Tol2 in zebrafish somatic assays and human cells—10-kb Tol2 transposons mediated gene transfer nearly as efficiently as 5-kb elements in HT1080 cells, demonstrating high cargo-capacity independent of size.

Tol2 lacks overexpression inhibition observed with Sleeping Beauty transposon—peak gene transfer rates were similar between systems despite Tol2's larger native size (5 kb vs 2 kb), with Tol2 maintaining activity across transposon:transposase ratios of 1:1 to 1:10 while SB activity decreased at high transposase concentrations.

Hydrodynamic injection of Tol2/FAHIL (8.5 kb) or miniTol2/FAHIL (5.8 kb) transposons corrected hereditary tyrosinemia type 1 in FAH-knockout mice—animals co-injected with transposase maintained body weight without NTBC treatment and showed 100-fold increase in liver luciferase expression over 2 months, with 36 integration sites confirmed via inverse PCR displaying 8-bp target duplications.