Improvement of Tol2 transposon for efficient large-cargo capacity transgene applications in culture cells and zebrafish

Most viruses and transposons serve as effective carriers for introducing < 11 kilobases (kb) of foreign DNA into vertebrate genomes, but its activity diminishes notably in the DNA payload larger than 11 kb. Expanding the payload capacity of transposons would enable the accommodation of more complex cargo designs, enhancing the ability to regulate expression and minimize mutagenic risks associated with these molecular therapeutics, metabolic engineering, and transgenic animals. In this study, we improved Tol2 transposon by increasing the protein expression level with a translational enhancer (QBI SP163, ST) and enhancing nucleus targeting ability with a nucleus localization protein H2B (SHT), respectively. Tol2 and the ST transposon efficiently integrated large DNA cargos into human cell cultures (H1299), comparable to the well-established super PiggyBac system. We characterized that the ST and SHT mRNA show significantly increased transgene efficiency of large DNA payloads (~8 kb, ~14 kb, ~24 kb) into zebrafish. This study introduces modified Tol2 transposon as an improved nonviral vector for delivering large DNA payloads in transgenic applications.