ChimericMos1andpiggyBactransposases result in site‐directed integration

Abstract

Genetic transformation systems based on Mos1 and piggyBac transposable elements are used to achieve stable chromosomal integration. However, integration sites are randomly distributed in the genome and transgene expression can be influenced by position effects. We developed a novel technology that utilizes chimeric transposases to direct integration into specific sites on a target DNA molecule. The Gal4 DNA binding domain was fused to the NH(2) terminus of the Mos1 and piggyBac transposases and a target plasmid was created that contained upstream activating sequences (UAS), to which the Gal4 DBD binds with high affinity. The transpositional activity of the Gal4-Mos1 transposase was 12.7-fold higher compared to controls where the Gal4-UAS interaction was absent and 96% of the recovered transposition products were identical, with integration occurring at the same TA site. In a parallel experiment, a Gal4-piggyBac transposase resulted in an 11.6-fold increase in transpositional activity compared to controls, with 67% of the integrations occurring at a single TTAA site. This technology has the potential to minimize nonspecific integration events that may result in insertional mutagenesis and reduced fitness. Site-directed integration will be advantageous to the manipulation of genomes, study of gene function, and for the development of gene therapy techniques.

piggyBac is a flexible and highly active transposon as compared to Sleeping Beauty , Tol2 , and Mos1 in mammalian cells

“…We have previously demonstrated that a GAL4-piggyBac transposase can direct integration to a single target site on a plasmid in mosquito embryos (27). In this study we show that piggyBac is more efficient than Tol2 and SB11 in different mammalian cell lines and is amenable to further molecular modification without reducing its activity in mammalian cells.…”

“…It appears that the most feasible way to achieve targeted transposition is to fuse a DBD recognizing a unique chromosomal sequence to the transposase (or possibly just the catalytic domain). Recently, a chimeric piggyBac transposase with GAL4 DBD attached to its N terminus resulted in targeting a specific site upstream of the UAS (GAL4-binding) site in a plasmid assay system in Aedes aegypti embryos (27). Herein we show that GAL4-piggyBac retains activity as compared with the wild type without changing its TTAA insertion preference in CHO cells.…”

“…Methods to target the transposon to a unique genomic site have been proposed (21). Theoretically, transposon site-specific integration can be achieved by increasing the DNA binding specificity of its transposase (21,27). It appears that the most feasible way to achieve targeted transposition is to fuse a DBD recognizing a unique chromosomal sequence to the transposase (or possibly just the catalytic domain).…”

“…To achieve target specificity, engineering a chimeric transposase fused with a DBD to specifically target a unique sequence was proposed (21) and later demonstrated in mosquito embryos by targeting a unique site in a plasmid (27). A transposon-based gene delivery system ideally requires a custom-engineered transposase with high integration activity and target specificity.…”

Active integration: new strategies for transgenesis

“…In the Gal4-piggyBac chimera 45/67 inserted into a single TTAA target site and 36 of them were in the 5¢-3¢ orientation. These results suggest that the Gal4-UAS limits the number of target sites at which integration can occur, likely due to the tethering of the transposase close to the UAS target (Maragathavally et al 2006). Mos1 does not appear to be functional in mammalian systems (Wu et al 2006).…”