A putative hyperacitve piggybac transposase in Dipteran systems

Transposons are mobile genetic elements that can integrate themselves as well as genetic cargo into host genomes. Currently the use of transposons is limited by low germ-line transformation frequencies and unpredictable integration sites. Previous studies have shown that increased transposition frequency of DNA transposons can be achieved through the mutagenesis of transposases. However, these studies failed to provide transposases with increased germ-line transformation rates in insects and thus these hyperactive mutants have had little application in subsequent genome manipulation. Aedes aegypti carries a high transposon load with 47% of the genome being composed of transposable elements and transposons introduced into it cannot be remobilized. In this study we are examining mutated transposable elements to test for increased or altered transposition frequency in order to create transposable elements that are more efficient gene vectors. One candidate hyperactive piggyBac transposase PB7 was selected after proving hyperactive in high throughput yeast assays. Transposition assays were performed in Drosophila melanogaster embryos and demonstrated a slightly higher transposition frequency than wild type piggyBac transposase. When PB7 was used in transformation assays however the frequency was greatly decreased with a significant increase in sterility from 6.8% to 38.2% and increased rates of sterility have been linked to hyperactivity. Transposition assays in Aedes aegypti embryos were performed and showed and increased transposition rate from 7.41x 10-5 to 1.35 x 10-4. We are now determining if there is any indication of germ-line activity of PB7 in Ae. aegypti.