Establishment and application of a low-leakage Tet-On inducible overexpression system based on the PiggyBac transposition system

Abstract:

In order to avoid the leakage expression of the Tet-on inducible expression system and to achieve easy, stable and efficient expression of exogenous gene when induced at a specific time, this study planned to add the destabilizing domain FK506-binding protein, regulated by the stable ligand Shield-1, to the N-terminal of the target gene to control its background expression at the protein level. To verify the performance of this system, the fluorescent protein TdTomato was used as the reporter gene in this study. The results of flow analysis showed that the leakage expression in the optimized system was reduced about 7 times compared with the original one at the protein level. This system was applied to the in vitro odontogenic induction system based on mouse embryonic stem cells. Under the synergistic effect of the inducible factor Dox and the stable ligand Shield-1, the induced overexpression of tooth development-related transcription factor Hand2 could improve the completion of odontogenic induction.