基于PiggyBac转座系统的低溢漏诱导型Tet-On过表达体系的建立及应用  

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

作　　者：张柏权 李中瀚 殷旖珂 ZHANG Bai-Quan;LI Zhong-Han;YIN Yi-Ke(College of Life Sciences,Sichuan University,Chengdu 610064)

机构地区：[1]四川大学生命科学学院,成都610064

出　　处：《四川大学学报（自然科学版）》2023年第3期165-172,共8页Journal of Sichuan University(Natural Science Edition)

基　　金：国家自然科学基金青年基金(31900900)。

摘　　要：为避免诱导基因稳定表达的Tet-On诱导表达系统溢漏表达,实现简便且高效的外源基因稳定诱导表达,本研究拟在Tet-On调控的转录水平基础上,将基于稳定配体Shield-1的不稳定结构域FK506结合蛋白引入目的基因的N端,从蛋白水平控制其本底表达水平.为验证该系统的效果,本研究以荧光蛋白TdTomato为报告基因,经流式分析结果证明优化后的体系较原体系的溢漏表达在蛋白水平上降低7倍左右.将该系统应用于基于小鼠胚胎干细胞的体外牙向分化模型,在诱导因子Dox和稳定配体Shield-1的协同作用下,诱导表达牙齿发育相关转录因子Hand2提高了牙向分化诱导的完成度.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.

关 键 词：PiggyBac转座系统 Tet-on诱导型过表达系统 溢漏表达 不稳定结构域 稳定配体Shield-1 

分 类 号：Q28[生物学—细胞生物学]