Establishment Of Tet-on Based Inducible RNAi System

RNA interference(RNAi) is a post-transcriptional gene regulation process involving the disruption of homologous m RNA by introduction of double-strand RNA or short hairpin RNA(sh RNA). RNAi has been widely used in the field of biological research due to its fast、high efficient and gene specific silencing effects. Ago2/c Ago2 has been reported to enhance the progress of RNAi,however, the expression of Ago2 could disturb the packaging of lentiviral vectors. To precisely regulate the RNAi process in the research of gene function, it is preferred to use the tetracycline-inducible expression system. In the present work, base on the sh RNA vector backbone in our lab, we further improved the Tet-inducible lentiviral expression system, aiming to build a new and high efficient tetracycline-inducible lentiviral expression system.Firstly, we compared the RNA interference efficient of mi R-451 backbone and two commercial mi R-30 a backbones(Esh R and Osh R). Secondly, based on the three sh RNA backbones, we inserted a reversed intron sequence around mi R30a-sh RNA. Thirdly, we compared the inducible expression efficient of two promoters of Tet O6 with TRE3 G, and rt TA3 with Tet ON3 G. Then we explored the function of IRES and P2 A in Tet-inducible expression system. Finally, we investigated the effects of positive/negative direction and different promoters in front of Tet ON3 G on the expression efficient of Tet-inducible system. The results are as follows:(1) compared with the two mi R-30 a backbones, the mi R-451 backbone had no better RNAi effect than the other two; while the Osh R backbone showed stronger interference than the Esh R;(2) a bit of RNAi enhancement was observed in Esh R backbone based plasmid that inserted with a reversed intron;(3) the inducible efficiency of rt TA3 that developed by Thermofisher was much more higher than the reverse inducible system of Tet ON3 G that developed by Clontech;(4) the Tet O6-Tet ON3 G backbone displayed the largest enhancement of inducible gene expression when we compared Tet O6/TRE3 G promoters and rt TA3/Tet ON3 G trans-acting factors;(5) the P2 A can enhance the expression level of protein compared with IRES;(6) among the screened efficient inducible systems, some background expression were observed in the reverse inducible expression system, while PGK and Ubc showed significant greater expression level with low background expression in the forward inducible system with different trans-acting factor.