动物造模 z-bio 2021-11-12 529

　　Purpose: Using Tet-On gene expression regulation system to construct a lentiviral vector pLVX-Alb-TetOne-TRE-puPA-T2A-CopGFP(pLATTPUTG ).

　　Method: First, using pAlb-Cre-GH/BS as a template, PCR amplification of the target gene Alb-enhancer/promoter, In-Fusion cloned into the Xho I/Xba I double digestion pLVX-TetOne, the Alb promoter was obtained to replace pLVX -TetOne's original PGK promoter plasmid pLVX-Alb-TetOne; then using pCD823A-1 as a template, PCR amplification of T2A-CopGFP, In-Fusion cloned into BamH I/Age I digested pLVX-Alb-TetOne , Obtain pLVX-Alb-TetOne-TRE-T2A-CopGFP (pLATTTG); finally use H8803 as template, PCR amplify puPA (3′ end with Flag tag), InFusion clones into pLATTTG, and finally obtain the lentiviral vector pLVX-Alb -TetOne-TRE-puPA-T2A-CopGFP (pLATTPUTG); the constructed plasmids are all identified by restriction enzyme digestion and sequencing. pLATTPUTG is transiently transfected into 293T cells, and after transfection for 24 hours, the green fluorescence is detected by an inverted fluorescence microscope; then a strong force is added to the six-well plate Dox, 48h later, detect CopGFP expression under an inverted fluorescence microscope (including wells without Dox), and then collect cells to extract total RNA and total protein for qRT-PCR to detect puPA and CopGFP expression and Westernblot detection Flag expression.

　　Results: Restriction digestion and sequencing confirmed that the lentiviral vector pLATTPUTG was successfully constructed. pLATTPUTG was transfected into 293T cells. After 24h, no green fluorescence was seen under the inverted fluorescence microscope. After Dox48h was added, almost all cells showed strong green fluorescence. There is still no green fluorescence in the wells of Dox; the expression levels of puPA, CopGFP and Flag on the cells in the wells with Dox are significantly increased, and the extremely low expression of the above genes is detected on the cells in the wells without Dox; the above data suggests that, The Tet-On gene expression regulation system was used to realize the inducible expression of puPA gene on 293T cells.

　　Conclusion: The successful construction of a lentiviral vector pLATTPUTG with the Alb promoter regulating the expression of puPA transgene based on the Tet-On gene expression regulation system has laid a solid foundation for related follow-up research.