Objective To develop a lentiviral vector containing a Tet-On system for inducible pig urokinase-typeplasminogen activator (puPA) transgene expression under the control of the albumin (Alb) promoter, and to confirm the invitro functionality of the resulting plasmid. Methods First, the fragment of the Alb-enhancer/ promoter was amplified frompAlb-Cre-GH/ BS and subsequently inserted into the XhoI and XbaI sites of pLVX-TetOne by In-Fusion cloning to generatepLVX-Alb-TetOne. Second, the T2A-CopGFP fragment was amplified from pCD823 A-1 and then subcloned into pLVXAlb-TetOne by In-Fusion cloning to construct pLVX-Alb-TetOne-TRE-T2A-CopGFP ( pLATTTG). Finally, the puPAfragment was amplified from H8803 and subsequently inserted into pLATTTG by In-Fusion cloning to produce pLVX-Alb-TetOne-TRE-puPA-T2A-CopGFP ( pLATTPUTG ). To validate the in vitro functionality of the resulting vector(pLATTPUTG), it was transiently transfected into 293T cells, followed by CopGFP assay under an inverted fluorescencemicroscope at 24 h after transfection in the absence of doxycycline (Dox-). Dox was then added into a 6-well plate, and theCopGFP assay was performed at 48 h after the addition of Dox, followed by testing the expression of puPA, CopGFP andFlag by qRT-PCR or western blot. Results The data from enzyme digestion and DNA sequencing demonstrated thatpLATTPUTG was successfully constructed. In the presence of Dox, most cells transfected with pLATTPUTG showed stronggreen fluorescence and a high-level expression of puPA, CopGFP and Flag, whereas in the absence of Dox, no cellstransiently transfected with pLATTPUTG displayed green fluorescence. Conclusions A lentiviral vector containing a Tet-On system for inducible puPA transgene expression under the control of an Alb promoter was successfully generated, which will lay a solid foundation for further study.