title: Construction of a single lentiviral vector containing tetracycline-inducible Alb-uPA for transduction of uPA expression in murine hepatocytes.
abstract: The SCID-beige/Alb-uPA mouse model is currently the best small animal model available for viral hepatitis infection studies [1]. But the construction procedure is often costly and time-consuming due to logistic and technical difficulties. Thus, the widespread application of these chimeric mice has been hampered [2]. In order to optimize the procedure, we constructed a single lentiviral vector containing modified tetracycline-regulated system to control Alb-uPA gene expression in the cultured hepatocytes. The modified albumin promoter controlled by tetracycline (Tet)-dependent transactivator rtTA2S-M2 was integrated into a lentiviral vector. The full-length uPA cDNA was inserted into another lentiviral vector containing PTight, a modified Tet-responsive promoter. Two vectors were then digested by specific enzymes and ligated by DNA ligase 4. The ligated DNA fragment was inserted into a modified pLKO.1 cloning vector and the final lentiviral vector was then successfully constructed. H2.35 cell, Lewis lung carcinoma, primary kidney, primary hepatic interstitial and CT26 cells were infected with recombinant lentivirus at selected MOI. The expression of uPA induced by DOX was detectable only in the infected H2.35 cells, which was confirmed by real-time PCR and Western blot analysis. Moreover, DOX induced uPA expression on the infected H2.35 cells in a dose-dependent manner. The constructed single lentiviral vector has many biological advantages, including that the interested gene expression under "Tet-on/off" system is controlled by DOX in a dose-depending fashion only in murine liver cells, which provides an advantage for simplifying generation of conditional transgenic animals.
authors.affiliation: Institute of Infectious Diseases, Southwest Hospital, Third Military Medical University, Chongqing, China.
authors.forename: Jiasi
authors.initials: J
authors.lastname: Bai
authors.forename: Jungang
authors.initials: J
authors.lastname: Li
authors.forename: Qing
authors.initials: Q
authors.lastname: Mao
mesh_terms: D000418:Albumins / Q000235:genetics*; D000818:Animals; D002461:Cell Line, Transformed; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism; D004318:Doxycycline / Q000494:pharmacology; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000821:virology*; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D016513:Mice, SCID; D008822:Mice, Transgenic; D011401:Promoter Regions, Genetic; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics*
chemical_list: D000418:Albumins; D014568:Urokinase-Type Plasminogen Activator; D011088:DNA Ligases; D000072481:DNA Ligase ATP; D004318:Doxycycline