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.
Highlights
Development of Alb-urokinase-type plasminogen activator (uPA)/SCID mouse model with humanized hepatic cells has become an important approach for studying the mechanism underlying infections by human hepatotropic viruses, like hepatitis B virus (HBV) and hepatitis C virus (HCV) [3,4,5]
Construction of Tet-Regulated Lentiviral Vector To establish a DOX-dependent gene expression system with lentiviral vector, we inserted the expression cassettes into two lentiviral vectors respectively (Figure 1); the uPA fragment cloned from the kidney of C57BL/J6 mouse was inserted into the downstream of pTight in the pLVX-Tight-Puro ‘‘CN361’’ (Figure 1A) while the albumin promoter and enhancer were inserted into the upstream of reverse transactivator TA (rtTA)-Advanced in pTet-On Advanced ‘‘CN360’’ (Figure 1B)
The vectors were ingested by Age I and Avr II restriction enzymes and recombined in the modified vector of pLKO.1 cloning vector to successfully construct a single lentiviral vector termed as plasmid CN362 (Figure 1C)
Summary
Development of Alb-uPA/SCID mouse model with humanized hepatic cells has become an important approach for studying the mechanism underlying infections by human hepatotropic viruses, like hepatitis B virus (HBV) and hepatitis C virus (HCV) [3,4,5]. The earliest study of substantial repopulation of a mouse liver with human hepatocytes was reported in 2001 by Dandri et al [6] and Mercer et al [7] In both reports, the liver failure was induced by the expression of urokinase-type plasminogen activator (uPA) that was driven by an albumin promoter. Plasmin is an enzyme that is able to degrade many proteins, fibrin clot In this model, uPA/SCID mice, suffering from uPA-induced liver disease, are transplanted early after birth with primary human hepatocytes. UPA/SCID mice, suffering from uPA-induced liver disease, are transplanted early after birth with primary human hepatocytes These human hepatocytes integrate in the parenchyma and progressively repopulate the diseased mouse liver without losing their normal metabolic functions. Transplanted mice can be infected with HBV and HCV
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