Institute of Infectious Diseases, Southwest Hospital, Third Military Medical University, Chongqing, China.
PLoS One. 2013 Apr 23;8(4):e61412. doi: 10.1371/journal.pone.0061412. Print 2013.
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.
SCID-beige/Alb-uPA 小鼠模型目前是用于病毒性肝炎感染研究的最佳小动物模型[1]。但是，由于后勤和技术困难，其构建过程通常昂贵且耗时。因此，这些嵌合小鼠的广泛应用受到了阻碍[2]。为了优化该过程，我们构建了一个包含修饰的四环素调控系统的单一慢病毒载体，以控制培养的肝细胞中 Alb-uPA 基因的表达。受四环素（Tet）依赖性转录激活子 rtTA2S-M2 控制的修饰白蛋白启动子被整合到慢病毒载体中。全长 uPA cDNA 被插入另一个包含 PTight 的慢病毒载体中，PTight 是一种修饰的 Tet 反应性启动子。然后，两个载体被特定的酶消化并用 DNA 连接酶 4 连接。连接的 DNA 片段被插入修饰的 pLKO.1 克隆载体中，最终成功构建了慢病毒载体。H2.35 细胞、Lewis 肺癌细胞、原代肾细胞、原代肝间质细胞和 CT26 细胞在选定的 MOI 下用重组慢病毒感染。只有在感染的 H2.35 细胞中，才可以通过实时 PCR 和 Western blot 分析检测到 DOX 诱导的 uPA 表达。此外，DOX 以剂量依赖性方式诱导感染的 H2.35 细胞中 uPA 的表达。构建的单一慢病毒载体具有许多生物学优势，包括感兴趣的基因表达受“Tet-on/off”系统控制，仅在鼠肝细胞中由 DOX 以剂量依赖的方式控制，这为简化条件性转基因动物的生成提供了优势。
