Construction of a highly-active, liver-specific transcriptional regulatory element through combination of the albumin promoter and α-fetoprotein enhancer
Construction of a highly-active, liver-specific transcriptional regulatory element through combination of the albumin promoter and α-fetoprotein enhancer
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DOI：
10.1016/j.plasmid.2010.11.006
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发表时间：
2011-03-01
期刊：
影响因子：
2.6
通讯作者：
Tang, Hong
中科院分区：
文献类型：
--
作者：
Chen, En-Qiang;Song, Xue-Qin;Tang, Hong
为了构建高活性的肝脏特异性转录调控元件，获得了小鼠白蛋白启动子（ALBp）和甲胎蛋白增强子（AFPe）。为了验证其肝特异性和活性，将含AFPe-ALB β的片段克隆到质粒pVAX-S和pGL 3-Luc中，去除原始启动子。将质粒pVAX-AFPe-ALBp-S转染小鼠肝细胞和非肝细胞，并分别通过静脉注射和肌肉注射给药。此外，将pGL 3-AFPe-ALB p-Luc转染到肝细胞和非肝细胞系中; pVAX 1、pVAX 1/S和pGL 3-ALB p-Luc用作对照。观察细胞表面B型肝炎表面抗原（HBsAg）的表达，并测定细胞内荧光素酶活性。对于质粒pVAX-AFPe-ALBp-S，在肝细胞系中观察到HBsAg的表达，但在非肝细胞系中未观察到。利用pVAX-S，在肝细胞和非肝细胞系中观察到HBsAg的表达。在表达pGL 3-AFPe-ALBp-Luc的细胞中，与非肝细胞系相比，肝细胞系中的荧光素酶活性水平显著更高。此外，表达pGL 3-AFPe-ALBp-Luc的细胞中荧光素酶活性水平明显高于表达pGL 3-ALBp-Luc的肝细胞，提示AFPe可在ALBp的调控下增强靶基因的表达。pVAX-AFPe-ALBp-S在小鼠肝脏中检测到HBsAg的表达，而在肌肉中检测不到。相反，用pVAX-S转染后，在小鼠肝脏和肌肉中均检测到HBsAg的表达。总之，AFPe-ALBp元件可用作诱导靶基因的肝脏特异性表达的工具。皇冠版权所有（C）2010由爱思唯尔公司出版。All rights reserved.
In an attempt to construct a highly active, liver-specific transcriptional regulatory element, the mouse albumin promoter (ALBp) and alpha-fetoprotein enhancer (AFPe) were obtained. To verify its hepatic specificity and activity, the AFPe-ALBp-containing fragment was cloned into the plasmids, pVAX-S and pGL3-Luc with original promoter removed. Plasmid pVAX-AFPe-ALBp-S was then transfected into hepatic and non-hepatic cells in vitro, and delivered into mouse by intravenous injection and intramuscular injection, respectively. In addition, pGL3-AFPe-ALBp-Luc was transfected into hepatic and non-hepatic cell lines; pVAX1, pVAX1/S, and pGL3-ALBp-Luc were used as controls. The expression of hepatitis B surface antigen (HBsAg) was observed, and luciferase activity in cells was measured. For plasmid pVAX-AFPe-ALBp-S, the expression of HBsAg was observed in hepatic cell lines, but not in a non-hepatic cell line. Using pVAX-S, the expression of HBsAg was observed in both hepatic and non-hepatic cell lines. In cells expressing pGL3-AFPe-ALBp-Luc, the level of luciferase activity was significantly higher in hepatic cell lines, compared with the non-hepatic cell lines. In addition, the level of luciferase activity in cells expressing pGL3-AFPe-ALBp-Luc was significantly higher than that of pGL3-ALBp-Luc in hepatic cell lines, suggesting that AFPe could enhance target gene expression under the control of ALBp. The expression of HBsAg was detected in mouse liver, but not muscle when using pVAX-AFPe-ALBp-S. In contrast, the expression of HBsAg was detected in both mouse liver and muscle upon transfection with pVAX-S. In conclusion, the AFPe-ALBp element could be used as a tool to induce liver-specific expression of a target gene. Crown Copyright (C) 2010 Published by Elsevier Inc. All rights reserved.