[The effect of HSV-tk/GCV on hepatic specific damage driven by murine ALB promoter/enhancer].
The recombinant vector (pLLTK), containing murine serum albumin (ALB) gene promoter/enhancer directing the herpes simplex virus thymidine kinase (HSV-tk) gene expression was constructed to study its effect on hepatic cell-specific damage. Firstly,in order to compare the hepatic cell-specific transcriptional activity, three vectors were constructed in which green fluorescent protein (GFP) gene was used as a reporter marker. Vector pLE was driven by murine serum ALB gene promoter,whereas pLLE contained not only murine ALB promoter but also an enhancer located at upstream of the promoter was included. In the meanwhile, the vector pLEL had a murine ALB promoter and enhancer placed at the downstream from GFP. After transfected into Hep-G2 (a human hepatic cell line) and HC-11 (a murine breast epithelial cell line), GFP expression was examined by using fluorescence microscope as well as flow cytometer. Secondly, the vector pLLTK was used to observe the killing effect on Hep-G2 cells. The results demonstrated that ALB promoter/enhancer was able to direct hepatic cell-specific GFP expression. Furthermore, HSV-tk expression in Hep-G2 cells was ganciclovir (GCV)sensitive. After seven days of culture with GCV, pLLTK-transfected Hep-G2 cells showed an obvious cell death (53%) when detected by 3-4,5 dimethylthiozol-2-yl-2, 5-diphenyl tetrazolium bromide colorimetry (MTT) assay. Compared to the untreated group, there were no obvious changes in cellular growth inhibition rate in the Hep-G2 cells transfected with a blank control vector pcDNA3. 1 (only 2% of cells appeared cell death). All these results indicated that the above constructs were hepatocyte-specific. It therefore paves a way for further creating a liver-specific damage animal model by transgenic approach using HSV-tk gene expression driven by the ALB promoter/enhancer.
Zhang
Yan
Y
Institute of Medical Genetics, Shanghai Jiao Tong University, Shanghai 200040, China. zhangyan20032003@hotmail.com
Huang
Shu-Zhen
SZ
Zeng
Yi-Tao
YT
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D002460:Cell Line; D003587:Cytomegalovirus / Q000235:genetics; D004742:Enhancer Elements, Genetic; D015774:Ganciclovir / Q000494:pharmacology*; D006801:Humans; D008099:Liver / Q000378:metabolism / Q000473:pathology*; D051379:Mice; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis; D012709:Serum Albumin / Q000235:genetics*; D018139:Simplexvirus / Q000201:enzymology*; D013937:Thymidine Kinase / Q000235:genetics*
D000998:Antiviral Agents; D012333:RNA, Messenger; D012709:Serum Albumin; D013937:Thymidine Kinase; D015774:Ganciclovir