Temporal and tight hepatitis C virus gene activation in cultured human hepatoma cells mediated by a cell-permeable Cre recombinase.
Conditional gene expression has greatly facilitated the examination of the functions of particular gene products. Using the Cre/lox P switching expression system, we plan to develop efficient conditional transgene activation of hepatitis C virus core protein (HCV-C) cDNA (nucleotide 342-914) in the transgenic mice to overcome "immune tolerance" formed during the embryonic period and "immune escape" against hepatitis virus antigen in our project. To use this system in vivo, the dormant transgenic construct, i.e., pApoE-SCS-EGFP-HCV-C, was generated using techniques of standard molecular biology. The liverspecific human apoE promoter was here used to target expression of genes of interest (EGFP and HCV-C) to murine liver. Prior to generating the transgenic mice, the availability of Cre/lox P system and construct functionality were successfully verified by a cell-free recombination system and via checking the expression of EGFP and HCV-C in the human hepatoma cells at the mRNA and protein levels. These results suggest that the Cre/lox P system could tightly control expression of EGFP and HCV-C in vitro, which laid a solid foundation to conditionally activate expression of target gene(s) in transgenic mice by Cre-mediated site-specific recombination.
Center of Experimental Animals, Zhongshan University, Guangzhou 510080, China.
Dong
D
Xiao
Kang
K
Xu
Ying
Y
Yue
Zhong-Min
ZM
Guo
Bing
B
Huang
Xin-Yan
XY
Deng
Huan
H
Tang
Xi-Gu
XG
Chen
D006528:Carcinoma, Hepatocellular / Q000201:enzymology* / Q000235:genetics*; D045744:Cell Line, Tumor; D002463:Cell Membrane Permeability / Q000502:physiology; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics*; D006801:Humans; D019426:Integrases / Q000235:genetics* / Q000378:metabolism*; D008249:Protein-Lysine 6-Oxidase / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000379:methods*; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism*
D016326:Extracellular Matrix Proteins; D014764:Viral Proteins; C077112:Lox protein, mouse; D008249:Protein-Lysine 6-Oxidase; C045073:Cre recombinase; D019426:Integrases