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ISSN 1672-9145 Acta Biochim Biophys Sin 2004, 36(10): 687-694 CN 31-1940/Q

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Temporal and Tight Hepatitis C Virus Gene Activation in Cultured Human Hepatoma Cells Mediated by a Cell-Permeable Cre Recombinase

Dong XIAO*, Kang XU¹, Ying YUE, Zhong-Min GUO, Bing HUANG, Xin-Yan DENG, Huan TANG², and Xi-Gu CHEN*

Center of Experimental Animals, Zhongshan University, Guangzhou 510080, China;

¹The first affiliated hospital of Sun Yat-sen University, Zhongshan University, Guangzhou 510080, China;

²The Third Military Medical University, Chongqing 400010, China

Abstract 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 342914) 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 liver-specific 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.

Key words hepatitis C virus; Cre/lox P switching expression system; cell-free and intracellular recombination systems; cell-permeable Cre recombinase; EGFP; human hepatoma cells

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Received: March 25, 2004 Accepted: September 10, 2004

This work was supported by the grants from the National Natural Science Foundation of China (No. 30271177, No. 39870676), Guangdong Province Natural Science Foundation of China (No. 021903) and Postdoctoral Fellowship Foundation of China (Series 29)

*Corresponding author: Tel, 86-20-87331393; Fax, 86-20-87331230; E-mail, [email protected]