Development of animal models with hepatitis C and hepatocellular carcinoma by transgenic techniques

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1. Production of transgenic miceTo establish an animal model of hepatitis C and hepatocellular carcinoma, we generated 31 transgenic mice carrying hepatitis C virus (HCV) genome : 3 lines of transgenic mice carrying a partial genome including the structural protein region under the control of the albumin promoter/enhancer (Alb-HN3.8), 19 lines with the whole genome of HCV (Alb-HN2) and 9 lines with the whole genome under the control of the SRalpha promoter (ME-HN2). HCV-specific RNA was little expressed within 8 weeks after birth.2. Induction of transgene expressionTo analyze the mechanism of transgene inactivation, we examined methylation inactivation of the transgene. Methylation-sensitive restriction enzyme fragment analysis and mapping of methylated cytosine by bisulfite-genome sequencing showed that the transgene was extensively methylated. A demethylating agent, 5-azacytidine, induced HCV gene expression in 4 of 6 lines examined. These results suggest that methylation of HCV cDNA is a cause of its suppressive expression in transgenic mice.3. Histopathological changes of transgenic liverWe investigated long-term pathological changes in liver of transgenic mice. Hepatitis such as semi-acute phase of fulminant hepatitis and liver atrophy with lymphocyte infiltration and ascitic accumulation was observed in mice with 7 to 22 months of age : I of 1 mouse in line Alb-HN3.8-l, 1 of 4 mice in line Alb-HN2-48, and 1 of 6 mice in line ME-HN2-5. The expression of HCV RNA was detected in these liver tissues. As for line Alb-HN2-44, dysplastic changes were observed in 2 of 17 transgenic livers. One of them developed adenoma. These results suggest that only the expression of transgene induces pathological change of liver. However, it is further necessarily examined whether these changes observed are transgene-associated.