Generation of a mouse model for the in vivo study of HCV-host interaction

The natural species tropism of hepatitis C virus (HCV) is limited to humans and higher primates. Due to the limited availability of chimpanzees, the establishment of a small-animal model for the study of HCV infection has a high priority. The recent identification of the human cellular receptors that are essential for HCV entry together with the advances in mouse genome engineering provides the bases for creating an inbred mouse model of HCV infection and propagation. In our study we designed a gene targeting strategy to produce novel humanized mouse strains expressing human-specific HCV entry receptors. Human genes for HCV essential entry factors were introduced in the mouse genome by homologous recombination in murine embryonic stem cells. To achieve the native expression patterns of their murine orthologues, we adopted a knock-in approach replacing the murine genes with human coding sequences. Three novel humanized mouse strains were generated, expressing respectively human CD81, scavenger receptor class B member 1 and occludin. Molecular analyses of mice genomic DNA and messenger RNA confirmed the presence and germ line propagation of the human genes, and their transcription. The native regulated expression of human-specific HCV entry receptors can overcome the major block to HCV replication in murine cells. To verify the virus entry into the cells of our humanized mice, we successfully optimized isolation and culture of murine primary hepatocytes and HCV pseudotype particles infection assay. Genetic humanization of mice for species-specific HCV receptors could allow HCV glycoproteins mediated cell entry and could be an important tool for in vivo studying of virus-host interactions.