| HIV-1 based lentiviral vectors have emerged as one of the most promising gene delivery tools because they are capable of transducing non-dividing cells and integrating into host genome for long-term transgene expression. However, there still exist some controversies concerning lentiviral transgene silencing, and much less is known about the underlying molecular mechanisms. To explore lentiviral transgene silencing, HIV-1 based self inactivating (SIN) vector was used to characterize the transgene expression in murine embryonic carcinoma cell line P19. We found that transgenes driven by either human EF1a promoter or CMV/CBA promoter were quickly silenced after lentiviral transduction. To investigate the lentiviral silencing mechanisms, we characterized DNA methylation and chromatin deacetylation status of the transgene promoter using pooled and single cell cloned P19 cells. Analysis of the transduced P19 cells revealed that DNA methylation of the lentiviral internal promoter was correlated with transgene silencing.; My second topic was to identify cancer-specific genes and characterize their functions. Using a murine hepatoma model, we applied a modified suppression subtractive hybridization method to isolate a small number of highly differentially expressed hepatoma-specific genes. For functional analysis of these hepatoma-specific genes, we employed the small interference RNA (siRNA) -mediated gene silencing technique using lentiviral vectors, which have the advantages of high delivery efficiency and long lasting effect. Among the few differentially expressed cancer-specific genes, stem cell antigen-2 (Sca-2) was identified as one of the highest differentially expressed hepatoma antigens. Together, our results suggest that Sca-2 is a signal transducer situated at the nexus of surface molecules regulating death receptor-mediated apoptosis. These technologies illustrated that a small number of highly differentially expressed cancer genes can be quickly identified that may have diagnostic and therapeutic potential. (Abstract shortened by UMI.)... |
