Abstract: Objective: To construct a lentiviral vector expressing a short hairpin RNA (shRNA) which targets the functional p100 gene and to construct a p100 stable knock-downed HepG2 cell line with this p100 shRNA expressing lentiviral system. Methods: A double-stranded shRNA targeting the p100 gene was designed, synthesized and cloned into the pLKO.3G vector. Positive clones were verified with double enzyme digestion and sequencing. Then the valid constructions were cotransfected with the other 2 packaging plasmids into 293T cells to package lentiviral particles carrying p100 shRNA expression element. The lentivirus was then harvested and used to infect HepG2 cells. Infection efficiency was indicated with GFP and detected with fluorescent microscope. The knock-down efficiency of p100 in infected HepG2 cells was verified with western blot. Results: A lentiviral vector carrying a shRNA targeting the p100 gene was successfully constructed and a p100 stable knock-downed HepG2 cell line was established by using this lentiviral system. After viral particles infection of HepG2 cells, strong green fluorescence was observed in HepG2 cells under fluorescent microscope, the infection efficiency exceeded 90%. Western blot analyses confirmed that the expression of p100 was significantly down-regulated in this infected HepG2 cell line. Conclusion: A lentiviral vector carrying an shRNA targeting the p100 gene was successfully constructed and a HepG2 cell line stably expressing p100 shRNA was established with this lentiviral system.

Key words: RNA interference, cell line, tumor, plasmids, lentiviral vector, p100 protein, HepG2 cell line