Abstract:

Objective   To construct a lentiviral vector carrying a short hairpin RNA (shRNA) targeting the HIF-1α gene and detect the silencing effect of the vector on mouse glioma cell line GL261. Methods   Four double-stranded shRNA targeting the HIF-1α gene were designed, synthesized and cloned. The resulting lentiviral vector containing HIF-1α shRNA was named as pLenti6.3-shRNA3. GL261 cells were transfected with pLenti6.3-shRNA3 lentivirus to obtain a cell line stably expressing HIF-1α shRNA. After the transfection, mRNA and protein expressions of HIF-1α in GL261 cells were detected by real-time PCR and Western blot, respectively. Results   A lentiviral vector carrying a shRNA targeting HIF-1α gene was successfully constructed. The GL261 cell line stably transfected with the vector was established. The recombinant lentivirus were harvested from 293T cells with titer of 1×108 TU/mL. Real-time PCR and Western blot analyses confirmed that the expressions of HIF-1α was down-regulated in GL261 cell line which stably transfected with the recombinant vector. Conclusion   shRNA targeting different sites of the HIF-1α gene exhibits different inhibitory effects. Specific shRNA could induce stable silencing of HIF-1α gene.

Key words:  Lentivirus;  HIF-1α;  RNA interference;  Mice;  GL261 cells