Construction of a lentiviral vector carrying shRNA against HIF-1α and generation of a GL261 cell line stably transfected with this vector

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.