Construction and functional identification of siRNA adenoviral vector targeting HIF-1α in mouse

Objective To construct an adenoviral vector targeting hypoxic inducible factor-1α (HIF-1α) gene in mouse and to identify the effect of the adenoviral vector,AdHIF-1α-shRNA-EGFP,on the expression of HIF-1α in cultured mouse embryonic neural stem cells,and provide a basis for investigating the contributiou of HIF-1 signaling pathway in neural stem cell biology.Methods The HIF-1α template DNA sequence was designed by using bioinformatics.DNA recombinant technique was used to construct recombinant adenoviral vector,AdHIF-1α-shRNA-EGFP.Polymerase chain reaction(PCR) and sequencing were performed.The neural stem cells from frontal cortex of the mouse were transfected with AdHIF-1α-shRNA-EGFP in vitro.The efficiency of transfection was detected by flow cytometry.The transfected cells were cultured under hypoxia condition.Double immunofluorescent staining of nestin and HIF-α was performed,and the expression of HIF-1α was quantified by reverse transcription - polymerase chain reaction (RT-PCR) and Western blot analysis.Results The sequence cloned into the adenoviral vector was identical with the one synthesized chemically.The neural stem cells were transfeeted effectively by AdHIF-1α-shRNA-EGFP.The results from RT-PCR and Western blot analysis showed that the expression of HIF-1α gene and protein in the neural stem cells transfected with AdHIF-1α-shRNA-EGFP decreased by 52.05％ and 67.27％ under hypoxia condition,respectively.Conclusion AdHIF-1α -shRNA-EGFP could effectively inhibit hypoxia - induced expression of HIF-1α gene and protein in the mouse neural stem cells in vitro. Key words: Neural stem cells; Mouse; Hypoxic inducible factor-1α; Small interference RNA; Adenoviral vector