Construction and identification of retroviral vector expressing hypoxia-inducible factor-1α and EGFP fusions

Objective To construct the recombinant retrovirus of human hypoxia-inducible factor-1α(HIF-1α) gene and to observe its ability to infect NIH3T3 fibroblasts.Methods The full-length human HIF-1α cDNA was cloned into the retroviral vector pLEGFP-N1 to express an amino-terminal fusion protein of HIF-1α and enhanced green fluorescent protein (EGFP) by the method of gene engineering. Then the recombinant retrovirus was transfected into NIH3T3 cells using Lipofectine DOTAP. The target gene was detected by polymerase chain reaction (PCR). The titer and its infection rate were determined using the EGFP expression with fluorescent microscope.Results Restriction endonuclease, sequencing and PCR analyses confirmed that the human HIF-1α cDNA was successfully inserted into the retroviral vector. The titer of recombinant retrovirus with HIF-1α gene was 1.2×10 6 pfu/ml and the retrovirus had a strong effect on NIH3T3 cells.Conclusion The recombinant retrovirus containing HIF-1α gene was successfully constructed by the method of gene engineering and it established a base for therapeutic angiogenesis.