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[Construction of bicistronic eukaryotic vector containing basic fibroblast growth factor and study of their functions
Li Shi1, Ming-min Dong, Wen-jie Shi
1Institute of Otorhinolaryngology, PLA General Hospital, Beijing 100853, China. shient@sina.com
Objective:
To construct basic fibroblast growth factor(bFGR) and enhance green fluorescence protein(EGFP) fusion gene eukaryotic expression vector internal ribosome entry site (pIRES)-bFGF-GFP and to evaluate the effect of transduction bFGF gene on noise induced hearing-loss in inner ear hair cells of guinea pigs.
Methods:
Human bFGF cDNA was inserted into mammalian expressed plasmid pIRES-EGFP. The recombinant expression plasmid pIRES-bFGF-EGFP was transfected into inner ear of guinea pigs, using lipofectin method. The transduced bFGF gene was mediated by SA lipidsome. IRES- bFGF-GFP was administered into the round window as rescue agent at the same time of noise exposure or as a protective agent 7 days before.
Results:
SA liposome-mediated bFGF expressed at a high level in the cochlea of guinea pigs, and in the rescue group, a significant lower hearing thresholds was displayed. pIRES- bFGF-EGFP could protect hair cells. It demonstrated that pIRES- bFGF-GFP could protect the inner ear both structurally and functionally. bFGF/EGFP gene could be transcripted and translated into inner ear hair cells of guinea pig. bFGF/EGFP gene could express a specific protein. The recombinant bFGF/EGFP had significant protective effect as well as manifestation of autonomous fluorescence.
Conclusion:
bFGF/EGFP fusion protein not only expressed in hair cells of guinea pig but also showed significant bFGF activity and autonomous fluorescence. IRES induced exogenous gene could enter the hair cells.