Construction of the eukaryotic expression vector pIRES2-EGFP-NT3 and expression in guinea pig cochlea fibroblast

Aim To construct the eukaryotic expression vector pIRES2 EGFP NT3 and observe its expression in guinea pig cochlea fibroblast. Methods The NT 3 cDNA was cloned by RT PCR from human liver tissue and was sequenced. The eukaryotic expression vector (pIRES2 EGFP NT3) was constructed and identified by PCR and with Eco R I and Bam H I digestion. The pIRES2 EGFP NT3 was transfected into guinea pig cochlea fibroblast by using lipofectamine. After transfection of 48 h, the NT 3 expression in transfected fibroblasts were observed and detected under fluorescence microscope and by immunor histochemical staining respectively. Results The size of PCR product of NT 3 gene was about 744 bp. Sequencing result revealed the sequence of amplified NT 3 gene was identical with that in GenBank. Restrictive enzyme( Eco R I/ Bam H I) digestion analysis showed that recombinant expression vector pIRES2 EGFP NT3 had been constructed successfully. The transfected fibroblasts displaying green fluorescence were observed under fluorescence microscope. The immunohistochemical staining showed that positive reactant of brown yellow could be seen in the cytoplasm of fibroblasts transfected by pIRES2 EGFP NT3, while the no NT 3 expression was observed in the fibroblasts transfected by empty vector. Conclusion The recombinant expression vector pIRES2 EGFP NT3 is constructed successfullu and can be expressed in the fibroblasts. The present study lay the foundation for the furhter test of treating deafened guinea pig by NT 3 gene transfection.