[Construction of bicistronic lentiviral vectors carrying HSV-tk and EGFP gene]
1Department of Ophthalmology, Eye and ENT Hospital, Fudan University, Shanghai 200031, China.
Objective:
To construct CMV-mediated Lentiviral vectors coexpressing EGFP and HSV-tk gene in order to establish a novel lentiviral vector platform for the suicide gene therapy of eye disease.
Methods:
The restriction endonuclease and T4 DNA ligase were used to construct the vector plasmid. HSV-tk fragments from pcDNA3-HSV-tk were cloned into the site of lenti-internal ribosomal entry site (IRES)-EGFP to construct the bicistronic lenti-HSV-tk-EGFP vector. Human embryonic kidney 293T cells were co-transfected with the lentiviral vector (three plasmids) by calcium phosphate DNA precipitation. HLEC, HXO-Rb(44), SH-SY-5Y and Hela cells were transfected with viral production and the expression of EGFP was examined under fluorescent microscope after transfection. The expression of HSV-tk and EGFP was examined by RT-PCR.
Results:
Lentivirus mediated stable integration and efficient expression of EGFP and TK genes in the cells tested. Coexpression of HSV-tk and EGFP in HLECs mediated by lentiviral vectors was confirmed by the result of RT-PCR. The transfection efficiency for HLECs was about 100% at MOI = 100, and kept the same level for at least 6 months.
Conclusion:
The bicistronic lentiviral vector platform carrying HSV-tk-EGFP is an efficient and stable gene transfer vector, it might be used for suicide genes therapy in the treatment some eye disease.
