[Construction of bicistronic lentiviral vectors carrying HSV-tk and EGFP gene].
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.


CONCLUSIONS
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.
Yang
Jin
J
Department of Ophthalmology, Eye and ENT Hospital, Fudan University, Shanghai 200031, China.
Lu
Yi
Y
Guo
Li-he
LH
Liu
Tian-jin
TJ
Mo
Xiao-fen
XF
D002478:Cells, Cultured; D004847:Epithelial Cells / Q000166:cytology; D017930:Genes, Reporter; D041722:Genes, Transgenic, Suicide; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006801:Humans; D007908:Lens, Crystalline / Q000166:cytology; D016086:Lentivirus / Q000235:genetics*; D013937:Thymidine Kinase / Q000235:genetics*
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D013937:Thymidine Kinase