Construction of TH-GDNF vector and its expression in a dopaminergic cell line MES23.5 / 第三军医大学学报

ABSTRACT

Objective To construct a vector carrying tyrosine hydroxylase (TH) and glial cell line-derived neurotrophic factor (GDNF) in order to establish a new gene therapy method in Parkinson's disease. Methods Human TH gene fragment from the plasmid pWAV2-TH was cloned into pIRES to construct pIRES-TH. The mouse GDNF gene, amplified by PCR was inserted into pIRES-TH to construct pIRES-TH-GDNF. Restriction analysis and nucleotide sequencing were used to confirm the structure of pIRES-TH-GDNF. Then MES23.5 cells were transfected with this eukaryon vector using Lipofectamine TM2000. The expression of TH and GDNF in mRNA and protein levels were detected by RT-PCR and immunofluorescence after G418 selection. Results The 2 objective fragments were inserted into pIRES correctly. RT-PCR and immunofluorescence results showed that TH and GDNF were highly expressed in MES23.5 cells. Conclusion The plasmid pIRES-TH-GDNF is constructed successfully and can express TH and GDNF in vitro.