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Human glial cell line-derived neurotrophic factor (GDNF) and vascular endothelial growth factor 165 (VEGF165) are essential genes for cell differentiation.To construct and identify pIRES2-GDNF-VEGF165 bicistronic eukaryotic expression vector.Human GDNF genes were obtained from the genomic DNA of human peripheral blood mononuclear cells by PCR. Then the GDNF cDNA fragment was inserted into the multiple cloning sites of pIRES2-EGFP, to generate the bicistronic eukaryotic expression plasmid pIRES2-GDNF-EGFP. The VEGF165 gene was obtained from pIRES2-VEGF165-EGFP plasmid by twin PCR. Then VEGF165 cDNA fragment was cloned into the pIRES2-GDNF-EGFP, instead of EGFP, to create a double gene co-expressing vector plasmid pIRES2-GDNF-VEGF165 containing internal ribosome entry sites. Then pIRES2-GDNF-VEGF165 was used to transfect HEK293 cells. RT-PCR and western blot analysis were performed to test the co-expression of double genes.DNA sequencing analysis demonstrated that the GDNF and VEGF165 were exactly consistent with the sequence recorded in the GenBank. The size of GDNF gene was 636 bp and the size of VEGF165 gene was 576 bp. Enzyme digestion analysis indicated that, pIRES2-GDNF-VEGF165 bicistronic eukaryotic expression vector inserted GDNF band by Bgl II/Bam HI, inserted IRES-VEGF165 fragment by Bam HI/Not I, and inserted GDNF-IRES-VEGF165 fragment by Bgl II/Not I. RT-PCR and western blot analysis showed that, after HEK293 cells were transfected with pIRES2-GDNF-VEGF165, double genes were expressed at the mRNA and protein levels. The pIRES2-GDNF-VEGF165 bicistronic eukaryotic expression vector is successfully constructed.glial cell line-derived neurotrophic factor; vascular endothelial growth factor; carrier proteins; tissue engineeringthe Tender Subject of Key Research Areas of Xinxiang Medical University in 2011, No. ZD2011-16; Key Projects in Scientific Research of Henan Provincial Education Department, No. 13A180850.Li BN, Li WD, Lin JT, Feng HG. Construction and identification of pIRES2-GDNF-VEGF165 bicistronic eukaryotic expression vector. Zhongguo Zuzhi Gongcheng Yanjiu. 2014;18(29): 4675-4682.