ABSTRACT. We used a simple and efficient method to construct a bicistronic eukaryotic expression vector pIRES 2 -LIF-NT-3. The leukemia inhibitory factor (LIF) and neurotrophin-3 (NT-3) genes were obtained from the genomic DNA of human peripheral blood mononuclear cells by polymerase chain reaction. The LIF cDNA fragment was inserted into the multiple cloning sites of a vector containing internal ribosome entry site and enhanced green fluorescent protein (EGFP) (pIRES 2 -EGFP) to generate the bicistronic eukaryotic expression plasmid pIRES 2 -LIF-EGFP. Next, the NT-3 cDNA fragment was cloned into pIRES 2 -LIF-EGFP in place of EGFP to create the plasmid pIRES 2 -LIF-NT-3. pIRES 2 -LIF-NT-3 was transfected into HEK293 cells and reverse transcription-polymerase chain reaction and Western blotting were used to test the co-expression of double genes. LIF and NT-3 genes were cloned and the DNA was sequenced. Sequencing analysis revealed that LIF and NT-3 were consistent with the sequence recorded in GenBank. Restriction analysis indicated that the LIF and NT-3 genes were inserted correctly into the expression vector pIRES 2 -EGFP. Following transfection of pIRES 2 -LIF-NT-3 into HEK293 cells, the double gene was expressed at the mRNA and protein levels. The LIF and NT-3 coexpression plasmid is a novel expression system that will enable further study of the functions of the LIF and NT-3 genes.

ABSTRACT. We used a simple and efficient method to construct the bicistronic eukaryotic expression vector pIRES2-NGF-VEGF 165 . The nerve growth factor (NGF) gene was obtained from the genomic DNA of human peripheral blood mononuclear cells by polymerase chain reaction. The NGF cDNA fragment was inserted into the multiple cloning sites of the pIRES2-EGFP vector to generate the bicistronic eukaryotic expression plasmid pIRES2-NGF-EGFP. The vascular endothelial growth factor 165 (VEGF 165 ) gene was obtained from the pIRES2-VEGF 165 -EGFP plasmid by polymerase chain reaction. Next, the VEGF 165 cDNA fragment was cloned into pIRES2-NGF-EGFP in place of enhanced green fluorescent protein creating the plasmid pIRES2-NGF-VEGF 165 . pIRES2-NGF-VEGF 165 was transfected into HEK293 cells and reverse transcriptionpolymerase chain reaction and Western blot analysis were used to test the co-expression of double genes. The NGF and VEGF 165 genes were cloned and the DNA was sequenced, which revealed that NGF and VEGF 165 were consistent with the sequence recorded in GenBank. Restriction analysis showed that the NGF and VEGF 165 genes were inserted into the expression vector pIRES2-EGFP. Transfection of pIRES2-NGF- 5675©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 13 (3): 5674-5685 (2014) Construction and identification of pIRES2-NGF-VEGF 165 VEGF 165 into HEK293 cells resulted in expression of the double gene at the mRNA and protein levels. The NGF and VEGF 165 coexpression plasmid provides a novel expression system, enabling further study of the functions of the NGF and VEGF 165 genes.