Construction and identification of pIRES2-LIF-NT-3 bicistronic eukaryotic expression vector
Genetics and Molecular Research
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
... ate 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 4692 ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 13 (2): 4691-4703 (2014) B.N. Li et al. 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.