Research Article

Construction and identification of pIRES2-LIF-NT-3 bicistronic eukaryotic expression vector

Published: June 18, 2014
Genet. Mol. Res. 13 (2) : 4691-4703 DOI: https://doi.org/10.4238/2014.June.18.12
Cite this Article:
(2014). Construction and identification of pIRES2-LIF-NT-3 bicistronic eukaryotic expression vector. Genet. Mol. Res. 13(2): gmr4475. https://doi.org/10.4238/2014.June.18.12
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Abstract

We used a simple and efficient method to construct a bicistronic eukaryotic expression vector pIRES2-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) (pIRES2-EGFP) to generate the bicistronic eukaryotic expression plasmid pIRES2-LIF-EGFP. Next, the NT-3 cDNA fragment was cloned into pIRES2-LIF-EGFP in place of EGFP to create the plasmid pIRES2-LIF-NT-3. pIRES2-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 pIRES2-EGFP. Following transfection of pIRES2-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.

We used a simple and efficient method to construct a bicistronic eukaryotic expression vector pIRES2-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) (pIRES2-EGFP) to generate the bicistronic eukaryotic expression plasmid pIRES2-LIF-EGFP. Next, the NT-3 cDNA fragment was cloned into pIRES2-LIF-EGFP in place of EGFP to create the plasmid pIRES2-LIF-NT-3. pIRES2-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 pIRES2-EGFP. Following transfection of pIRES2-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.