title: Construction and identification of pIRES2-LIF-NT-3 bicistronic eukaryotic expression vector.
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.
authors.affiliation: Department of Life Sciences and Technology, Xinxiang Medical University, Xinxiang, He'nan, China bingnanli120@yeah.net.
authors.forename: B N
authors.initials: BN
authors.lastname: Li
authors.affiliation: Department of Life Sciences and Technology, Xinxiang Medical University, Xinxiang, He'nan, China.
authors.forename: W D
authors.initials: WD
authors.lastname: Li
authors.affiliation: Department of Life Sciences and Technology, Xinxiang Medical University, Xinxiang, He'nan, China.
authors.forename: J T
authors.initials: JT
authors.lastname: Lin
authors.affiliation: Department of Life Sciences and Technology, Xinxiang Medical University, Xinxiang, He'nan, China.
authors.forename: H G
authors.initials: HG
authors.lastname: Feng
authors.affiliation: Department of Life Sciences and Technology, Xinxiang Medical University, Xinxiang, He'nan, China.
authors.forename: Z Q
authors.initials: ZQ
authors.lastname: Yuan
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D053605:Leukemia Inhibitory Factor / Q000235:genetics* / Q000378:metabolism*; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D009414:Nerve Growth Factors / Q000235:genetics* / Q000378:metabolism*; D020933:Neurotrophin 3; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection
chemical_list: C507837:LIF protein, human; D053605:Leukemia Inhibitory Factor; C000595808:NTF3 protein, human; D009414:Nerve Growth Factors; D020933:Neurotrophin 3; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins