title: Enhanced expression of bioactive recombinant VEGF-111 with insertion of intronic sequence in mammalian cell lines.
abstract: Among all VEGF-A isoforms, VEGF-111 is particularly important in molecular biology research owing to its potent angiogenic properties and its remarkable resistance to proteolysis. These features make it a potential candidate for therapeutic use in ischemic diseases. VEGF-111 is not expressed in normal cells, but expression is induced by UV-B irradiation and exposure to genotoxic agents. Here, to increase expression at the transcriptional and translational levels, we synthesized and cloned recombinant VEGF-111 cDNA. Two fragments encoding exons 1-4 and intron 4/5 plus exon 8a were amplified and cloned into the pBud.CE4.1 vector using a class IIs restriction enzyme-based method. The expression of VEGF-111 in CHO-dhfr - and HEK 293 cell lines was evaluated with real-time PCR, dot blotting, and ELISA. VEGF expression was increased about 10- and 18-fold in transfected CHO-dhfr - and HEK 293 cells, respectively. Dot blotting and ELISA confirmed successful production of VEGF-111 in both cell lines.
authors.affiliation: Division of Genetics, Biology Department, Faculty of Sciences, University of Isfahan, Isfahan, Iran, z.hojati@sci.ui.ac.ir.
authors.forename: Zohreh
authors.initials: Z
authors.lastname: Hojati
authors.forename: Fariba
authors.initials: F
authors.lastname: Dehghanian
mesh_terms: D000818:Animals; D016466:CHO Cells; D003412:Cricetulus; D018076:DNA, Complementary; D005091:Exons / Q000235:genetics; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D007438:Introns / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D018076:DNA, Complementary; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A