The expression of recombinant eukaryotic expression vector of wild type and c.454C>T mutant human VEGFA gene in vitro

Objective: To explore whether there is a difference between the expression of recombinant eukaryotic expression vector of wild type and c.454C>T mutant human VEGFA gene(pEGFP-VEGFA) in vitro.Methods: The wild-type and mutant-type pEGFP-VEGFA were transfected into the HEK293T cells by lipofectin reagents,and the expressions of fusion proteins(EGFP and VEGFA)were observed under the fluorescence microscopy after 24 and 48 h post-transfection.VEGFA mRNA expressions were detected by real-time fluorescence quantitative PCR.Results: Green fluorescences were observed in the HEK293T cells transfected with wild-type and mutant-type pEGFP-VEGFA.The intensity of fluorescence in HEK293T cells transfected with mutant-type pEGFP-VEGFA was lower than that transfected with wide-type pEGFP-VEGFA at 48 h post-transfection.The mRNA level of VEGFA in HEK293T cells transfected with mutant-type pEGFP-VEGFA was significantly lower than that transfected with wide-type pEGFP-VEGFA(P<0.05).Conclusions: The recombinant eukaryotic expression vectors of human wild-type and mutant-type pEGFP-VEGFA have transiently expressed in HEK293T cells.Our findings preliminarily show that VEGFA mutant leads to down-regulation of VEGFA mRNA and protein expression level in HEK293T cells,which lays a foundation to further study the role of VEGFA c.454C>T mutant in the development of congenital left ventricular outflow tract obstruction.