Construction of Eya1S298A overexpressed lentiviral vector and its influence on MES23.5 cell apoptosis

Objective To construct Eya1S298A overexpressed lentiviral expression vector and investigate the effect of site 298 mutation of EYA1 on the apoptosis of MES23.5 dopaminergic cells. Methods A recombinant expression plasmid of lentivirus carrying Eya1S298A gene was constructed by PCR amplification, Golden Gate and LR reaction. MES23.5 cells with stable expression of Eya1S298A were screened by purinomycin. The cells stably expressing Eya1S298A or Eya1 were exposed to 6-hydroxydopamine for 2 h. The effects of site 298 mutation on cell viability and Bcl-2 protein expression were detected by CCK8 assay and Western blot. Results According to DNA sequencing and PCR analysis, the site 298 S of Eya1 gene was mutated into A, and Eya1S298A gene was successfully linked to the lentiviral vector. CCK8 results showed that the cell viability of the Eya1S298A mutant group was significantly lower than that of the Eya1 wild-type group (P<0.05). Western blot results showed that Bcl-2 protein expression of the mutant group was significantly lower than that of the wild-type group (P<0.05). Conclusions The lentiviral expression plasmid of Eya1S298A gene is successfully constructed and the stable expression cell line of Eya1S298A is established. Moreover, the 298 mutation of Eya1 reduces the antiapoptotic effect of Eya1 on MES23.5 cells.