Construction of recombinant adenovirus vector of Uhrf1 gene and its role in DNA damage repair of cardiomyocytes
AIM:To construct a recombinant adenovirus vector carrying the mouse ubiquitin-like with plant homeodomain and RING finger domains 1(Uhrf1)gene,validate the expression of Uhrf1 in neonatal mouse cardiomyo-cytes and explored its role in hydrogen peroxide(H2O2)-induced DNA damage.METHODS:The mouse Uhrf1 gene cod-ing sequence was amplified by polymerase chain reaction(PCR),digested,and inserted into the pADM-CMV-C-FH vec-tor to create the recombinant adenoviral plasmid ADM-Uhrf1.Following transfection into HEK293T cells,we generated re-combinant adenoviral particles,amplified,purified,and determined the titer.Neonatal mouse cardiomyocytes were infect-ed at an multiplicity of infection(MOI)of 50,UHRF1 protein expression was validated via Western blot and immunofluo-rescence staining.H2O2-induced DNA damage was explored along with adenovirus-mediated Uhrf1 overexpression to inves-tigate its role in DNA damage repair.RESULTS:ADM-Uhrf1 virus titer,determined by capsid immunofluorescence as-say,was 1.8×1013 pfu/L.Western blot confirmed a significant increase in UHRF1 protein expression(P＜0.05),with im-munofluorescence indicating predominant nuclear localization.Uhrf1 overexpression effectively inhibited the expression of the DNA damage marker,phosphorylated H2AX protein(γH2AX)(P＜0.01).CONCLUSION:We successfully con-structed a recombinant adenoviral vector carrying the mouse Uhrf1 gene,facilitating Uhrf1 overexpression in neonatal mouse cardiomyocytes.Furthermore,this overexpression effectively alleviated DNA damage in cardiomyocytes.
国家科技期刊平台
NSTL
万方数据
维普
