Abstract： Objective To construct a recombinant short-hairpin RNA (shRNA) lentiviral vector targeting the β-catenin gene in cochlear precursor cells (CPCs) in mice,and to investigate its inhibitory effect.Methods PCR was used for the multiplication of the β-catenin gene,and shRNA oligo was designed based on the β-catenin gene to construct an interference vector.Gateway Technology was used to construct shRNA lentiviral vector which carried the β-catenin gene,and then 293FT cells were transfected with the constructed lentiviral vector and helper plasmids pLV/helper-ST3,pLV/helper-ST4,and pLV/helper-SL5.The virus supernatant was collected to obtain viral particles,and then mouse CPCs were transiently infected with the recombinant lentivirus with four different concentrations (0,5,10,and 20 μl).The shRNA control group was established.Quantitative real-time PCR and Western blot were used to investigate the inhibitory effect of shRNA β-catenin lentiviral vector on β-catenin.Results The recombinant shRNA β-catenin lentiviral vector was successfully constructed,and the virus titers of shβ-catenin and shβ-catenin-control were 5.05×107 and 4.34×107,respectively.The results of in vitro experiments showed that in CPCs transfected with four different concentrations of recombinant lentivirus,the content of β-catenin protein gradually decreased with the increase in concentration,and there was a significant difference between groups (P＜0.05);the CPCs transfected with shβ-catenin had significantly lower mRNA expression of β-catenin than those in the shβ-catenin-control group (P＜0.05).Conclusion The constructed lentiviral vector targeting the β-catenin gene has a high infection efficiency,and the successful construction of lentiviral vectors provides a technical support for analyzing the role of β-catenin in the differentiation of CPCs into auditory hair cells.