[Construction of cochlear progenitor cells with recombinant short-hairpin RNA lentiviral vector inhibiting the expression of the β-catenin gene].

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-SL3, pLV/helper-SL4, 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 5.05×10(7) and 4.34×10(7), 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.