Objective To construct desmuslin (DMN) gene recombinant retroviral vector and establish its package cell lines.Methods DMN gene (-3700 bp) was amplified by using polymerase chain reaction (PCR) and cloned into retroviral vector (pRetroQ-AcGFP1-C1).Recombinant retroviral vector pRetroQ-AcGFP1-C1-DMN was constructed and identified by sequencing,and transfected into PT67 package cells by LipofectamineTM LTX and PLUSTM.Mter 48 h,cells stably expressing pRetroQ-AcGFP1-C1-DMN retroviral vector were screened by puromycin,and the viral titer was detected by infecting 293 cells.Results The recombinant retroviral vector pRetroQ-AcGFP1-C1-DMN was successfully constructed,and the cloning site and reading frame of the objective gene were confirmed by enzyme digestion and sequencing.PT67 package cell lines were successfully established and average viral titer was (2.80 ± 1.46) ×106 cfu/mL.Conclusion DMN gene retroviral vector has been successfully constructed and its package cell line PT67-DMN has been established.