【Abstract】Objective To construct dual-functioning lentiviral vector(lv-dual) encoding both zinc transporter SLC39A8(ZI-P8)targeting shRNA and growth differentiation factor 5 (GDF5) and detect its expression level, induce pluripotent stem cells (iPSCs) stable cell line overexpressing GDF5 and silencing ZIP8 and provide seed cells for cell therapy for osteoarthritis. Methods The lv-dual was constructed by sequentially incorporating GDF5 encoding fulllength sequence amplified from human GDF5 cDNA and shRNA sequence of mouse ZIP8 to double enzyme digested pRNAU6.2 vector. The lvdual vector and negative control plasmid cotransfected HEK293T cells with lentivirus packaging plasmids, respectively. Viral supernatant was collected to infect iPSCs and protein overexpression and silencing effect of GDF5 and ZIP8 were detected by realtime quantitative PCR and Western blot. G418 treatment was used to screen stable iPSCs clones. Results The recombinant lentivirus pRNAU6.2-CMV-hGDF5-mshZIP8-U6 was successfully constructed. The iPSCs cells with stable over expression of GDF5 and suppress expression of ZIP8 was obtained by viral infection and G418 screening. Conclusion Successfully constructed pRNAT U6.2-GDF5-msh ZIP8-U6 lentiviral vector can both effectively promote GDF5 synthesis and suppress the expression of ZIP8 by the release of corresponding shRNA. The successful establishment of a stable iPSCs cell lines can provide a reliable cellular platform for cell therapy for osteoarthritis.