›› 2010, Vol. 30 ›› Issue (12): 1501-.doi: 10.3969/j.issn.1674-8115.2010.12.013

• Original article (Basic research) • Previous Articles     Next Articles

Construction of mouse osteoinductive factor gene retroviral vector and its expression in 293T cells

ZHENG Cui-xia1, ZHANG Xiao-na2, HAN Bing1, SONG Huai-dong2, MA Qin-yun2   

  1. 1.Department of Respiratory Medicine, The Ninth People's Hospital,Shanghai Jiaotong University School of Medicine, Shanghai 200011, China；2.Molecular Medicine Center, Shanghai Institute of Endocrinology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China
  • Online:2010-12-25 Published:2010-12-31
  • Supported by:

    Shanghai Natural Science Foundation, 10ZR1418300

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Abstract:

Objective To construct mouse osteoinductive factor (OIF) gene recombinant retroviral vector, and investigate its expression in 293T cells. Methods OIF-3FLAG gene was amplified with PCR and cloned into retroviral vector (Puro-IRES-GFP) after sequencing, and recombinant retroviral vector pMSCV PIG-OIF-3FLAG was constructed and identified by sequencing. PMSCV PIG-OIF-3FLAG retroviral vector with helper vectors of VSAG and GAG-POL were co-transfected into 293T cells by lipofectamine2000. Forty-eight hours after transfection, the expression of green fluorescent protein (GFP) was examined by fluorescence microscopy. The supernatant of 293T cells, which were tansfected with pMSCV PIG-OIF-3FLAG retroviral vector and helper vectors of VSAG and GAG-POL, was used to reinfect 293T cells. After 48 h, 293T cells stably expressing pMSCV PIG-OIF-3FLAG retroviral vector were screened by 3 μg/mL puromycin for 7 d, and the expression of OIF mRNA and protein of 293T cells was detected by Real-Time PCR and Western blotting, respectively. Results OIF gene retroviral vector pMSCV PIG-OIF-3FLAG was successfully constructed, and the cloning site and reading frame of objective gene were confirmed by enzyme digestion and sequencing. Then, 293T cells were infected by retrovirus supernatant, and 293T cells with stable expression of mouse OIF were obtained by puromycin screening. Real-Time PCR and Western blotting revealed high expression of OIF mRNA and protein in 293T cells. Conclusion Mouse OIF gene retroviral vector has been successfully constructed, and 293T cells with stable expression of OIF gene are obtained.

Key words: retroviral vector, mouse osteoinductive factor, 293T cell