Chinese Journal of Tissue Engineering Research ›› 2011, Vol. 15 ›› Issue (6): 1009-1014.doi: 10.3969/j.issn.1673-8225.2011.06.014

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Construction and transfection of lentiviral eukaryotic expression vector carrying basic fibroblast growth factor

Hu Yang1, Sheng Lei2, Ma Ying1, He Hui-yu1, Abulizi•Abudula2, Aerziguli1   

  1. 1First Affiliated Hospital, Xinjiang Medical University, Urumqi  830054, Xinjiang Uygur Autonomous Region, China
    2Key Laboratory of Endemic Disease and Molecular Biology, Xinjiang Medical University, Urumqi  830054, Xinjiang Uygur Autonomous Region, China
  • Received:2010-09-10 Revised:2010-10-12 Online:2011-02-05 Published:2011-02-05
  • Contact: He Hui-yu, Doctor, Professor, Chief physician, First Affiliated Hospital, Xinjiang Medical University, Urumqi 830054, Xinjiang Uygur Autonomous Region, China
  • About author:Hu Yang★, Master, Physician, First Affiliated Hospital, Xinjiang Medical University, Urumqi 830054, Xinjiang Uygur Autonomous Region, China joe98344@sina.cn
  • Supported by:

    the Science and Technology Project of Xinjiang Uygur Autonomous Region, No. 200533118*; the Key Project of Scientific Research for Scientific Research Planning of Higher Education of Xinjiang Uygur Autonomous Region, No. xj EDU2009I22*; the Science and Technology Project of Urumqi City, No. Y09131002*

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Abstract:

BACKGROUND: Previous studies mainly used plasmid vector. Due to its low transfection efficiency, liposome transfection reagent is needed. The transfection has cytotoxicity, with complex operation.
OBJECTIVE: To construct lentiviral vector carrying human basic fibroblast growth factor (bFGF) gene, to transfect ossification-induced bone marrow mesenchymal stem cells (BMSCs) and to identify the expression of bFGF gene.
METHODS: Experimental group: bFGF gene primers were designed, total RNA was extracted from placental tissue using TRIzol. The bFGF gene amplified by RT-PCR, and the PCR product was connected to the pLenti6/V5-D-TOPO ® expression plasmid, which proved correctly constructed by the Xho-Ⅰand BamH-Ⅰ double digestion and DNA sequencing. With the promotion of Lipofectamine 2000, bFGF-pLenti6/V5 plasmid and packaging plasmid pLP1, pLP2, pLP/VSVG cotransfected 293FT cell line, bFGF-lentivirus supernatant was collected and infected the passageⅡ BMSCs. Control group: the green fluorescent protein (GFP) gene was amplified by PCR from GFP-PMSLV-Plazmid, GFP gene was connected to pLenti6/V5-D-TOPO ® expression plasmid, and transfected into the BMSCs. The bFGF and GFP gene expression was detected by RT-PCR and Western-blot assay.
RESULTS AND CONCLUSION: At 48 hours after transfection, GFP of the control BMSCs was visible. At 15 days after transfection, the bFGF expression of the experimental BMSCs was detected by RT-PCR and Western-blot. These indicated that lentiviral vectors carrying human bFGF and GFP were successfully constructed, and a method of transfecting rabbit BMSCs was constructed.

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