Construction of Lentivirus Vector Carrying HGF and Exploration of Transfection Condition of Rat Adipocyte-derived Mesenchymal Stem Cells.

Jia-ming Zhang; Fei-er Feng; Qian-ming Wang; Xiao-lu Zhu; Chen-cong Wang; Bo-yang Pei; Xiao-hui Zhang

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Construction of Lentivirus Vector Carrying HGF and Exploration of Transfection Condition of Rat Adipocyte-derived Mesenchymal Stem Cells.

Author: Jia-Ming ZHANG ¹ ; Fei-Er FENG ¹ ; Qian-Ming WANG ¹ ; Xiao-Lu ZHU ¹ ; Chen-Cong WANG ¹ ; Bo-Yang PEI ¹ ; Xiao-Hui ZHANG ²
Author Information

1. Peking University Institate of Hematology, Peking University People Hospital, Beijing 100044, China.
2. Peking University Institate of Hematology, Peking University People Hospital, Beijing 100044, China. E-mail: zhangxh100@sina.com.
Publication Type:Journal Article
MeSH: Adipocytes; Animals; Cell Line; Gene Expression; Genetic Vectors; Hepatocyte Growth Factor; Humans; Lentivirus; Mesenchymal Stromal Cells; Plasmids; Rats; Transfection
From: Journal of Experimental Hematology 2015;23(2):490-494
CountryChina
Language:Chinese
Abstract:
OBJECTIVEThis study was to construct the lentivirus vector carrying hepatocyte growth factor (HGF) gene and to explore the condition for transfecting the adipocyte-derived mesenchymal stem cells (ADSC) by HGF lentivirus.
METHODSThe target gene was obtained from plasmid carrying HGF gene by PCR and was cloned into GV287 vector. The recombinant GV287-HGF vector plasmid and lentivirus-packing plasmid were co-transfected into 293 T cells to generate HGF lentivirus, and the virus titer was assayed, then the ADSC were transfected by using recombinant HGF lentivirus, and the optimal multplicity of infection (MOI) was detected.
RESULTSThe PCR product of HGF gene was consistent with expectant sizes, suggesting that the electrophoretic result of recombinant GV287-HGF plasmid PCR product was correct. The sequencing analysis of cleaved product showed consistance of obtained results with the sequences of target gene, suggesting correct construction of recombinant lentivirus carrying HGF gene. The ELISA showed that the virus tilter was 5×10(8) TU/ml. The optimal MOI for transfecting ADSC with recombinant lentivirus carrying HGF gene was 50.
CONCLUSIONThe lentivirus vector expressing human HGF gene has been constructed, and transfected the ADSC succesfully. This study lays a foundation for further stadying the ADSC over-expressioning HGF, treating the radiation damage of bone marrow and impartant internal organs.