Chinese Journal of Tissue Engineering Research ›› 2012, Vol. 16 ›› Issue (1): 11-16.doi: 10.3969/j.issn.1673-8225.2012.01.002

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Construction and expression of pHSV1-TK- IRES2-EGFP eukaryotic vector in mouse bone marrow mesenchymal stem cells

Wu Li-chuan1, Yang Kun1, Liu Yong-zhe1, Chen Peng1, Xu Wen-gui2   

  1. 1Department of Toxicology, School of Public Health, Tianjin Medical University, Tianjin  300070, China; 2Tianjin Medical University Cancer Institute and Hospital, Tianjin  300060，China
  • Received:2011-07-19 Revised:2011-09-16 Online:2012-01-01 Published:2012-01-01
  • Contact: Correspondence to: Yang Kun, Associate professor, Master’s supervisor, Department of Toxicology, School of Public Health, Tianjin Medical University, Tianjin 300070, Chinayangkun@tijmu.edu.cn Correspondence to: Xu Wen-gui, Chief physician, Master’s supervior, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, ChinaWenguixy@tom.com
  • About author:Wu Li-chuan★, Studying for master’s degree, Department of Toxicology, School of Public Health, Tianjin Medical University, Tianjin 300070, Chinaxihuzi0217@163.com
  • Supported by:

    Supported by: the Natural Science Foundation of Tianjin, No. 08JCZDJC23700*; the Science and Technology Research Foundation of Tianjin Education Committee, No. 20080133*

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Abstract:

BACKGROUND: Up to date, thestudies regarding the construction of herpes simplex virus 1-thymidine kinase (HSV1-TK) and enhanced green fluorescent protein (EGFP) reporter gene co-expression vector and its expression in mouse bone marrow mesenchymal stem cells are few. OBJECTIVE: To construct a pHSV1-TK- IRES2-EGFP eukaryotic vector, and to observe the expression of HSV1-TK in mouse bone marrow mesenchymal stem cells. 
METHODS: The HSV1-TK cDNA fragments were obtained by polymerase chain reaction (PCR) from pHSV106 and Bgl Ⅱ, Sal Ⅰ two restriction sites were added, after T vector cloning, the recombinant plasmid pHSV1-TK/18T was digested by two restrictive endonucleases, and then HSV1-TK cDNA was collected and recombined with eukaryotic vector pIRES2-EGFP by using gene recombination technique. The recombinant plasmid pHSV1-TK- IRES2-EGFP was transfected into mouse bone marrow mesenchymal stem cells in vitro with lipofectamine transfection reagent.
RESULTS AND CONCLUSION: A length of 1 130 bp with Bgl Ⅱ and Sal Ⅰ target gene sequences was obtained by PCR. And the pHSV1-TK- IRES2-EGFP expression plasmid was constructed successfully. The TK/GFP green fluorescent protein was detected after transfection. At the same time, the expression of HSV1-TK mRNA was determined. The eukaryotic vector pHSV1-TK- IRES2-EGFP was successfully constructed, and effectively expressed HSV1-TK mRNA in mouse bone marrow mesenchymal stem cells in vitro.

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