Chinese Journal of Tissue Engineering Research ›› 2011, Vol. 15 ›› Issue (41): 7706-7710.doi: 10.3969/j.issn.1673-8225.2011.41.026

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Construction of pTIE2-CREG-EGFP-N1 eukaryotic expression plasmid and its expression in mouse artery endothelial cells

Li Jie1, 2, Yan Cheng-hui1, Zhang Xiao-lin1, Liang Zhen-yang1, Feng Xue-yao1, Bai Jing1, 2, Han Ya-ling1   

  1. 1Department of Cardiology, Institute of Cardiovascular Disease of Chinese PLA, General Hospital of Shenyang Military Area Command of Chinese PLA, Shenyang  110016, Liaoning Province, China
    2Department of Cardiology, Xijing Hospital, Fourth Military Medical University of Chinese PLA, Xi’an   710032, Shaanxi Province, China
  • Received:2011-01-12 Revised:2011-04-25 Online:2011-10-08 Published:2011-10-08
  • Contact: Han Ya-ling, Doctor, Professor, Doctoral supervisor, Department of Cardiology, Institute of Cardiovascular Disease of Chinese PLA, General Hospital of Shenyang Military Area Command of Chinese PLA, Shenyang 110016, Liaoning Province, China hanyaling029@163. com
  • About author:Li Jie★, Master, Attending physician, Department of Cardiology, Institute of Cardiovascular Disease of Chinese PLA, General Hospital of Shenyang Military Area Command of Chinese PLA, Shenyang 110016, Liaoning Province, China; Department of Cardiology, Xijing Hospital, Fourth Military Medical University of Chinese PLA, Xi’an 710032, Shaanxi Province, China jjetli@163.com
  • Supported by:

    the National Natural Science Foundation of China (General Program), No. 30770793*, 30971218*, 81070097*; the National Natural Science Foundation of China (Youth Program), No. 30800465*; the Natural Science Foundation of Liaoning Province, No. 20092088*;
    High-Tech Research and Development Program of Liaoning Province, No. 2009225009-9*

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Abstract:

BACKGROUND: The molecular mechanism of blood vessel endothelium formation is the primary premise for cellular and gene replacement therapy.
OBJECTIVE: To construct an eukaryotic expression plasmid for cellular repressor of E1A-stimulated genes (CREG) driven by an endothelial cell specific promoter TIE2 and reported by enhanced green fluorescence protein (EGFP), and then to observe its expression in mouse artery endothelial cells in vitro.
METHODS: TIE2 promoter DNA sequence was synthesized according to GenBank, and was attached with AseⅠ and NheⅠrestriction enzyme recognition sites at both ends, respectively. The TIE2 promoter sequence was released from pUC57-TIE2 plasmid by AseⅠ and NheⅠ digestion enzymes, and then was inserted into pEGFP-N1 plasmid to form pTIE2-EGFP-N1 recombinant plasmid. CREG gene fragment was released from pcDNA3.1 myc-His/hCREG plasmid by BamHⅠ and EcoRⅠdigestion enzymes, and then was subcloned into pTIE2-EGFP-N1 plasmid in order to construct pTIE2-CREG-EGFP-N1 plasmid. The recombinant pTIE2-CREG-EGFP-N1 plasmid was then transfected into mouse artery endothelial cells by liposome. The EGFP expression was observed by fluorescence microscopy and the CREG expression was detected by western blot analysis.
RESULTS AND CONCLUSION: Identification of pTIE2-CREG-EGFP-N1 by enzyme digestion and sequencing analysis showed that lengths of the target genes which were inserted into the recombinant plasmid were correct, and that the recombinant plasmid was transfected into mouse artery endothelial cells successfully. The strong expression of EGFP was observed by fluorescence microscopy, and the expression of CREG protein was detected by western blot analyis. The results showed that the pTIE2-CREG-EGFP-N1 eukaryotic expression plasmid was successfully constructed and the recombinant vector provides a practical tool in investigating the function and regulation of CREG in the development of cardiovascular diseases and also in producing transgenic mice with endothelial cell specific over-expression of CREG.

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