CRCU

Chemical Research in Chinese Universities ›› 2009, Vol. 25 ›› Issue (5): 677-680.

• Articles • Previous Articles     Next Articles

Effect of Regulation of HSV-tk Gene Expression and Tumor Killed Activity with a Single Tetracycline-regulatable Plasmid Vector on HeLa Cells

WANG Qian1,2, DU Zhen-wu1,3, MA Qing-shan4, ZHANG Yu-cheng1, WU Xiao-dong1, YANG Shao-juan1, WANG Ya-li1 and ZHANG Gui-zhen1*   

  1. 1. Central Laboratory,
    2. Department of Urinary Surgery, China-Japan Union Hospital, Jilin University, Changchun 130033, P. R. China;
    3. School of Pharmaceutical Sciences,
    4. Department of Pediatrics, The First Hospital, Jilin University, Changchun 130021, P. R. China
  • Received:2009-03-11 Revised:2009-03-23 Online:2009-09-25 Published:2009-12-07
  • Contact: ZHANG Gui-zhen. E-mail: zhangguizhenjlu@yahoo.com
  • Supported by:

    Supported Partly by the Program of Jilin Provincial Science & Technology Department, China(Nos.200705335 and 200504057) and the Program of Development and Reform Commission of Jilin City, China(No.2004987).

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Abstract:

To construct a single tetracycline-regulatable plasmid vector based on the double tetracycline-regulatable plasmid vector system for regulating HSV-tk gene expression so as to effectively kill HeLa cells. Two tetracycline operator(TetO2) was cloned into pcDNA3.1 and a cassette was made for a cytomegalovirus-type 2 tetracycline operator(CMV-TetO2) promoter, and the obtained vector was named pcDNA3.1-CMV-TetO2. Herpes simplex virus thymidine kinase(HSV-tk) gene and tetracycline repressor(TR) gene were cloned into pcDNA3.1-CMV-TetO2 and the two genes were linked with internal ribosome entry site(IRES) to gain a vector named pcDNA3.1-CMV-TetO2- HSV-tk-IRES-TR. The HeLa cells were stablly transfected with pcDNA3.1-CMV-TetO2-HSV-tk-IRES-TR plasmid. The expression of HSV-tk and TR were detected by RT-PCR, the tumorcidal activity of HSV-tk/GCV was determined by MTT assay. In Hela cells transfected with the above plasmid vector, HSV-tk gene and TR gene can be expressed lowly and the concentration of GCV producing a 50% decrease in cell viability was about 50 ?g/mL without adding deoxycycline; in contrast, the expessions of HSV-tk gene and TR gene increased significantly and the concentration of GCV producing a 50% decrease in cell viability was about 5 ?g/mL with adding deoxycycline. Therefore      tetracycline can regulate the expression and tumorcidal activity of HSV-tk gene in HeLa cells with this single plasmid vector.

Key words: HSV-tk gene; Tetracycline; HeLa cells; Gene expression