›› 2013, Vol. 33 ›› Issue (4): 404-.doi: 10.3969/j.issn.1674-8115.2013.04.005

• Original article (Basic research) • Previous Articles     Next Articles

Study on activity of GAL4/VP16 fusion artificial transcription factor

ZHANG Ping1, YING Lei1, QIAN Guan-xiang1, XU Rang2   

  1. 1.Department of Biochemistry and Molecular Cellular Biology, Basic Medical College, Shanghai Jiaotong University, Shanghai 200025, China； 2.Scientific Research Center, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200092, China
  • Online:2013-04-28 Published:2013-05-03
  • Supported by:

    National Natural Science Foundation of China, 81070135

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Abstract:

Objective To construct GAL4/VP16-up-stream activating sequence (UAS) system, which is composed of expression vector containing GAL4/VP16 fusion transcriptional factor and expression vector containing UAS promoter driving reporter gene enhanced green fluorescent protein (EGFP), and evaluate the activity of GAL4/VP16 fusion transcriptional factor. Methods The expression vector pcDNA3-GAL4/VP16 containing GAL4/VP16 fusion transcriptional factor was constructed, and the expression vector pGL3-G5E1b-TATA-EGFP was also constructed, in which the reporter gene EGFP was driven by G5E1b-TATA promoter consisted of five tandem-repeat copies of UAS and E1b core promoter. Hep3B and HEK 293 cells were transiently transfected by these two vectors. After 48 h, the expression of EGFP was determined by RT-PCR and flow cytometry, and the effect of GAL4/VP16 fusion transcriptional factor on the G5E1b-TATA promoter activity was observed. Results The activity of G5E1b-TATA promoter was significantly up-regulated by GAL4/VP16 fusion transcriptional factor, and might even reach the level of cytomegalovirus (CMV) promoter. Conclusion In transcriptional level, the GAL4/VP16 fusion transcriptional factor can significantly up-regulate the activity of G5E1b-TATA promoter, and the G5E1b-TATA promoter can drive the efficient expression of downstream target genes in the presence of GAL4/VP16 protein. It is expected to make a valuable contribution in the study of gene function and gene therapy.

Key words: GAL4/VP16, artificial transcription factor, enhanced green fluorescent protein