JGG 2002, 29(11): 983-989 DOI:   ISSN: 1673-8527 CN: 11-5450/R           

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A Novel System fro Characterization of the Transctiption Activating Proteins in Mammalian Cells

ZHANG Hao;ZHOU Jian-Guang;LI Jie-Zhi;WANG Jian;SUN Yu-Long; CHEN Shan; HUANG Cui-Fen

Institute of Biotechnology,Academy of Military Medical Sciences, Beijing 100850,China


A system used for detecing the transcriptional activating activity of the function-unknown gene products in mammalian cells was developed, Based on the plasmid pTet-Off and the eukaryotic expressing vector pCDNA3.1B(-)/myc-his, firstly, we constructed a sct of recombinant plasmids namely pZHO1 (for cloning into the foreign gene fragment and as a negtive control)、pZHO2 (as a positive control). The system also includes the plasmids pTRE-luc(encoding the Firefly luciferase reporter gene) and pRL-TK (encoding Renilla luciferase gene as blackground control). To confirm the feasibility of the system, the plasmids pZHO1, pZHO2 and pZHO3 (encoding p53 transcriptional activating domain, containing 73 amino acids in its N terminal) was cotransfected into such mammlian cells as C4-2,MCF-7,COS-7 respectively, each with pTRE-luc and pRL-TK plasmids, the feasibilty of the system was determined by comparing the relative activity of Firefly luciferase activity ratio of and Renilla in different transfecting panels. Our research result showed that the system we constructed can be used for detecting the transcriptional activating activity of the target protein molecules in mammalian cells.

Keywords transcriptional activation   dual-luciferase reporter system   Tet-Off  
Received 1900-01-01 Revised 1900-01-01 Online 2002-11-10 
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