[A novel system for characterization of the transcription activating proteins in mammalian cells].
A system used for detecting 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 set of recombinant plasmids namely pZHO1 (for cloning into the foreign gene fragment and as a negative 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 background 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 contransfected into such mammalian cells as C4-2, MCF-7, COS7 respectively, each with pTRE-luc and pRL-TK plasmids, the feasibility 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.
Zhang
Hao
H
Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100850, China. zhoujgx@public.bta.net.cn
Zhou
Jian-Guang
JG
Li
Jie-Zhi
JZ
Wang
Jian
J
Sun
Yu-Long
YL
Chen
Shan
S
Huang
Cui-Fen
CF
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics*; D011506:Proteins / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
D011506:Proteins; D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53; D008156:Luciferases