[Establishment of estrogen receptor alpha trans-activation system].
OBJECTIVE
To construct estrogen receptor alpha (ERalpha) trans-activation system.


METHODS
The full length ERalpha and its different function regions [(transcriptional activation function 1 (AF1), DNA binding domain (DBD), and transcriptional activation function 2 (AF2)] were amplified from pcDNA3-ERalpha by PCR and cloned into the pGAL vector. The expressions of the recombinant plasmids constructed were detected via immunoblotting. The 293T cells transfected with recombinant plasmids of full length ERalpha, its different function regions and empty vector were divided into 5 groups; each group was divided into 2 parts which were treated with or without estrogen (E(2)). The transcriptional activity of each group was detected in 293T cells after the recombinant plasmid was co-transfected with 0.2 microg of estrogen receptor element luciferase (ERE-LUC) and 0.1 microg of plasmid expressing beta-galactosidase and treated with or without 10 nmol/L E(2) for 24 hours.


RESULTS
The full length ERalpha and its different function regions were expressed in the 293T cells. Compared with the empty pGAL vector, the transcription activities of full length ERalpha, AF1, AF2 and DBD recombinant plasmids were raised about 20.44 +/- 1.01, 2.09 +/- 0.11, 8.09 +/- 0.30 and 1.05 +/- 0.09 fold, respectively, with the induction of E(2) after transfection in the 293T cells.


CONCLUSIONS
The trans-activation system of ERalpha has been successfully established.
Li
Jie-Ping
JP
Beijing Institue of Biotechnology, Beijing 100850, China.
Xiong
Zhi-Hong
ZH
Yang
Zhi-Hong
ZH
Wang
Zhao-Yun
ZY
Yang
Li-Ping
LP
Ye
Qi-Nong
QN
D002460:Cell Line; D047628:Estrogen Receptor alpha / Q000235:genetics* / Q000378:metabolism; D004967:Estrogens / Q000494:pharmacology*; D005822:Genetic Vectors; D006801:Humans; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
D047628:Estrogen Receptor alpha; D004967:Estrogens; D011994:Recombinant Proteins