Establishment of a reporter gene-based cell screening model for discovering new agonists of estrogen receptor beta subtype.
- Author:
Li-min CHEN
1
;
Qiu-jun LÜ
;
Inoue SATOSHI
;
Guang-xing BIAN
;
Zhen-hua CHEN
;
Li-qing WEN
Author Information
- Publication Type:Journal Article
- MeSH: Alkaline Phosphatase; genetics; metabolism; Cell Line; Estradiol; pharmacology; Estrogen Receptor beta; agonists; genetics; metabolism; Gene Expression Regulation; drug effects; Genetic Vectors; Humans; Immunohistochemistry; Promoter Regions, Genetic; genetics; Recombinant Fusion Proteins; genetics; metabolism; Response Elements; genetics; Transfection
- From: Acta Pharmaceutica Sinica 2006;41(8):721-726
- CountryChina
- Language:Chinese
-
Abstract:
AIMTo establish a sensitive and efficient reporter gene-based screening model for finding agonists of estrogen receptor beta subtype.
METHODSA recombinant vector pTAL-ERE-SEAP was constructed by inserting a synthetic sequence composed of five estrogen responsive elements in front of promoter of pTAL-SEAP vector. pTAL-ERE-SEAP was then transfected into human embryonic kidney (HEK293) cells. G418 (200 microg x mL(-1)) was added to select positive clones that can be induced by E2 to express reporter gene SEAP. The speciality was tested by several ligands of relative nuclear receptors of the same family. The stability of the model, the time-effect relationship, the dose-response relationship, and the immunocytochemistry staining of ERbeta expression after transfection were observed. 2 622 compounds were screened by using this model.
RESULTSStably transfected clones were obtained. The expression levels of reporter gene SEAP of positive clones was induced by E2 in a dose-response and time-effect relationship manners. The Z' factor value was 0.7. The expression levels of dexamethasone and other ligands were low. The result of immunocytochemistry staining showed the expression of ERbeta. E2 had no proliferating effects on stably transfected clones.
CONCLUSIONStably transfected positive clones transfected with recombinant vector pTAL-ERE-SEAP were obtained. The positive clones may be used to screen for agonists of estrogen receptor beta subtype by measurement of luminescent value of expressed SEAP in wells of microlitre plate.
