[Establishment of a reporter gene-based cell screening model for discovering new agonists of estrogen receptor beta subtype].
OBJECTIVE
To establish a sensitive and efficient reporter gene-based screening model for finding agonists of estrogen receptor beta subtype.


METHODS
A 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.


RESULTS
Stably 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.


CONCLUSIONS
Stably 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.
Chen
Li-min
LM
Institute of Radiation Medicine, Academy of Military Medical Sciences, Beijing 100850, China.
Lü
Qiu-jun
QJ
Satoshi
Inoue
I
Bian
Guang-xing
GX
Chen
Zhen-hua
ZH
Wen
Li-qing
LQ
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004958:Estradiol / Q000494:pharmacology; D047629:Estrogen Receptor beta / Q000819:agonists* / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D005822:Genetic Vectors; D006801:Humans; D007150:Immunohistochemistry; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D020218:Response Elements / Q000235:genetics; D014162:Transfection
D047629:Estrogen Receptor beta; D011993:Recombinant Fusion Proteins; D004958:Estradiol; D000469:Alkaline Phosphatase