Establishment of recombinant cell line stably expressing humanα2A-adrenoceptor
10.3867/j.issn.1000-3002.2016.05.015
- VernacularTitle:稳定表达人源α2A-肾上腺素受体细胞系的建立
- Author:
Yi YANG
;
Yulei LI
;
Meng LIU
;
Peilan ZHOU
;
Ruibin SU
;
Zehui GONG
- Publication Type:Journal Article
- Keywords:
Chinese hamster ovary cells;
receptors,adrenergicα2A;
protein kinase A;
cAMP
- From:
Chinese Journal of Pharmacology and Toxicology
2016;30(5):576-581
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE To establish a new cell line that can stably express humanα2A-adrenoceptor (α2A- AR). METHODS Recombinant plasmid of α2A- AR with hygromycin B (Hygro) resistance (pcDNA3.1/Hygro-HA-α2A-AR)was stably transfected into Chinese hamster ovary(CHO)cells which had expressed protein kinase A catalytic subunits(PKAcat) with labeling of enhanced green fluorescent protein(EGFP)by a Lipofectamine based method. A single positive clone expressingα2A-AR was selected through cultivation in the presence of 200 mg · L-1 hygromycin B followed by PKA redistribution assay. The transcriptional expression ofα2A-AR was detected by quantitative real-time PCR(qRT-PCR). Time-resolved fluorescence resonance energy transfer immunoassay was used to identify the function of inhibiting cAMP accumulation of α2A-AR. RESULTS The CHO-PKAcat-α2A-AR cell line No.7 exhibited stable response in PKA redistribution assay. qRT-PCR analysis demonstrated that the high expression ofα2A-AR in the cell line remained stable after a few generations compared with CHO-PKAcat-EGFP cells (P<0.01). The cAMP accumulation caused by forskolin was significantly inhibited by α2A-AR agonist in CHO-PKAcat-α2A-AR cells(P<0.01). CONCLUSION CHO-PKAcat-α2A-AR cell line is constructed successfully, which provides an effective model for drug screening and studies of mechanisms.
