Development of a cell-based assay for screening of phosphodiesterase 10A (PDE10A) inhibitors using a stable recombinant HEK-293 cell line expressing high levels of PDE10A.
The cDNA encoding PDE10A (phosphodiesterase 10A) was cloned and a stable recombinant HEK-293 (human embryonic kidney-293) cell line expressing high levels of PDE10A was generated. Transient transfection of pCRE-Luc plasmid, harbouring the luciferase reporter gene under the control of CRE (cAMP-response element)-binding sequence, into the stable recombinant cell line, followed by treatment with PDE10 inhibitor, resulted in a dose-dependent increase in luciferase activity. This method provides a simple and sensitive cell-based assay for screening of PDE10 inhibitors for development of novel therapeutics for the treatment of neurological disorders.
Bora
Roop Singh
RS
Department of Biotechnology, Ranbaxy Research Laboratories, Plot No. 20, Sector 18, Udyog Vihar Industrial Area, Gurgaon-122 001, Haryana, India. roop.singh@ranbaxy.com
Gupta
Dikshi
D
Malik
Renu
R
Chachra
Sonia
S
Sharma
Pratibha
P
Saini
Kulvinder Singh
KS
D001681:Biological Assay / Q000379:methods*; D015374:Biosensing Techniques / Q000379:methods*; D002460:Cell Line; D006801:Humans; D007668:Kidney / Q000187:drug effects* / Q000378:metabolism*; D008163:Luminescent Measurements / Q000379:methods; D010726:Phosphodiesterase Inhibitors / Q000008:administration & dosage* / Q000032:analysis; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*
D010726:Phosphodiesterase Inhibitors; D011994:Recombinant Proteins; C119843:PDE10A protein, human; D010727:Phosphoric Diester Hydrolases