RAPID GENERATION OF STABLE CELL-LINES EXPRESSING CORTICOTROPIN-RELEASING HORMONE-RECEPTOR FOR DRUG DISCOVERY

Human HEK293 cells that stably express the Epstein Barr nuclear antige n 1 (EBNA1) support the episomal replication of plasmids containing th e Epstein Barr virus origin of replication (EBV oriP). A 293EBNA (293E ) cell line expressing the human corticotropin-releasing hormone recep tor subtype I (CRHR1) from an episomal plasmid was generated (293CR1s) , analyzed, adapted to spinner culture, and scaled-up for production i n less than 6 weeks, Forty-seven stable CHO cell lines transfected wit h CRHR1 were also isolated. Expression of the receptor in the best of these lines (as judged by CRH-induced cAMP production), CHO-R22, was c ompared to that in 293CR1s cells. Results indicate that the CRHR1 epis omal expression vector in 293E cells (1) rapidly generates stable cell lines suitable for scale-up; (2) is stably maintained during 3 months in culture; (3) expresses high levels of CRHR1 mRNA; and (4) expresse s significantly more CRHR1 than the CHO-R22 line. Coexpression of addi tional G protein alpha subunit (G(alpha s)) with CRHR1 in 293E cells c onverts a higher percentage of receptor to the agonist high-affinity G -protein-coupled state. Our data support the idea that using the EBV o riP-driven episomal system for gene expression results in greater prod uction of protein in a relatively short period of time. (C) 1997 Acade mic Press.