Rapid generation of stable cell lines expressing corticotropin-releasing hormone receptor for drug discovery.
Human HEK293 cells that stably express the Epstein Barr nuclear antigen 1 (EBNA1) support the episomal replication of plasmids containing the Epstein Barr virus origin of replication (EBV oriP). A 293EBNA (293E) cell line expressing the human corticotropin-releasing hormone receptor subtype I (CRHR1) from an episomal plasmid was generated (293CR1s), analyzed, adapted to spinner culture, and scaled-up for production in less than 6 weeks. Forty-seven stable CHO cell lines transfected with CRHR1 were also isolated. Expression of the receptor in the best of these lines (as judged by CRH-induced cAMP production), CHO-R22, was compared to that in 293CR1s cells. Results indicate that the CRHR1 episomal 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) expresses significantly more CRHR1 than the CHO-R22 line. Coexpression of additional G protein alpha subunit (G alpha s) with CRHR1 in 293E cells converts a higher percentage of receptor to the agonist high-affinity G-protein-coupled state. Our data support the idea that using the EBV oriP-driven episomal system for gene expression results in greater production of protein in a relatively short period of time.
Horlick
R A
RA
Applied Biotechnology Department, DuPont Merck Pharmaceutical Co., Wilmington, Delaware 19880-0400, USA.
Sperle
K
K
Breth
L A
LA
Reid
C C
CC
Shen
E S
ES
Robbins
A K
AK
Cooke
G M
GM
Largent
B L
BL
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D015195:Drug Design; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D004854:Herpesvirus 4, Human / Q000235:genetics; D006801:Humans; D010957:Plasmids / Q000235:genetics; D012313:RNA / Q000235:genetics / Q000378:metabolism; D018019:Receptors, Corticotropin-Releasing Hormone / Q000096:biosynthesis / Q000145:classification / Q000235:genetics*; D018741:Replication Origin
D019309:Epstein-Barr Virus Nuclear Antigens; D018019:Receptors, Corticotropin-Releasing Hormone; D012313:RNA