TY - JOUR
T1 - A hybrid system using both promoter activation and gene amplification for establishing exogenous protein hyper-producing cell lines
AU - Dong, Xiao Yan
AU - Teruya, Kiichiro
AU - Katakura, Yoshinori
AU - Zhang, Yingpei
AU - Miura, Takumi
AU - Daimon, Yoshihito
AU - Mori, Tetsuya
AU - Ohashi, Hideya
AU - Shirahata, Sanetaka
PY - 2003
Y1 - 2003
N2 - We previously developed a promoter-activated production (PAP) system using amplified ras oncogene to activate the cytomegalovirus (CMV) promoter controlling the foreign gene in mammalian cells. CHO cells were demonstrated to be suitable for the PAP system. Here, we show that very high-level production of a recombinant protein was achieved when the human CMV promoter was inserted into a glutamine synthetase (GS) minigene expression plasmid, pEE14. A highly productive host CHO cell line, ras clone I containing amplified ras oncogene, was further transfected with the plasmid expressing both hIL-6 gene and GS minigene, and selected with methionine sulphoximine. We were able to establish a hIL-6 hyper-producing cell line, D29, which exhibited a peak productivity rate of approximately 40 μg hIL-6 10-6 cells day-1 through a combination of the PAP system and the GS gene amplification system. The cellular productivity of D29 cells was about 13-fold higher than control hIL-6-producing cells derived from CHO cells whose hIL-6 gene was amplified by the GS gene amplification system, and about 5-fold higher than the I13 cells established by the PAP system, which contains amplified ras oncogene and non-amplified hIL-6 gene. When D29 cells were cultured for a month, an accumulation rate of approximately 80 μg hIL-6 ml-1 per 3 days was achieved on the 9th day. These results indicate that this PAP and GS hybrid system enables the efficient and rapid establishment of recombinant protein hyper-producing cell lines.
AB - We previously developed a promoter-activated production (PAP) system using amplified ras oncogene to activate the cytomegalovirus (CMV) promoter controlling the foreign gene in mammalian cells. CHO cells were demonstrated to be suitable for the PAP system. Here, we show that very high-level production of a recombinant protein was achieved when the human CMV promoter was inserted into a glutamine synthetase (GS) minigene expression plasmid, pEE14. A highly productive host CHO cell line, ras clone I containing amplified ras oncogene, was further transfected with the plasmid expressing both hIL-6 gene and GS minigene, and selected with methionine sulphoximine. We were able to establish a hIL-6 hyper-producing cell line, D29, which exhibited a peak productivity rate of approximately 40 μg hIL-6 10-6 cells day-1 through a combination of the PAP system and the GS gene amplification system. The cellular productivity of D29 cells was about 13-fold higher than control hIL-6-producing cells derived from CHO cells whose hIL-6 gene was amplified by the GS gene amplification system, and about 5-fold higher than the I13 cells established by the PAP system, which contains amplified ras oncogene and non-amplified hIL-6 gene. When D29 cells were cultured for a month, an accumulation rate of approximately 80 μg hIL-6 ml-1 per 3 days was achieved on the 9th day. These results indicate that this PAP and GS hybrid system enables the efficient and rapid establishment of recombinant protein hyper-producing cell lines.
UR - http://www.scopus.com/inward/record.url?scp=23944527075&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=23944527075&partnerID=8YFLogxK
U2 - 10.1023/B:CYTO.0000039901.92984.7a
DO - 10.1023/B:CYTO.0000039901.92984.7a
M3 - Article
C2 - 19003202
AN - SCOPUS:23944527075
VL - 43
SP - 11
EP - 17
JO - Cytotechnology
JF - Cytotechnology
SN - 0920-9069
IS - 1-3
ER -