Limitations to the development of recombinant human embryonic kidney 293E cells using glutamine synthetase-mediated gene amplification: Methionine sulfoximine resistance.
To investigate the feasibility of glutamine synthetase (GS)-mediated gene amplification in HEK293 cells for the high-level stable production of therapeutic proteins, HEK293E cells were transfected by the GS expression vector containing antibody genes and were selected at various methionine sulfoximine (MSX) concentrations in 96-well plates. For a comparison, CHOK1 cells were transfected by the same GS expression vector and selected at various MSX concentrations. Unlike CHOK1 cells, HEK293E cells producing high levels of antibodies were not selected at all. For HEK293E cells, the number of wells with the cell pool did not decrease with an increase in the concentration of MSX up to 500μM MSX. A q-RT-PCR analysis confirmed that the antibody genes in the HEK293E cells, unlike the CHOK1 cells, were not amplified after increasing the MSX concentration. It was found that the GS activity in HEK293E cells was much higher than that in CHOK1 cells (P<0.05). In a glutamine-free medium, the GS activity of HEK293E cells was approximately 4.8 times higher than that in CHOK1 cells. Accordingly, it is inferred that high GS activity of HEK293E cells results in elevated resistance to MSX and therefore hampers GS-mediated gene amplification by MSX. Thus, in order to apply the GS-mediated gene amplification system to HEK293 cells, the endogenous GS expression level in HEK293 cells needs to be minimized by knock-out or down-regulation methods.
Department of Biological Sciences, KAIST, 373-1 Kusong-Dong, Yusong-Gu, Daejon 305-701, South Korea.
Da Young
DY
Yu
Department of Biological Sciences, KAIST, 373-1 Kusong-Dong, Yusong-Gu, Daejon 305-701, South Korea.
Soo Min
SM
Noh
Department of Biological Sciences, KAIST, 373-1 Kusong-Dong, Yusong-Gu, Daejon 305-701, South Korea. Electronic address: gyunminlee@kaist.ac.kr.
Gyun Min
GM
Lee
D000818:Animals; D016466:CHO Cells; D060846:Cell Engineering / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D004351:Drug Resistance / Q000235:genetics*; D005784:Gene Amplification / Q000235:genetics*; D005974:Glutamate-Ammonia Ligase / Q000235:genetics* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008717:Methionine Sulfoximine / Q000378:metabolism / Q000494:pharmacology*
D008717:Methionine Sulfoximine; D005974:Glutamate-Ammonia Ligase
CHO cells; Glutamine synthetase; HEK293 cells; Methionine sulfoximine