Glutamine synthetase gene knockout-human embryonic kidney 293E cells for stable production of monoclonal antibodies

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Previously, it was inferred that a high glutamine synthetase (GS) activity in human embryonic kidney (HEK) 293E cells results in elevated resistance to methionine sulfoximine (MSX) and consequently hampers GS-mediated gene amplification and selection by MSX. To overcome this MSX resistance in HEK293E cells, a GS-knockout HEK293E cell line was generated using the CRISPR/Cas9 system to target the endogenous human GS gene. The GS-knockout in the HEK293E cell line (RK8) was confirmed by Western blot analysis of GS and by observation of glutamine-dependent growth. Unlike the wild type HEK293E cells, the RK8 cells were successfully used as host cells to generate a recombinant HEK293E cell line (rHEK293E) producing a monoclonal antibody (mAb). When the RK8 cells were transfected with the GS expression vector containing the mAb gene, rHEK293E cells producing the mAb could be selected in the absence as well as in the presence of MSX. The gene copies and mRNA expression levels of the mAb in rHEK293E cells were also quantified using qRT-PCR. Taken together, the GS-knockout HEK293E cell line can be used as host cells to generate stable rHEK293E cells producing a mAb through GS-mediated gene selection in the absence as well as in the presence of MSX.
Publisher
WILEY
Issue Date
2018-05
Language
English
Article Type
Article
Keywords

PROTEIN-PRODUCTION; EXPRESSION; LINES

Citation

BIOTECHNOLOGY AND BIOENGINEERING, v.115, no.5, pp.1367 - 1372

ISSN
0006-3592
DOI
10.1002/bit.26552
URI
http://hdl.handle.net/10203/241403
Appears in Collection
BS-Journal Papers(저널논문)
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