Selection of transfected cells with stably integrated GOI is of great interest in the #Biosimilar and #Biologics industry currently. #Metabolic markers give a very precise control over cell behavior as well as taking care of #GeneAmplification requirements. Glutamine synthetase (GS) system of selection is the current favorite, because of its ability to pull down developmental timelines.
Principle
GS converts #glutamate to #glutamine, an essential element for many cells (#NS0 as a native trait, #CHOK1SV GS-KO as an engineered trait) with the help of ammonium ion. Cells required here have to be GS -ve. Complementation of glutamine auxotrophy is used as a basis of GS selection system.
Untransfected control cells survive because they are supplied with #glutamine necessary to grow.
Untransfected (although transfected) cells die because they are supplied with media not containing #glutamine. #MSX, a drug similar to glutamate, binds to GS, thereby inhibiting the production of glutamine, which is necessary for cell to grow.
Transfected cells survive because they produce GS as well as downstream cloned GOI. Although MSX blocks GS, by challenging the cells with increasing concentration of MSX we can amplify the GS+GOI cassette and finally achieve a stable cell pool with multiple copies of GS+GOI cassette. This would yield us humongous amount of protein of interest.
Once selected, transfected cell lines derived from the GS negative host do not require MSX in the culture medium.
Merits
The GS expression and selection system does not typically require multiple rounds of amplification to isolate high-producing clones (Brown, 1992), shortening the developmental timeline.
This system mitigates ammonia accumulation in the culture medium because the overexpressed GS catalyzes the conversion of glutamate and ammonia into glutamine.
Demerits:
Although a June,2006 study shows gene amplification with increasing concentrations of MSX, but many of the amplified clones did not stably maintain their enhanced productivity even when cultured in the presence of high concentrations of MSX.
Many non-producing clones have also been shown to survive the amplification process.
More to munch on:
#https://www.nature.com/articles/s41598-018-23720-9
#https://www.sigmaaldrich.com/IN/en/technical-documents/technicalarticle/genomics/advanced-gene-editing/msx-amplification
#https://bioscience.lonza.com/lonza_bs/IN/en/mammalian-protein-expression
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