Selection of transfected cells with stably integrated and amplified GOI fragments 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.
Principle
Cells required here have to be DHFR -ve.
Untransfected control cells survive because they are supplied with hypoxanthine and thymidine containing media. So they synthesize nucleotides via salvage pathway.
Untransfected (although transfected) cells die because they are supplied with media not containing hypoxanthine and thymidine. Also, supplemented with #Methotrexate (MTX) which will block DHFR products produced by the transfected neighboring cells.
Transfected cells survive because they produce DHFR as well as upstream cloned GOI. Although MTX blocks DHFR, by challenging the cells with increasing concentration of MTX we can amplify the DHFR+GOI cassette and finally achieve a stable cell pool with multiple copies of DHFR+GOI cassette. This would yield us humongous amount of protein of interest.
MTX resistance in cells happen through one or more of the following ways: (a) alteration in affinity of MTX for DHFR, (b) altered transport of MTX, (c) overproduction of DHFR as a result of gene amplification. Once selected, transfected cell lines derived from the DHFR negative host do not require MTX in the culture medium.
Merits
The DHFR system results in very high levels of amplification (up to 1000 copies per cell in some cases) and expression.
Demerits:
DHFR amplification process is lengthy and may require several months to isolate and characterize a stable, amplified line.
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