How the DHFR System works
The diagram to the left shows how the DHFR enzyme catalyses the conversion of folate to tetrahydrofolate. This precursor is necessary for de-novo synthesis of purines and pyrimidines (Goeddel, 1990), allowing cells to replicate.
When the protein expression system in CHO cells has the DHFR deletion, researchers transfect CHO cells with recombinant DNA consisting of the gene of interest closely linked to the gene for DHFR. The methotrexate (MTX) selection system is used to select CHO cells producing the protein of interest. MTX, a drug similar to folate, binds to DHFR, thereby inhibiting the production of tetrahydrofolate, which is necessary for the de-novo synthesis of purines and pyrimidines (Goeddel, 1990).
During the gene amplification process, CHO cells are cultured in increasingly higher levels of MTX. CHO cells that have increased copies of the DHFR gene, combined with the gene of interest, will be selected.
CHO cells with insufficient levels of DHFR are deprived of nucleoside precursors (hypoxanthine and thymidine) and die. Once selected, transfected cell lines derived from the CHO DHFR negative host do not require MTX in the culture medium.