I am trying to express proteins in CHO DG44 cells. I am following the methods in the Freedom™ DG44 Kit USER GUIDE. After transfection, I recovered viability to 90% and adapted cells to 1 µM MTX after treating them with 50 nM MTX. I confirmed protein expression through SDS-PAGE.
These cells are currently stored as stocks in liquid nitrogen. To express the protein later, I have freshly cultured the stocks. How is protein expression typically confirmed in such cases?
I cultured the stocks and checked for protein expression by SDS-PAGE after about 5 days, but found minimal protein expression. My hypothesis is either that I did not sub-culture initially, so the cells were not stable, or that protein expression was low due to insufficient MTX treatment. That's what I think. Could you tell me how protein is usually obtained after adapting to MTX?
Dear Eunyoung Seo
Did you perform transient tranfection?
Differntelly from E.coli or other bacteria for which the presence of replication origin and antibiotic resistance in plasmid allow it to propagate during cell replication, and therefore once you have transfromed cells you can froze it, propagate and express again the protein at similar level, this do not happen in mammalian cells. In mammalian cells when you are perfroming trasfections the plasmid is manitaned for limited times only in the trasfected cells but do not propagate and replicate therefore is progressivelly lost once the cells duplicates, for these reason normally the cells are duplicated until and cell density close to the maximun and then trasfected for some days, in this way if the transfection rate is high, ah nigh % of cells contain the vector and therefore are able to express the protein of interest, but to reproduce the protein we do not store the trasfecterd cells and propagated again but we have to perform a new trasfection.
On the contrary if from a trasfection, you have produced a stable cell lines with the gene integrated in the genome, you can store and propagate it for a long time but it do not seems that this is your case.
best regards
Manuele
Thank you for your answer. If I explain the experimental process, after transfection, we subcultured in CD OptiCHO medium (+Gln) without hypoxanthine and thymidine until the viability reached over 90%, and through this, we determined that we had secured a stable cell line. We treated these cells with MTX and adapted them to 1 uM MTX. After that, we stored the cells in liquid nitrogen, and when we cultured the stock again, there was a problem that the protein expression was much lower than before. Do you know the reason for this?
Since you first used 50nM MTX and then adapted your cells in 1uM MTX, to my understanding you made stable cell line and used MTX to create selection pressure to get high protein producers.
Loss of protein expression happens over time (although GOI has integrated into the cell genome and high copy no. of the gene would have been achieved due to MTX addition, MTX assures DHFR gene amplification but over time GOI amplified copies can be lost/kicked out of the cell due to metabolic burden). Also, in a mixed culture low producers can just outgrow the high producers ultimately leading to reduction in protein expression.
A way out is to develop single cell clones from this MTX adapted culture (preferably in absence of MTX) and to check the stability of cells/clones in maintaining the protein expression over few generations (atleast 30-40) depending upon the work that you do.
I faced a similar problem while working with Dictyostelium since there too stable clones were made and often expression is lost upon revival of the stocks.
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