Question about GFP - (Feb/13/2012 )
I cloned my interested gene by a 3FLAG-GFP tag vector. The cells were selected with puromycine, and the stable cell line were sorted one more time by FACS sorting to have GFP+ population cells. Now, after several months using the GFP+ cell line, I recognize that the GFP signal becomes weaker and weaker even though the WB still show the expression of my interested gene and GFP protein. The cells are resorted by FACS and the result is 3%. Only 3% of the assumed GFP+ cells is really positive. What is happening? Why the GFP becomes faded over time and so should I believe all the results I had with the assumed GFP+ cells that I had worked for more than 6 months?
I think you should maintain the stable cell line in the media containing the puromycine so as to select the GFP positive cells always.
Mybe your interested proteins have some toxicity or negetive effect on the cell growth, so they cannot compete with the cells without GFP and your interested genes.
If you have the strict and logical control, your results are still available and useful and can be published in journals.
I always maintain my cells in medium containing puromycin. My main problem is that I cannot enrich my GFP positive cells. For example,the 1st time I did the FAC sorting I got 50% cells GFP positive. I grew the cells and 1 week later, the FAC sort gave 30% positve, and now after 4 time FAC sort I got 3% cell GFP positive. So where were all my GFP positive cells? I selected my cells in puromycin for more than 4 weeks to have the stable cell line and FACs afterwards. I cannot understand, my cells become resistant to puromycine but did not express GFP?
It is common for protein expression to decrease with subculturing. As protein expression stresses the cells and reduces the cell growth rate, cells with spontaneous mutations that reduce protein expression will have a growth advantage and gradually dominate the population. For example, it has been shown that the amount of functional T7 RNA polymerases in BL21(DE3) strain decreases after multiple subculturing due to spontaneous mutation (http://www.microbialcellfactories.com/content/4/1/3). This in turn reduces overexpression of the target protein from a pET vector. Even if it's not a pET system, mutations to the promoter that reduces overexpression will give the same effect. Since the antibiotic resistance gene is usually constitutively expressed and independent of the protein expression, your cells would still be resistant to puromycine but the protein expression ability is impaired.
It happens to my GFP stable cells too. I construct a LNCaP/EGFP stable cell lines by cloning pickup. Everything goes well. The cells have very good green fluorescence first. but the fluorescence of my cells suddenly become weaker and weaker. I thought this clone might be unstable clone, so I picked another 3, but all have the similar result. I almost gave up, but I find that the GFP expression of the cell can be recovered by increasing the G418 concentration. When I increase the concentration to 1000 ug/ml and culture them for around 1 week, all the cells show very very strong fluorescence again and cells are also healthy. The only problem is the cell growth rate, very slow. BTW, I use the high pure G418. So I think the GFP gene is not kicked out, might be in hybernation. You can wake it up by G418.