I am trying to create stable GFP CHO cells using G418 as a selection agent. I carried out a dose response curve and determined 0.6 mg/ml would completely kill cells by day 7. I used 0.8 mg/ml for my study. After 24 hours 90% of cells were producing GFP. I added the G418 24 hours after transfection. Within a week my negative control contained no cells, so the drug was at a good concentration. At 1-2 weeks my cells regained their viability (>95%), but were not producing GFP accordingly. The more days that pass, the fewer cell are producing GFP. Now only 5% of cells are GFP positive. I wondered if the positive cells supplied enough resistance for all the cells, so i seeded at a very low density to try and combat this but it didnt work. Does anyone have any idea what might be happening?
How is your GFP expressed? What is the promoter? How do you detect GFP expression FACS or IF? Some promoter get methylated which affects expression. and/or your GFP expression might be low. FACS is more sensitive than IF. Might want to sort out your GFP+ population by FACS. This is not an uncommon result. What you see after 24 hours is transient expression which is typically stronger than stable expression.
I am checking for GFP positive cells via flow cytometry. The GFP gene is under a CMV promoter. Surely it's unlikely that the GFP promoter is silenced when the resistance gene isn't? This is a common and very expressive promoter. I have just carried out a FACS sort, but my worry is that once I have got a population of GFP positive cells, the G418 might not keep it this way. I am happy to FACS sort to acquire an expressing population, but I would be reluctant to do this during my work because it introduces a huge population bottleneck which would surely impact my work.
Is your resistance gene under the control of a CMV promotor? If not, it is likely, that the GFP is silenced whereas the restance gene is not. And additionally, you just need some cell finding out the trick only to silence the CMV promotor of the GFP and not the one of the resistance gene for surviving and this will overgrow your GFP-expressing population.
So the best thing you can do is to change your cellular system (e.g. U2OS cells should tolerate the CMV promotor of the GFP), or to change your vector system, either with GFP under the control of a different promotor, or a bi-cistronic expression vector (contains the GFP ORF and the resistance gene ORF on the same transcript, separated by an internal ribosomal entry site (IRES)).
Another posibility is that your G418 in the media is used up, but i guess you frequently perform a media change or passage the cells, so this is very unlikely.
Best,
GAD
hello Jow
I made a stable line of CHO cells transfected with EGFP-C1 (Clontech). Then i was selectingt though cells for sorting. Now I keep the selection pressure using 0.8 mM G418 from sigma.
I hope to help you and best wishes with your experiments.
The high GFP expression, which you see is from transient expression, not from genomic integration. In case you use an construct with the GFP and the resistance under the same promoter methylation would not matter. When working years ago with CHO cells, we observed that some are very resistant to G418. Probably they overgrow your transfected cells. The best way to avoid this was to seed to very low density and to pick clones growing from single cells afterwards.
I would strongly recommend using a bi-cistronic vector. See Biotechniques. 20(1):102-4, 106, 108-10, 1996, for the reasoning. Any initial transient expression might be exaggerated by the very long half life of some GFP protein. Using pIRES type vectors you should be able to work with pools that express stably for many weeks without even needing to clone out. That's not possible for every protein, but it certainly should be possible with something as simple as GFP that is unlikely to create its own negative selection by over expression. Best to clone out anyway, but keep the pool going as well just to see if this is an easier route for next time.
I would agree with Kay-Dietrich, transient transfection generates high expression from a non integrated episomal plasmid, so expression will start off high, but will diminish in time as the plasmids are ejected after a while. If you want a stable cell line try linearising the plasmid before transfecting-the overall expression will be lower one or two days after transfection, but the number of stable clones will be higher. Try selecting them and then performing limiting dilution cloning and select the best clones by checking GFP expression.
Your G418 concentration is correct. After transfection try to add first antibiotic in different time period early before 24 hrs. it will work out
Please, take into consideration that a DNA vector, a transfection reagent, expression of an antibiotic resistance (trans)gene, expression of a reporter (trans)gene, and selection by acute/chronic antibiotic treatment may evoke cellular responses that affect the biochemical processes under investigation. In the following review, we demonstrate that an assumption that empty vector-transfected cells preserve the cytogenetic and phenotypic characteristics, and represent the adequate control in transfection experiments is not universally valid. We exemplify a number of studies, which reported obvious genomic, transcriptomic and phenotypic changes of tumor cells after transient/stable transfection of an empty vector. Finally, we conceptualize that the diverse experimental manipulations (e.g., transgene overexpression, gene knock out/down, chemical treatments, acute changes in culture conditions, etc.) may act as a system stress, promoting intensive genome-level alterations (chromosomal instability, CIN), epigenetic and phenotypic alterations, which are beyond the function of manipulated genes.
Please see the following manuscript for details:
Stepanenko AA, Heng HH. Transient and stable vector transfection: Pitfalls, off-target effects, artifacts. Mutat Res. 2017 Jul;773:91-103. doi: 10.1016/j.mrrev.2017.05.002.
Im super late, but maybe someone in the future will read this and find it helpful. If you are just transfecting your GFP plasmid into the cells then its possible they are losing the GFP gene with time. Do a lenti transduction to integrate the plasmid into the host genome.
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