I am trying to obtain a stably transfected B16F10 cell line of floxed GFP plasmid (http://www.addgene.org/8389/). The plasmid structure is pCMV-lox-GFP-lox-RFP. Thus it is expected to express GFP in normal conditions. Cre recombinase expression should induce recombination and RFP expression.
I tried to transfect through both amexa transfection and lipofection. In both cases I was not able to obtain stable cell lines. Basically, after more than 1 month of antibiotic selection, most of the cells died. However, remaining cells which will express GFP lose their GFP expression after a few cell divisions. So forming colonies look patched.Even I tried to flow sort the cells for GFP expression after 6 weeks the sorted cells started to lose GFP expression in few cell divisions. Again I obtained patched colonies.
Moreover, after having problems with transfections I decided to use lentivirus for trunsduction.
I cloned the fragment into lentiviral vector and did transduction.
Unfortunately, same result. Cells are losing GFP expression.
As a note, they don't become red when they lose GFP expression
What is going on here? Any suggestions?
May I ask why you are using ampicillin and not geneticin as slective marker?
Ampicillin will not have any affect on mammalian cells as a selective antibiotic. Selectig for stable transfectants requires killing those cells that do not have the plasmid DNA so that they do not out-compete those that have it. Transientlytransfected cells will lose the plasmid ovber time unless it is integrated into a chromosle, a rare event. Those that lose it can be selected against, but only by an agent that normally kills the cells. If the plasmid has a resistence gene for that antibiotic that will be expressed in mamma\lian cells, usually having a viral promoter like CMV or SV40 and a polyA sequence, then those incorporating the plasmid will survive selection. Ampicillin resistence genes in plasmids have only bacterial promoters, will not be expressed, and will not matter as ampicillin will not kill the cells.
The plasmid has kanamycin/neomycin resistance markers, so I guess you should use neomycin rather than ampicillin as selection marker for generating a stable cell line
The plasmid has kanamycin resistance gene so I used neomycin for selection but it does not matter since even trunsduction is not working. I also flow sorted them to collect GFP expressing cells however they lose the expression with cell division..
Something else should be going on.
At least that clears that you didn't use ampicillin to select. One month selection is a long time, I usually select for about 14 days at highest dose determined for cells and then reduce the amount of antibiotic by 1/2 to just keep some selective pressure on the cells. The long term high exposure to the antibiotic may be putting the cells under too much pressure so worth trying to lower the dose; however, doubt that will sort the problem.
Alternatively try another vector with different promoter (NOT CMV). This is not liked by some cell lines but have not read about it for B16F10.
I think it is not related to antibiotic selection. Since with trunsduction I am having the same problem. May be it is being silenced by methylation but not sure...
The question is solved! The problem was related to the cell line. In this cell line somehow the gene is being suppressed or not integrated at all (or removed by some mechanism).
I used same virus to transduce a human melanoma cell line and they formed perfect colonies without loosing the expression.
Dear Omer,
I'm an student in PhD in immunology and it is my first year.
I also tried to transfect B16F10 cells ... and I have troubles too! I would like to know if your B16F10 cells were alredy luciferace + before transfections? What was your G418 selei concentrtion? Because, my well with B16F10 without resistance (control) are still alive with a concentration of 8mg/ml!
Thank you in advance for your answer
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