I have developed 4 stable clones with GFPs and RFPs none of them show up after 20 or 25 passages though they are in selection media? Is it slowly losing the plasmids or is the florescence not enough to visualize? Anybody have any suggestions?
Thanks
Did you do subcloning and selection of stable clones with some functional assay besides fluorescence?
Stable clones mean in this case that your plasmid has integrated into the genome of your cells?
Dear Dr. Lehotzky, I actually verified the expression of my protein of interest in those cells. As Dr. Praetorius said, these plasmids (not linearized) are supposed to get into the genome in stable clones
Without subcloning, you can get a mixture of cells likely containing real clones and the original cells helped by the resistant others. Add the selection antibiotic always from stock. Dns built-in from a plasmid are usually a rear event, depends on cell line, too. However, it is rear too to loss four clones during few months... Is the target the same protein in the four constructs?
Anyway, plasmid containing cells were lost slowly during the first few week of selection. So it looks you should have some clones but they are instabile for some reason.
Hi Arunkumar,
did you consider silencing of the promotor driving your FP-GOI Fusion? Which cell line and which promotor are you using?
I concurr with Christopher: 20-25 passages is too much; your cells will not be the 'same' as at the start anymore. After isolation of your stable lines freeze cells and start from those stocks when you reached passage 10 or so.
hm, even a human fibroblast primary culture is able to live about 50 passage. so, stable cell line is something different. especially is a newly isolated one. first of all, it has to be revive after freezing. it is a not certain behaviour for an isolated clone. etc.
for FACS: i know that is a quick and good method to get clear sample from transient transfection, or even for sorting clonal cells (ie fluo ones). this still does not mean subcloning.
Dear Lehotzky,
Thanks for your suggestions. I have sub cloned in 2 different cell lines and selected with selection media and maintaining in the selection media only. the antibiotic concentration is very high and that is not tolerable to the wild type cells (using 300ug/ml and the IC 50 for WT cells is 75-100ug/ml). I am using the antibiotic from 100x stock. when I check the cells for the expression of my protein of interest it is over expressed but when I look it under the microscope I do not see any fluorescence, which I suppose to see Green or Red. the GFP/RFP and ORF are under CMV promoter. the target is different protein in all the cases. in fact I was doing 8 diff. clones.
I can see the fluorescence in first 10 - 15 passages but slowly it looses the fluorescence but the expression of protein is not affected.
Is there any possibility that GFP fragment alone get lost during integration, because the GFP comes after my ORF and in between Pme I Not I/Xho I restriction enzymes.
Thanks for your suggestions.
Dear Dr. Drexler, thanks for the suggestion but I am not so clear about the silencing of the promoter. please make it little clear because I do not have much exposure in this area. I am using CMV promoter and MCF10A and MDA MB 231 cells to overexpress and suppress my protein of interest
Thanks
Dear Dr.Duntsch and Dr. Metzendorf,
Thanks for the suggestion and I do have a backup frozen cells. again If I take the cells they tend to loose the fluorescence more quickly in 10 passages.
Do these vectors work as tagged fluo constructs? Unfortunately, i do not know this cell lines.
Yes. The vector contains GFP and antibiotic resistant genes along with ORF under CMV promoter. The cell lines are breast tumor and non tumor cell lines
I have no clue. Actually, i have a clonal line behaved very similarly. In my case the protein perturbed the dividence of cells. So we more or less thought and accepted that the loss was generated by clonal loss that was unbeatable albeight the loss looked to be slower than in your case.
In general, GFP variants are sensitive to pH or redox change. Try to variate culture condition or use some additives like NAC if the loss only flourescence, and the proteins are still there. On my opinion, the changes are too fast to be genetic. But i have no more idea, unfortunately.
The GFP is being turned of due to chromosomal modification of histones. To test this, take your cells and add ~5 uM Sodium Butyrate and check you cells daily for GFP expression.
As Daniel said its pretty possible that the region containing the fluorescent protein is silenced by the cell. Its not necessary for survival (as is the region of the antibiotic resistance) so this does no harm to the cell.
Dear Drs. That is an acceptable explanation. Let me try this first. Thanks
GFP silencing in your experiment is expected. CMV promoter is highly sensitive to methylation and not suitable for stable transfection. It is best to choose another vector with another promoter i.e. EF1
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