Hello,
I'm generating stable cell lines by transfecting plasmid DNA with lipofectamine 3000.
The cells went through antibiotics selection for 3 weeks (polyclonal selection) and when I analyzed mRNA expression, GOI was abundantly expressed (~1000 fold)
However, when I performed Western blot, protein of interest was not detected.
I used the antibody against the protein of interest, as well as the antibody against FLAG tag.
I've tried generating stable cell line using lentiviral vector (with exactly same sequences for the GOI) and this time, the cell abundantly expressed both mRNA and protein of interest.
Does anyone have similar experiences?
I'm just curious why the protein is not expressed, although mRNA is expressed when using DNA plasmid.
Thanks in advance!
Hello Hyun-Keun Kim ,
We have the same problem with the neomycin resistance protein from our pcDNA3.1 vector.
We see it when we transfect in the U2OS cell line in mass spec proteomic.
But never see it in the HEK293 cell line (no signal in Western blot nor proteomic).
Still both cell population can be selected through antibiotic G418.
Hi Hyun-Keun Kim ,
That's interesting. I would like to share a "similar" experience, so I hope it helps.
In our laboratory we had problems with an adenovirus engineered with a transgene of interest that expressed in mRNA level but not in protein level (we confirmed it both in WB and also seeing GFP fluorescence because our gene was fused with a GFP fraction).
However, when we transfected a plasmid pcDNA3.1-Hygromicin carrying this same gene in HEK293T cells (similar as Philippe Paget-Bailly's vector) our GOI was properly expressed in both transcriptional and proteomic levels, which in fact contrasts with Philippe experiment.
Can you think about why does it happen??
Regarding your problems, I think that a feasable explanation of these differences would be both the used technique and the cell line you work with in your experiment. Because, taking all into consideration:
1) Could be that the lentiviral vector promotes the integration in the genome and this promotes it expression (could be that if it is in a transcient vector the resulting mRNA might be rapidly degraded --> by recruiting decapping factors, the transcript is not properly poli-Adenilated in the poly-A terminal as it is transcribed in the citosol (instead of inside the nucleus), union of destabilizing proteins or exonucleases...
2) As far as I am concerned, HEK293 and U2O2 have a very different cell linage, and therefore, might be reasonable to assume they have not the same expression patterns --> for example, due to a different biased codon usage (f.ex. in our laboratory we have been focused on studying differences of codon usage and tRNA pools between cell lines). However, Philippe Paget-Bailly I don't understand why you don't obtain mRNA, so can be also something in the transcription.
By the way Hyun-Keun Kim , how long is your gene of interest? And does it have any peculiarities (apart from the FLAG epitope)?
What do you think about it guys?
Best regards,
Francesc
Hello Philippe Paget-Bailly
Do you see both protein and mRNA expression when HEK293 cells are transiently transfected?
Does the protein disappear only after going through antibiotics selection?
Francesc Estrany
Thanks for your thorough answer.
1) Regarding polyadenlyation issue, I do have poly-A sequence in my plasmid and for my qRT-PCR, theoretically, only those with poly-A are converted into cDNA so I think this is not the major issue.
2) I also think it might be cell-context or cell-lineage dependent. I've only tested it in 3 different cell lines.
My GOI is about 2400 bp, and it does not have any pecularities but I can't find any commercial stable cell lines expressing this gene. Perhaps, many CROs have failed making such stable cell lines.
Thanks in advance.
Hello Hyun-Keun Kim Francesc Estrany ,
sorry if i was not clear.
I have mRNA expression in both cell lines, but protein expression in only one cel line.
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