The plasmid contains two selectable markers, puromycin and EGFP, but in spite of this the cultures continue to be resistant and green, we loose the KD efficiency after several passages. Could it be a problem with promoter methylation?
What promoter is being used in this system to drive shRNA expression? Is the EGFP part of the shRNA gene cassette or instead controlled by a different promoter?
to drive shRNA expression is U6, hPGK for puromycin resistance and CMV for EGFP. Is the Mission pLKO.1-CMV-turboGFP from Sigma Aldrich. Thank you
CMV can undergo methylation-dependent silencing, but PGK and U6 are not usually susceptible. It might help to monitor GFP mRNA over time. Since the protein is exceptionally stable, it makes it somewhat difficult to assess whether transcription from the transgene is falling off with time, especially at very high levels of expression. So if silencing is occurring over the entire lentiviral cassette, it might be evident in your shRNA before you detect a chance in GFP intensity.
What cell type are you using?
I had a year's worth of trouble using lentiviral transduced shRNA trying to generate stable KD cell lines. We could get a solid KD that lasted maybe 2-3 weeks, after that, no matter how much antibiotic pressure, the KD would slowly go away. I've had much more success lately by switching to transient KD using Origene's Trilencer siRNA and electroporation as the transfection method.
I was told that cell lines being polyploid allows them the ability to perhaps lose or silence whole parts of chromosomes, so we may have lost the KD that way. Another caveat is that using the lentiviral transduction, we had no idea where in the genome our shRNA was being inserted, perhaps it was in a area not highly transcribed. Whatever the reason, we had the same problem you had, but switched technologies and overcame it.
Thank you very much for the answers. We had have this problem with three different lentiviral transduced shRNAs used by three different persons in the group. We thought that this could be explain as methylation silencing of the promoters. While methylation of the promoter driving antibiotic resistance should be negative selected (if adding the antibiotic and the same happens withe EGFP if you sort the culture), methylation of the U6 promoter (driving the shRNA transcription) could be positive selected if the lack of this protein is important for the cell well being. So in the long term you will have antibiotic-resistant, EGFP-positive cells, in which shRNA is not transcribed and this combination could overgrowth the culture.
If this (survival pressure to select against knockdown) is the case, Leonardo, you should consider using an inducible promoter instead such as the H1/tetO promoter.
@ Leonardo. That is exactly what we had. Cells that grew fine in Puromycin, but our protein of interest was no longer knocked down.
Mr.Leonardo,
Did you find the reason for your problem. We are facing a problem like this with Crisper system mediated KD.
Mr.Leonardo,
Did you find out what was it? I got puromycin resistance but no GFP in microscope with CRISPR All in one Lentiviral particles.
Has anybody solved this issue? I'm having the same problem where I've selected with puromycin but when I run my Western I do not have much knockdown once I take my cells off of the puromycin.
I have found this pdf that talks about a feedback loop and the loss of the T antigen causing loss of signal and transduction.
https://www.mdanderson.org/education-and-research/departments-programs-and-labs/programs-centers-institutes/center-for-biological-pathways/files/faq---protocols-revised-by-12-20-2013.pdf
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