Hi all.. I am using a mouse cell line with a silenced GFP promoter in it... I have been doing RNAi screening and have identified a candidate gene that might be involved in silencing.. Once I made the stables, I checked for % of GFP cells and it was significantly higher than my scrambled shRNA.. On doing qPCR, GFP expression of my candidate gene was again significantly higher than my scrambled shRNA... After a month, I took the cells for FACS again and the % of GFP cells were reduced and they were all almost equal.. I thought they might have lost the stable integration and so checked it with Puromycin and there were very few dead cells in the case of stables and my non-transduced cells were all dead!!! Any reason why there could be a reduction in GFP expression?? Thank you
Hi da. After a month you said. So were they in culture in the meantime or were frozen and thawed? I'm assuming Puromycin is the selection in the shRNA vector; is the GFP still integrated? Did you check the transcript levels of GFP and your shRNA. And, is the unsilencing of the GFP promoter reversible?
Hi devdu.. They were in the culture and yes Puro selection.. Unfortunately I cant check the expression levels of GFP because they are silenced in the cell line.. But what I did was induce the expression of GFP with a drug and analyze it by FACS.. That gave a good % of GFP cells which means they GFP is still integrated.. Well, I read another thread where they were discussing the same issue and it pretty much answers my question!! They say that CMV promoter is prone to methylation after a lot of passages which could explain what is happening in my case because the promoter in my case is also CMV.. I think it might be reversible but I am not so sure about that!!!
Oh. I thought GFP was unsilenced by the shRNA. And, good you got the answer. I have also heard from people that CMV is not very versatile and it doesn't work in many cell lines. A vague guess: Does your shRNA target some deacetylase?
Hi da.. I have a number of shRNAs targeting different types of epigenetic modification!!! So its a mix of everything I would say.. But just curious, why did you think it is deacetylase??
If CMV promoter is getting silenced by methylation during passaging, your shRNA may not be a methyltransferase because you would have otherwise altered the methylation trends in the cells. Acetylation predominantly causes activation. Since silencing a gene is causing activation of GFP, you might be targeting a deacetylase I thought. Vague. I could have been wrong!
Well yeah that is a good way of looking at it.. But I would say it might not be that simple because of redundancy and other types of modifications that might occur.. When we get time, probably we can discuss about this in detail!!
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