RNAi confusion - More expression! (Mar/18/2006 )

Hi everyone

I am having a real problem in creating a stable line of knockdown myoblasts.

It seems that whenever I compare protein levels of my selected (not clonal) cells, they are expressing significantly MORE protein than my WT controls. Has anyone heard of this happening before? Is it possible that some compensation mechanism in my cells is bypassing the RNAi machinery?

Any advice would be gratefully recieved!

Thanks all,

Oliver

Hi Oliver,

I would say this is an interesting observation regardless of its mechanism. The compensation mechanism you mentioned is one possibility, the siRNA could also regulate gene expression at transcriptional level by binding to its cognate DNA. Lastly, off-target effect and interferon response are also possible mechanisms.

Could you elaborate on the interferon response you mention? My cells contain the large T-antigen and require IF-gamma in the media. Might this have some influence?

Thanks,

Oliver

Hi Oliver,

Reports have shown that either shRNA or siRNA can induce interferon response. Since you have added IF-gamma in your media which alone may induce the expression of your gene. Have you tested that? Is IF-gamma necessary for the growth of your cells? If not, omitting it will make things much simpler. If you can rule out interferon response, this finding worths further exploration.

hi
ifn g is used to activate interferon response in hela cells.
For checking interferon response, you can check if STAT1 is phopshorylateed, or at mRNA level, if you can detect OAS RNA, it shows great possibility of activate interferon response.

Thanks for all the responses everyone.

I add IFN-gamma to stimulate the large T-antigen in my cells to keep them immortalised. Although I havent checked my gene's expression levels in untreated cells, the IFN really has to be there to keep the cells from fusing into myotubes.

hi
large T is a protein of SV40 virus, if i'm not wrong, and should be under the control of a constitutive promoter (CMV or sthg like that)

The myoblast line I am using is expressing the large-T under the control of the H2kb promoter. IFN-gamma increases transcription of thermolabile Large-T protein, thus the need for this in the media.