To get much higher efficiency, I did something that they didn't do. I did cell sorting and isolated GFP-highly-expressing cells. Because this gene has been shown to be critical for cell growth, so after sorting, when I found that knockdown cells didnt grow well, I thought that the knockdown went quite well. However, after 1-week culture (to collect more cells), I did real time PCR and found the mRNA level was not affected at all, for both shRNAs. I also did FACS and confirmed the GFP expression was 100% and very high.
Here are some thoughts of mine but I dont know whether they are correct. I suspect that in the GFP-highly-expressing cells (among all the GFP positive cells, I picked those with even higher expression), the expression of shRNA might be hindered. It seems a little rediculous but when I knocked down another gene with a different system in which GFP is driven by hPGK promoter, I didnt have this strange thing. Could it be possible that in some case, CMV promoter is competitive to U6? I know knockdown a gene that is critical for cell growth might generate some unexpected results but the successful case that I just mentioned above is also knockdown of a cell growth promoting gene.
Have you ever had such problem? Can you provide some explanations? Thank you very much.
Edited by gyma, 21 November 2012 - 04:34 AM.