trouble with establishing a stable cell line - (May/20/2009 )
I have been working on establishing a stable cell line through retroviral infection. After selection and expanding, I got a population of surviving cells. When I checked the total RNA with RT-PCR, my gene of interest was overexpressed at the mRNA level significantly. But when I did Western Blot to check the protein level, the protein was not overexpressed much (-- very little). I don't know why. Can anyone give me some hints? I'd really appreciate it!!!
No translation signal from the transfected DNA? Over expression is toxic to the cells?
I've seen the same thing here, but unfortunately, i cannot aswer your question. I found my protein was 139 fold higher than the wild type in RT-PCR, but I couldn't detect it in WB. I'm about to try in a new, easier to transfect cell line. I'll tell you about it.
It could be beacuse of your target cells, which somehow might not be liking your GOI or the method of generating stable cell lines is not letting your GOI to express well because of the promoter controlling it.