Problem with pooled stable clones: tale of unstable clones :( - (May/12/2011 )

Hi all

I have been struggling for more than a year with my project which has truly been a "project from hell". I made stable pooled clones expressing protein of my interest. I chose the optimal plasmid concentration based on transient transfection that showed fairly good expression of my protein. I moved on to creating pooled clones by transfecting about 9 plates. After selecting for about 2 weeks, I pooled 3 plates into one and continued selection. I did a western blot to confirm the expression of my protein. After few failed attempts I finally managed to get clones to express my protein stably. Meanwhile I did several biological assays using the clones. However, when I tried checking the expression level of protein with western blot, there was no expression to my disappointment. Surprisingly another lab member was able to demonstrate the expression of protein in the stable cell lines. So we moved forward with the project. A microarray analysis was done using those cell lines. My misfortune returned when after qPCR validation (using same batch of RNA that was used for microarray), we picked few genes for protein level validation. Unfortunately I couldn't show any correlation between the qPCR and western blot data for the different genes I picked up. We tried several approaches, however none seemed to work. This has frustrated me immensely not to mention my PI. We decided to hold off on it for a while. However, I am still attached to the project and very curious to know what went wrong. I would really appreciate if you could give me your expert opinions on this issue. Also, I don't seem to get any protein expression in my clones anymore. Could the clones have lost the expression of proteins? I am still maintaining them in G418 containing media, and my cells are alive and healthy.

I'm presuming it wasn't co-transfection?


Perhaps your lines weren't as well selected as you thought... there could have been some cells in there which didn't have the plasmid in them, but they continued to grow and have out-competed the transfected cells.

The transfected DNA can be methylated by the cells, just leaving the resistance gene intact.

Well it is actually quite common for viral promoters to become silenced after culturing stably transfected cell for a few weeks

Did you use insulator to flank your gene of interest? Insulator like CHS4 do work to keep high level of expression going for longer period of time. (although they are species specific).

Thanks for your replies.
This wasn't a cotransfection. It was just one plasmid. It was a plasmid that was used in the lab previously and I am not sure if it has the CHS4 element.
I am guessing the most plausible explantation for my variable gene expression is promoter methylation or the resistant cells outcompeting the ones with the plasmid. Also since I had pooled clones, it must have heterogenous population of cells expressing different levels of my protein. I guess I can't really rescue my project at this point

If you have any populations frozen down that did express highly you could do clonal selection with them to isolate single clones and test them separately. I don't get why you pooled them in the first place... Good luck! I know all about cell lines that suddenly decide to stop making your protein...