Reduction in EGFP after sorting - (Sep/06/2007 )
I recently sorted primary human fibroblasts stably transfected with a vector carrying EGFP. Immediately after sorting up to 24 hrs I did not see GFP expression which started showing up only after 48hrs and yet not all the cells are green!(~60% green) . Is this a common observation or there is something needs to be done? Thanks
How did you transfect these cells? transient ? or viral stable method?
It was a lentivirus mediated STABLE transfection.
THere could be a few things, IMO:
- You said that you sort them for GFP signal, so you used FACS, right? Did you include controls for the sorting? At least a normal, untransfected cell line as neg.?
- Did you read the histogram before/during/after sorting? How strong is your signal?
- Did you sort for all the positive cells (so long as it has GFP, as compared to negative), or you only sort for the strong/medium/weak ones?
- How many time did you infect the cells?
- After infection, did you select (using antibiotic) before sorting or you sort immediately?
- How did you check for the GFP signal after sorting? By eyes (under microscope), by IF, by FACS? Did you induce the cells?
- You said the signal is not 100% right after sorting, I take it as you check after you let the cells recover and then look at them after they already adhered and spread out, but before you split them? Or you meant something else?
The reason for so many questions is because there could be a number of reasons for your result:
- If you sort for all cells expressing GFP signal, just so that they are differ from the original (based on histograph), and your GFP strength is not homogenous (which I guess is the case), then there is a possibility that you did sort some uninfected (no GFP) together with your "real GFP cells".
- It also could be that the GFP gene is not yet stable (insert into the genome and stay there) after your infection. Sometimes they get lost, you only got the antibiotic resistant gene there. But they lost after you sort the pool.
- It could also be that the GFP is there but too weak so you cannot see by your eyes. This is very common, either when you do transfection or infection. Thus, IF or FACS to double check is much better than just look under microscope. There is also possibility that you have to induce cells before you can see the really weak signal. But FACS is more sensitive than your eyes, so the cells could be positive for FACS and negative for your eyes.
- Even if you sort for stronger expression of GFP, there are still chance that they will get weaker. And so, I think that the phenomenon is not too strange, but do need to verify by better observation methods.
I've had something similar happen to me - EGFP with a CMV promotor. I've shown that my single-cell sorted clone which is <30% green is a pure clone and that the loss of fluorescence is due to gene downregulation by methylation.
You can try and recover fluorescence temporarily by incubation with sodium butyrate, TSA or azadoexycytidine.