Hi Haroon,

I have experienced this with different proteins and lentiviral vectors. I think that it really depends on the nature of your overexpressed protein. Consider post-translational modifications and negative feedback loop. Depending on the cellular context your protein for instance could be constantly modified to be degraded by several enzymes. Unfortunately I do not have a solution for this problem, unless you are able to identify the regulatory mechanisms that lead to protein destabilisation. In that case you can try to overexpress mutants of your GOI that cannot be post-translationally modified inside the cell (for instance phosphorylation sites mutants). However as I said, it depends on how much literature is available on that topic.

Another explanation is protein secretion (it happened to me once). In this case you will notice that, under fluorescence microscope imaging of living cells, the background of your transduced cells will be higher than control ones. If this happens take a look again at your construct to check if you accidentally inserted some protein export signals to your protein. However this is very very rare.

As a control you can check your GOI overexpression in cells selected with Puro at 10 days post transduction by qRT-PCR. If o/e is detectable by qRT-PCR it means your transgene is still expressed and, as I was suggesting, post-translational modification are preventing your GOI accumulation inside the cells. 

My last suggestion is, depending on your experimental need, to analyse your phenotype at short time points, as troubleshooting of this problem could be extremely tedious and time-consuming. 

Hope it helps

Best

Francesco

Hi Haroon,

What promoter is driving your pLVX lentiviral construct? Is it CMV? What is driving your expression of mEOS2 and PH-Akt? If it was a CMV promoter, it can be DNA-methylation induced silencing? You could try a human promoter such as hPGK or EF1a to see if you are still seeing loss of mEOS2 signal.

Do have other ways of measuring your lentiviral transduction such as qPCR based methods? If so that will help you to determine whether your are losing lentiviral transduced cells because they have a decreased survival advantage compared with untransduced Jurkats.

Best Wishes,

Dave

Hi Francesco, thank you for your suggestions. I also thought in the same direction. As other cells (HEK293T and NIH-3T3) transduced with the same virus had stable expression and the expression of the protein was distributed, which was not the case in Jurkat's where the fusion protein tends to localize even in unstimulated cells. So it might be Jurkat cells are not a good model for this type of study. However other people have these kinds of probe by electroporating T cells.

Moreover you also mentioned about the protein secretion. So the protein is not secreted out of the cells it just accumulates at different position or a single position in a cells (ideally it should be distributed throughout).

About performing thr qRT-PCR of GOI, my idea was that the stable line gets established if even there is expression of GOI in qRT-PCR it won't solve the problem.

Thanks again for your suggestions.

Had a similar issue on a couple of occasions. One time, I believe that overexpression of my oncogene was too toxic, thus the cells with the highest integration (and brightest GFP) eventually got selected out and I was left with only Dim-GFP cells. Also, certain cells are sensitive to high amounts of fluorescent proteins. Had a breast cancer cell line once die from overexpression of RFP, and another leukemia die of too much GFP. Lastly, there's a good chance the promoter driving expression is getting silenced by methylation.

Hi David, yes the pLVX vectors are driven by CMV promotor and both the genes mEOS2 and PH-Akt are under CMV promotor.

Yes I believe with qRT-PCR I will know whether the transgene is still in the cells or not but as cells are resistant to puromycin that might not be the case.

Thanks for your suggestions. :)