These are likely stress granules and in the case of primary cells, stress can lead to replicative senescence (hence the enlarge and non-growing phenotype---you could test this by doing a b-gal assay). I would suggest doing both a titer to decrease MOI and a curve with polybrene to use minimal amount if you are using any. The polybrene is not required for infection per se but will usually increase efficiency. At the end of the day, some cells just don't like being infected with viruses... 

Hey,

we are using the same backbone with some modifiactions. I observe more granular cells every time I am transducing them, however for me they are still proliferating (slower but proliferating). So I guess this granularity is due to transduction. The growth arrest is for me not explainable by transduction itself. Have you tired to transduce other cell types like HT-1080? Or try a lower MOI for our cells. Do you use Polybrene oder protaminsulfate?

Regards

Sven

These are likely stress granules and in the case of primary cells, stress can lead to replicative senescence (hence the enlarge and non-growing phenotype---you could test this by doing a b-gal assay). I would suggest doing both a titer to decrease MOI and a curve with polybrene to use minimal amount if you are using any. The polybrene is not required for infection per se but will usually increase efficiency. At the end of the day, some cells just don't like being infected with viruses... 

I agree with Richard, you may have infected the cells with a number of copies of the virus that lead to such expression levels the cell cannot cope with. If you have images of the fluorescent cells check the sub cellular distribution of the fluorescent protein, my gut feeling would tell me that you will find it accumulated in the Golgi and endoplasmic reticulum. If indeed you are overexposing it you may be causing overload of the ER and Golgi followed by ER stress, proteasome stress and potentially autophagy. This to me would explain the presence of vesicles and granules in the cells. Reducing the viral load used for te transfection should solve the problem. For your own safety it is worth also following the lead from Moritz. Good luck.

Hi all,

Thanks for you answers! I think I am indeed dealing with stressed cells. I will have a look at my GFP signal at sub cellular distribution whether it is primarily located in the ER and Golgi. We haven't titrated the viral load as of yet, so that will be my next step. We just wanted to see how well the transduction would be with pure virus supernatant. With respect to the possibility of having a replication competent virus, I can indeed do a WB or an ELISA to make sure. 

 @Sven, I am using polybrene. 

 Bram

In my experience, there is one more thing you may consider that transduction slightly  lead the cells go towards the differentiating state. if they got slower in proliferation is kind of a normal event but you should know that do not use too much titer of virus or even do not use a very concentrated volume of virus to your cells. We usually remove the half of the media and replace it with concentrated media containing virus and add it drop by drop to the cells not at once and nor onto one location (put every drop of viruses in different location of the well.