I have performed Lentiviral transduction in Jurkat cells with a plasmid encoding the mEOS fused with the PH domain of Akt. My experimental plan is to visualize T cell synapse by stimulating the transduced cells. However 24 hours post transductions I observe mEOS expression under fluorescent microscope only at a localized single position in Jurkat cells whereas in HEK293T cells, mEOS is distributed across the member as well as in cytoplasm in few cell.
What is the localized position in Jurkat cells? Did you identify it via colocalization? Maybe mEOS expression is too strong for Jurkat cells, so it aggregates and the cell collects them somewhere.
Btw, did you transduce the cells with a packaged virus or transfected a plasmid encoding mEOS? The question is not clear to me. I guess your viral RNA is encoded on a plasmid. In case of virus, does your effect also happen when the virus does not carry any transgene?
mEOS is localized at the peripheral part of the jurkat cells. As mEOS is fused to PH domain of Akt, it might be possible if the cells are activated by transduction PH domain have guided mEOS to the peripheral area of the cells but I am not sure about it. Also I did not perfomed colocalization.
I transduced the Jurkat cells from packaged virus (pLVX transfer vector and 3rd generation packaging vectors).
It is interesting that you find fluorescence only at a single spot, as I would expect that aktivated PH domain localises mEOS at the periphery equally distributed. Did you consider adding tyrosine kinase inhibitor(s) during transduction to inhibit T cell receptor signalling?
yes I was expecting the same and I did observed this in HEK293T cells. Addition of tyrosine kinase inhibitors during transductions is an interesting approach, I will consider it in my future viral infections. Thanks.
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