Hi Ahmed Eltokhi

There is a limit to expression in HEK293 cells. HEK293T cells are better at expressing protein due to presence of a plasmid encoding a temperature-sensitive mutant of the SV40 large T antigen - plasmids that contain the SV40 origin of replication will thus be maintained. Alternatively, HEK293f cells have also been designed to grow in at high density and produce vast amounts of protein.

ThermoFisher is a great place to start for designing this setup: https://www.thermofisher.com/uk/en/home/life-science/protein-biology/protein-expression/mammalian-protein-expression/transient-mammalian-protein-expression/expi293-expression-system.html

Finally, follow this guide to help maximise your protein expression: https://www.profacgen.com/recombinant-protein-expression-in-mammalian-cells.htm

Hope this helps!

Hi Jack Hopkins ,

Thanks a lot for your answer. The Thermofisher option looks promising. However, do you think this can work with proteins that are not needed to be harvested. I would like to perform electrophysiological recording from these cells. So for me, it is more important to increase the level of protein/ cell rather than the whole amount of proteins produced by the whole dish. By reading the two links that you showed, I have the feeling that they are mainly concerned about the total amount of protein and how to increase the density of cells to increase the protein production. What are your thoughts about that ?

Thanks again.

If you need to increase the expression of your transduced gene in an individual cell, it might be worth attempting to use a higher MOI so that your cells end up with multiple copies of the viral cassette. You might also want to look into the promoter that is driving your transgene, as some are more efficient than others.

Hi John, Thanks for your answer. I am using a maximum concentration of the virus. From my experience, there is a limit of increasing the expression after a specific concentration. Tat is why I think I have alredy reached the limit of the amount of virus used.