Hi all,

I am transducing murine CD8+ T cells (OT-Is) with concentrated lentivirus at various MOIs. (We calculated the MOIs by using a lentivirus quantification kit). I'm transducing the activated T cells on day 2 with our lentivirus using Rectronectin-coated plates and following the rectronectin-virus bound method as described by Takara bio. This lentivirus construct should result in the co-expression of GFP and gene of interest *GOI) in T cells.

We originally transduced HEK293 cells and got excellent co-expression of the GFP and GOI.

However, when we tested transduction efficiencies in murine T cells we saw that co-expression was lost at the various MOIs. There was either expression of GFP or GOI or nothing at all in the murine T cells. I was wondering whether anyone could suggest a reason for the loss of co-expression upon transducing the T cells. We were planning to use the GFP to track the cells for future in-vivo experiments.

I am aware that most people have used retroviruses to transduce murine T cells, but equally, there are some articles that have successfully used lentivirus, despite murine T cells having mechanisms to prevent adequate lentiviral integration into T cells. Could epigenetic silencing upon transduction be an issue, DNA methylation silencing the GFP?

It sounds like I have answered my own question, but we wanted to see if we could use lentivirus to transduce murine T cells or whether we may be required to swap to Retrovirus.

Any help will be greatly appreciated!

thanks.

Scott