NKp30 and CD244(2B4) overexpression by mRNA transfection with CART ONA in human primary NK cells

We are investigating the function of natural killer (NK) cell receptors, NKp30 and CD244 (2B4) in the functional response of NK cells to HIV-infected autologous CD4+ T cells. We will overexpress NKp30 or CD244 in human primary NK cells by transfecting them with charge-altering releasable transporters (CARTs) to deliver the mRNA that encodes NKp30 or CD244. To distinguish NK cells that have translated the exogenous mRNA from the ones that naturally express these NK cell receptors, we will co-transfect NK cells with GFP mRNA and GFP expression will indicate the transfected NK cells. In this experiment, we are testing if the co-transfection will result in co-expression of the NK cell receptor and GFP.
Conclusion:
We transfected NK cells isolated from PBMCs of 10 healthy adult donors. In all the donors that we tested, 30-55% of the NK cells express GFP. We also observed higher expression levels of NKp30 and CD244 in GFP+ cells in NKp30+GFP and CD244+GFP co-transfection groups, respectively. This indicates that the transfection resulted in higher levels of NKp30 and CD244 expression compared to the untransfected NK cells.
Notes:
In this experiment, we tested the co-transfection of NKp30+GFP mRNA at 1:1 mass ratio, where 50 ng of NKp30 mRNA and 50 ng of GFP mRNA was mixed and complexed with CART ONA and added into 500,000 NK cells for transfection. The mass ratio of CD244 mRNA and GFP mRNA in CD244+GFP co-transfection group is also 1:1, and prepared as in NKp30+GFP group mentioned above, except that CD244 mRNA was used instead of NKp30. The expression of NKp30 or CD244 and GFP was analyzed 18 hours after transfection.