Construction of NKG2D genetically modified
CD4+ T cells
To understand the regulation of the NKG2D on CD4+ T
cells, NKG2D genetically modified CD4+T cells were
constructed by transfection with lentiviral (LV) vectors encoding the
over-expression of NKG2D or NKG2D siRNA, or with blank control vector.
Three siRNA sequences targeting the human NKG2D sequence were
transfected into 293T cells and their interference efficacy was examined
by RT-PCR. NKG2D-siRNA-3 5’-CAGCAAAGAGGACCAGGAUUUACtt-3’ displayed the
highest efficacy (Figure 3A) and was selected for the subsequent
construction of LV-NKG2D-siRNA. LV-NKG2D was constructed and was
confirmed by sequencing. Transfection of 293T cells with LV-GFP and
LV-NKG2D was successful, with expression of GFP detected by fluorescence
microscopy (data not shown). NKG2D expression was assessed by flow
cytometry and real-time quantitative RT-PCR to confirm the success of
NKG2D genetically modified CD4+ T cells (Figure 3B,
3D).