Figure 3 Construction of NKG2D genetically modified
CD4+ T cells and NKG2D expression assay. Three NKG2D
small interfering RNA (siRNA) sequences were designed and transfected
into 293T cells. After 24 h, mRNA expressions of NKG2D were assayed by
RT-PCR. (A) NKG2D siRNA efficiency tested by RT-PCR revealed the highest
interference for NKG2D siRNA-3. Lentiviral vectors encoding NKG2D
(LV-NKG2D), NKG2D siRNA-3 (LV-siRNA) and empty blank control lentivirus
(LV-BC) were constructed and titrated, then added to
CD4+ T cells at a multiplicity of infection of 100 in
polybrene (5 μg/mL) to produce NKG2D genetically modified and controlled
CD4+ T cells. (B) Percentages of NKG2D positive CD4+ T
cells transfected with LV-BC, LV-NKG2D or LV-siRNA determined by flow
cytometry. (C) Bar chart of relative mRNA expression of NKG2D by
genetically modified CD4+ T cells detected by
real-time quantitative RT-PCR (n = 6 each group). * P < 0.01
and # P < 0.05 for within group interaction.