RNAi down-regulation of Nck1 adaptor protein in Jurkat T cells

RNA interference (RNAi) is a potent gene delivery system for studying the regulation of gene expression in a wide variety of eukaryotic cells.In the present study, different RNAi approaches, namely, synthetic small interfering RNA (siRNA) and plasmid-and lentivirus-based short hairpin RNA (shRNA) were investigated to assess the down-regulation of Nck1 protein efficiency in the Jurkat T cell line.Jurkat T cells treated with these three different systems substantially and specifically reduced the expression of the Nck1 protein but not that of the Nck2 protein.Although the three systems showed a similar Nck1 knockdown efficiency, they led to different T cell activation outcomes.After stimulation, CD69 expression and IL-2 production were impaired in Nck1-siRNA and plasmid-based Nck1-shRNA transfected Jurkat cells.However, these T cell activation outcomes were increased in lentiviral vector based Nck1-shRNA transfected cells.These data suggest that the outcomes from transfection with the shRNA based lentiviral vector differ from those of siRNA and shRNA-based plasmids although they provide the same gene silencing efficiency.The verification of suitable RNAi strategies to silence target genes is therefore necessary before using it in experiments.