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

PussadeeÿPaensuwan^a, JatupornÿNgoenkam^a, DonruedeeÿSanguansermsri^a, BoonruangÿKhamsri^a, IchayaÿYiemwattana^b, ApirathÿWangteeraprasert^c, SutatipÿPongcharoen^c,d,*

 
ABSTRACT:     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.

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* Corresponding author, E-mail: sutatipp@nu.ac.th

Received 25 Apr 2014, Accepted 29 Sep 2014