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Research articles

ScienceAsia 40 (2014): 340-347 |doi: 10.2306/scienceasia1513-1874.2014.40.340

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

Pussadee˙Paensuwana, Jatuporn˙Ngoenkama, Donruedee˙Sanguansermsria, Boonruang˙Khamsria, Ichaya˙Yiemwattanab, Apirath˙Wangteeraprasertc, Sutatip˙Pongcharoenc,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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a Department˙of˙Microbiology˙and˙Parasitology, Faculty˙of˙Medical˙Science, Naresuan˙University, Pitsanulok˙65000˙Thailand
b Department˙of˙Preventive˙Dentistry, Faculty˙of˙Dentistry, Naresuan˙University, Pitsanulok˙65000˙Thailand
c Department˙of˙Medicine, Faculty˙of˙Medicine, Naresuan˙University, Phitsanulok˙65000˙Thailand
d Centre˙of˙Excellence˙in˙Medical˙Biotechnology, Faculty˙of˙Medical˙Science, Naresuan˙University, Pitsanulok˙65000˙Thailand

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

Received 25 Apr 2014, Accepted 29 Sep 2014