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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