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KMID : 0545120120220020190
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Journal of Microbiology and Biotechnology
2012 Volume.22 No. 2 p.190 ~ p.198
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Short-Hairpin RNA-Mediated Gene Expression Interference in Trichoplusiani Cells
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Kim Na-Young
Baek Jin-Young
Choi Hong-Seok
Chung In-Sik
Shin Sung-Ho
Lee Jung-Ihn
Choi Jung-Yun
Yang Jai-Myung
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Abstract
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RNA interference (RNAi) is rapidly becoming a valuable tool in biological studies, as it allows the selective and transient knockdown of protein expression. The shortinterfering RNAs (siRNAs) transiently silence gene expression. By contrast, the expressed short-hairpin RNAs induce long-term, stable knockdown of their target gene. Trichoplusia ni (T. ni) cells are widely used for mammalian cell-derived glycoprotein expression using the baculovirus system. However, a suitable shRNA expression system has not been developed yet. We investigated the potency of shRNA-mediated gene expression inhibition using human and Drosophila U6 promoters in T. ni cells. Luciferase, EGFP, and β-Nacetylglucosaminidase (GlcNAcase) were employed as targets to investigate knockdown of specific genes in T. ni cells. Introduction of the shRNA expression vector under the control of human U6 or Drosophila U6 promoter into T. ni cells exhibited the reduced level of luciferase, EGFP, and β-Nacetylglucosaminidase compared with that of untransfected cells. The shRNA was expressed and processed to siRNA in our vector-transfected T. ni cells. GlcNAcase mRNA levels were down-regulated in T. ni cells transfected with shRNA vectors-targeted GlcNAcase as compared with the control vector-treated cells. It implied that our shRNA expression vectors using human and Drosophila U6 promoters were applied in T. ni cells for the specific gene knockdown.
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KEYWORD
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shRNA, T. ni cells, U6 promoter, β-N-Acetylglucosaminidase, glycoprotein
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