Short-Hairpin RNA-Mediated Gene Expression Interference in Trichoplusia ni Cells

RNA interference (RNAi) is rapidly becoming a valuable tool in biological studies, as it allows the selective and transient knockdown of protein expression. The short-interfering 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 beta-N-acetylglucosaminidase (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 beta-N-acetylglucosaminidase 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.