Department of Life Science, Sogang University, Seoul 121-742, Korea.
J Microbiol Biotechnol. 2012 Feb;22(2):190-8. doi: 10.4014/jmb.1108.08045.
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.
RNA 干扰（RNAi）在生物研究中迅速成为一种有价值的工具，因为它允许蛋白质表达的选择性和短暂抑制。短干扰 RNA（siRNA）短暂沉默基因表达。相比之下，表达的短发夹 RNA 会诱导其靶基因的长期稳定抑制。家蚕（T. ni）细胞广泛用于使用杆状病毒系统表达哺乳动物细胞衍生的糖蛋白。然而，尚未开发出合适的 shRNA 表达系统。我们研究了 shRNA 介导的基因表达抑制在 T. ni 细胞中使用人源和果蝇 U6 启动子的功效。荧光素酶、EGFP 和 β-N-乙酰氨基葡萄糖苷酶（GlcNAcase）被用作靶标，以研究 T. ni 细胞中特定基因的敲低。与未转染细胞相比，在 T. ni 细胞中转染受人类 U6 或果蝇 U6 启动子控制的 shRNA 表达载体后，荧光素酶、EGFP 和 β-N-乙酰氨基葡萄糖苷酶的水平降低。shRNA 在我们的载体转染的 T. ni 细胞中被表达并加工成 siRNA。与对照载体处理的细胞相比，转染靶向 GlcNAcase 的 shRNA 载体的 T. ni 细胞中的 GlcNAcase mRNA 水平下调。这意味着我们使用人源和果蝇 U6 启动子的 shRNA 表达载体可在 T. ni 细胞中用于特定基因的敲低。
