J. Microbiol. Biotechnol. (2012), 22(2), 190–198 http://dx.doi.org/10.4014/jmb.1108.08045 First published online December 15, 2011 Short-Hairpin RNA-Mediated Gene Expression Interference in Trichoplusia ni Cells Kim, Na Young 1 , Jin Young Baek 1 , Hong Seok Choi 1 , In Sik Chung 2 , Sungho Shin 1 , Jung Ihn Lee 3 , Jung Yun Choi 4 , and Jai Myung Yang 1 * 1 Department of Life Science, Sogang University, Seoul 121-742, Korea 2 Department of Genetic Engineering, Kyung Hee University, Suwon 449-701, Korea 3 Department of Computer Science and Information, Hanyang Women’s University, Seoul 133-817, Korea 4 Hazard Substances Analysis Division, Seoul Regional Food and Drug Administration, Seoul 158-050, Korea Received: August 18, 2011 / Revised: November 22, 2011 / Accepted: November 23, 2011 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 β- 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 β- 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. Keywords: shRNA, T. ni cells, U6 promoter, β-N- acetylglucosaminidase, glycoprotein RNA interference (RNAi) is a valuable tool in biological studies, as it enables the selective, transient knockdown of protein expression [7, 9, 37]. The RNAi mechanism is based on the strongly conserved post-transcriptional gene silencing function mediated by double-stranded molecules [10, 32]. There are two types of RNAi mechanisms: RNA- based RNAi and DNA-based RNAi. In RNA-based RNAi, a synthetic short-interfering RNA (siRNA) duplex is introduced into the cells. In DNA-based RNAi, a plasmid carrying sequences encoding short-hairpin RNA (shRNA) is delivered to the cells [37]. The siRNA method, which transiently blocks protein expression, can be achieved by delivering synthetic double-stranded RNA into cells. siRNA is incorporated into a RNA-induced silencing complex (RISC) that causes the sequence-specific degradation of the corresponding mRNA [4, 47]. siRNA is also produced by the delivery of a plasmid encoding a shRNA that generates the 21 mer nucleotides of siRNA [33]. Compared with siRNA, shRNA leads to a long-term gene silencing effect. Thus, the plasmid DNA system is effective for generating stable RNAi in target cells. For shRNA expression, a RNA polymerase III (PolIII) promoter is used to transcribe shRNA that contains a double- stranded stem of 19-25 bp with sequences complementary to the target gene, and a 5-10 base loop sequence connecting them [31]. shRNAs can be effectively transcribed in mammalian cells by PolIII promoters, such as U6 and H1 [5, 33, 35, 40]. The human U6 promoter is composed of a TATA-like element located approximately 30 bp upstream of the transcription initiation site and a proximal sequence element (PSE) located upstream of the TATA box [26]. A distal sequence element (DSE) located further upstream from the PSE enhances transcription from the core promoter [25]. This promoter has been widely used in mammalian systems for the expression of shRNA [6, 14]. The use of the Drosophila melanogaster U6 promoter for *Corresponding author Phone: +82-2-705-8457; Fax: +82-2-701-8550; E-mail: jaimyang@sogang.ac.kr # Supplementary data for this paper are available on-line only at http://jmb.or.kr.