title: Short-hairpin RNA-mediated gene expression interference in Trichoplusia ni cells.
abstract: 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.
authors.affiliation: Department of Life Science, Sogang University, Seoul 121-742, Korea.
authors.forename: Na Young
authors.initials: NY
authors.lastname: Kim
authors.forename: Jin Young
authors.initials: JY
authors.lastname: Baek
authors.forename: Hong Seok
authors.initials: HS
authors.lastname: Choi
authors.forename: In Sik
authors.initials: IS
authors.lastname: Chung
authors.forename: Sungho
authors.initials: S
authors.lastname: Shin
authors.forename: Jung Ihn
authors.initials: JI
authors.lastname: Lee
authors.forename: Jung Yun
authors.initials: JY
authors.lastname: Choi
authors.forename: Jai Myung
authors.initials: JM
authors.lastname: Yang
mesh_terms: D000118:Acetylglucosaminidase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D019677:Artificial Gene Fusion; D016367:Baculoviridae; D004330:Drosophila / Q000235:genetics; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques / Q000379:methods*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007915:Lepidoptera / Q000235:genetics*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism*
chemical_list: D034741:RNA, Small Interfering; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D008156:Luciferases; D000118:Acetylglucosaminidase