Application of the BC1 RNA gene promoter for short hairpin RNA expression in cultured neuronal cells.
BC1 RNA is a neuronal cell-specific non-messenger RNA transcribed by RNA polymerase III (Pol III). We previously reported that the transcription of BC1 RNA is controlled both by intragenic promoters for Pol III and by a 5'-flanking region containing several unique cis-elements that are possible members of the Pol II transcription system. In this study, we chose beta-secretase (BACE1) as a target and applied the promoter to produce a short hairpin RNA (shRNA) for RNA interference (RNAi) in cultured neuronal cells. A plasmid vector in which the promoter was linked to a target sequence functioned in rodent NG108-15 cells and suppressed BACE1 protein expression, but did not function in non-neuronal NIH3T3 cells. It was also effective in rat primary hippocampal neurons. We further showed that the promoter can be active in human neuroblastoma SH-SY5Y cells and reduced expression of targeted protein, although the BC1 RNA gene is a rodent-specific gene. The use of this vector-based system for shRNA expression may be an important component of future development of neuronal cell-selective RNAi in both transgenic and therapeutic applications.
Kobayashi
Shunsuke
S
Department of Biochemistry, College of Pharmacy, Nihon University, Narashinodai, Funabashi, Chiba 274-8555, Japan. kobashun@pha.nihon-u.ac.jp
Higuchi
Takashi
T
Anzai
Kaijiro
K
D053829:Amyloid Precursor Protein Secretases; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D010450:Endopeptidases; D020868:Gene Silencing; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D009474:Neurons / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D034622:RNA Interference; D020733:RNA, Small Cytoplasmic / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics*; D051381:Rats; D014162:Transfection / Q000379:methods*
C117734:BC1 RNA; D020733:RNA, Small Cytoplasmic; D034741:RNA, Small Interfering; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human; C509282:Bace1 protein, mouse