title: Sequence-specific gene silencing in murine muscle induced by electroporation-mediated transfer of short interfering RNA.
abstract: BACKGROUND
  Post-genomic biomedical research requires efficient techniques for functional analyses of poorly characterized genes in living organisms. Sequence-specific gene silencing in mammalian organs may provide valuable information on the physiological and pathological roles of predicted genes in mammalian systems. Here, we attempted targeted gene knockdown in vivo in murine skeletal muscle through the electroporation-mediated transfer of short interfering RNA (siRNA).
  
  
  METHODS
  siRNA duplexes corresponding to the firefly luciferase (Luc), green fluorescent protein (GFP), or glyceraldehyde-3-phosphate dehydrogenase (GAPD) genes were delivered by electroporation into the tibial muscle of normal or enhanced GFP (EGFP) transgenic mice. Plasmid vectors carrying the Luc, hRluc or beta-galactosidase (beta-gal) reporter genes were also delivered. The Luc and hRluc activities in the muscle lysates were assayed. The EGFP and GAPD expression was detected by fluorescence microscopic observation and RT-PCR, respectively.
  
  
  RESULTS
  When Luc-specific siRNA was co-delivered with the Luc expression vector into the tibial muscle, the reporter gene expression was markedly suppressed (less than 1% of the control level) for 5 days. As little as 0.05 micro g of siRNA almost completely blocked the reporter gene expression from 10 micro g of the plasmid. To examine whether siRNA can also suppress expression of an endogenous gene, transgenic mice carrying the EGFP gene received intramuscular transfection of a mixture of beta-gal plasmid and GFP-specific siRNA. beta-Gal-positive cells failed to express detectable levels of EGFP, while EGFP expression was not inhibited in control mice that received nonspecific siRNA. Expression of GAPD was also suppressed by the specific siRNA.
  
  
  CONCLUSIONS
  The present system may provide a useful means of phenotypic analysis of genetic information in mammalian organs for basic research as well as therapeutic molecular targeting in the post-genomic era.
authors.affiliation: Department of Microbiology, Kyoto Prefectural University of Medicine, Kyoto 602- 8566, Japan.
authors.forename: Tsunao
authors.initials: T
authors.lastname: Kishida
authors.forename: Hidetsugu
authors.initials: H
authors.lastname: Asada
authors.forename: Satoshi
authors.initials: S
authors.lastname: Gojo
authors.forename: Suzuyo
authors.initials: S
authors.lastname: Ohashi
authors.forename: Masaharu
authors.initials: M
authors.lastname: Shin-Ya
authors.forename: Kakei
authors.initials: K
authors.lastname: Yasutomi
authors.forename: Ryu
authors.initials: R
authors.lastname: Terauchi
authors.forename: Kenji A
authors.initials: KA
authors.lastname: Takahashi
authors.forename: Toshikazu
authors.initials: T
authors.lastname: Kubo
authors.forename: Jiro
authors.initials: J
authors.lastname: Imanishi
authors.forename: Osam
authors.initials: O
authors.lastname: Mazda
mesh_terms: D000818:Animals; D005260:Female; D005544:Forecasting; D020869:Gene Expression Profiling; D020868:Gene Silencing; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000096:biosynthesis / Q000235:genetics; D049452:Green Fluorescent Proteins; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D008822:Mice, Transgenic; D018482:Muscle, Skeletal / Q000166:cytology / Q000502:physiology; D010641:Phenotype; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D008156:Luciferases; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases