Sequence-specific gene silencing in murine muscle induced by electroporation-mediated transfer of short interfering RNA.
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.
Kishida
Tsunao
T
Department of Microbiology, Kyoto Prefectural University of Medicine, Kyoto 602- 8566, Japan.
Asada
Hidetsugu
H
Gojo
Satoshi
S
Ohashi
Suzuyo
S
Shin-Ya
Masaharu
M
Yasutomi
Kakei
K
Terauchi
Ryu
R
Takahashi
Kenji A
KA
Kubo
Toshikazu
T
Imanishi
Jiro
J
Mazda
Osam
O
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
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D008156:Luciferases; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases