Sequence-specific suppression of mdr1a/1b expression in mice via RNA interference.
OBJECTIVE
RNA interference (RNAi) is a powerful tool for silencing gene expression posttranscriptionally. The purpose of this study was to examine whether in vivo RNAi can be induced against endogenous mdr1a/1b in adult mice and to assess the feasibility of generating P-glycoprotein (P-gp) knockdown mice based on RNAi by a very simple intravenous injection of synthetic small interfering RNA (siRNAs) or siRNA-expressing plasmid DNAs.


METHODS
The targeted sequences for silencing mdr1a specifically or mdr1a/1b simultaneously were examined in an in vitro study using a mouse colon carcinoma cell line, colon26 cells, in culture. Mice were repeatedly treated with intravenous synthetic siRNAs or siRNA-expressing plasmid DNAs in naked form administered via a large-volume and high-speed injection, i.e., the hydrodynamics-based procedure. The amount of targeted mRNA and P-gp in the liver were determined by real-time polymerase chain reaction and Western blot analysis, respectively.


RESULTS
Among several targeted sequences, two and one optimized sequences were selected for mdr1a and mdr1a/1b, respectively, in the in vitro study. Following administration of synthetic siRNAs or siRNA-expressing plasmid DNAs directed against mdr1a, the mRNA level in the liver was significantly reduced to approximately 50-60% of that in control mice. Furthermore, a slight reduction was observed at the protein level. Similar results were obtained in the experiments using siRNA-expressing pDNA directed against mdr1a/1b.


CONCLUSIONS
Our results demonstrate that sequence-specific suppression of mdr1 gene expression is possible at the mRNA level as well as the protein level in mice following intravenous delivery of siRNA effectors.
Matsui
Yumi
Y
Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan.
Kobayashi
Naoki
N
Nishikawa
Makiya
M
Takakura
Yoshinobu
Y
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D045744:Cell Line, Tumor; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D005260:Female; D007275:Injections, Intravenous; D051379:Mice; D008807:Mice, Inbred BALB C; D034622:RNA Interference / Q000187:drug effects*; D012333:RNA, Messenger / Q000096:biosynthesis; D034741:RNA, Small Interfering / Q000494:pharmacology*; D014162:Transfection; D000097202:ATP-Binding Cassette Sub-Family B Member 4
D018435:ATP Binding Cassette Transporter, Subfamily B; D018528:ATP-Binding Cassette Transporters; D004247:DNA; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D000097202:ATP-Binding Cassette Sub-Family B Member 4; C095122:multidrug resistance protein 3