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Sequence-Specific Suppression of mdr1a/1b Expression in Mice via RNA Interference

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Purpose

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.

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Abbreviations

pDNA:

plasmid DNA

RNAi:

RNA interference

siRNA:

small interfering RNA

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Acknowledgments

The authors are grateful to Drs. Makoto Miyagishi and Kazunari Taira, Department of Chemistry and Biotechnology, School of Engineering, the University of Tokyo, for their valuable help in determining the target sites for RNAi using the algorithm developed in their laboratory. This work was supported partly by a Grant-in-Aid for Scientific Research from the Ministry of Education, Cultures, Sports, Science, and Technology, Japan.

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Authors and Affiliations

  1. Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto, 606-8501, Japan

    Yumi Matsui, Naoki Kobayashi, Makiya Nishikawa & Yoshinobu Takakura

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  1. Yumi Matsui
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  2. Naoki Kobayashi
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  3. Makiya Nishikawa
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  4. Yoshinobu Takakura
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Correspondence to Yoshinobu Takakura.

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Matsui, Y., Kobayashi, N., Nishikawa, M. et al. Sequence-Specific Suppression of mdr1a/1b Expression in Mice via RNA Interference. Pharm Res 22, 2091–2098 (2005). https://doi.org/10.1007/s11095-005-8178-8

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  • DOI: https://doi.org/10.1007/s11095-005-8178-8

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