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Nucleic Acids Research 2005 33(9):e88; doi:10.1093/nar/gni088
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Published online 26 May 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oupjournals.org

Methods Online

Targeted disruption of transcriptional regulatory function of p53 by a novel efficient method for introducing a decoy oligonucleotide into nuclei

Masakiyo Sakaguchi, Takamasa Nukui, Hiroyuki Sonegawa, Hitoshi Murata1, Junichiro Futami1, Hidenori Yamada1 and Nam-ho Huh*

Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shikatachou, Okayama 700-8558, Japan 1Department of Bioscience and Biotechnology, Okayama University Graduate School of Natural Science and Technology Tsushimanaka, Okayama 700-8530, Japan

*To whom correspondence should be addressed. Tel: +81 86 235 7393; Fax: +81 86 235 7400; Email: namu{at}md.okayama-u.ac.jp

Received April 5, 2005. Revised May 16, 2005. Accepted May 16, 2005.

Decoy oligonucleotides have been used for functional sequestering of transcription factors. Efficient introduction into cells is a prerequisite for the oligonucleotides to exert their blocking function. Lipofection is the most widely used technique for that purpose because of its convenience and relatively high efficiency. However, the transduction efficiency of lipofection largely depends on cell types and experimental conditions and the introduced nucleotides are not specifically directed to nuclei where they exert their major function. In the present study, we designed a new system for transporting oligonucleotides into cell nuclei. The vehicle is composed of glutathione-S-transferase, 7 arginine residues, the DNA-binding domain of GAL4 and a nuclear localization signal, which are linked with flexible glycine stretches. The p53-responsive element linked to the GAL4 upstream activating sequence was efficiently transferred by the vehicle protein into nuclei of primary cultures of neuronal cells, embryonic stem cells and various human normal cells. Transcriptional activation of p21WAF1/CIP1 and Bax by p53 on exposure to cisplatin was completely blocked by introducing the p53 decoy oligonucleotide. Thus, the system developed in the present study can be a convenient and powerful tool for specifically disrupting the function of DNA-binding proteins in culture.


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