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 Murata, Junichiro Futami, Hidenori Yamada, Nam Ho Huh

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)1-9
Number of pages9
JournalNucleic acids research
Volume33
Issue number9
DOIs
Publication statusPublished - 2005

ASJC Scopus subject areas

  • Genetics

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