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Please use this identifier to cite or link to this item: http://hdl.handle.net/10119/13690

Title: 人為的RNA Editingを利用した遺伝コード修復法の開発
Other Titles: Development of genetic code restoration by using artificial RNA editing
Authors: 塚原, 俊文
Authors(alternative): Tsukahara, Toshifumi
Keywords: Deaminase
RNA editing
MS2
活性ドメイン
guide RNA
遺伝子修復
Issue Date: 1-Jun-2016
Abstract: RNA editing機構を模倣して細胞内で人為的かつ部位特異的な塩基の脱アミノ化を誘導するため、ADAR1の活性中心部位とguide RNAをMS2システムを介して結合させた。レポーター遺伝子として、EGFPのNonsense変異体を使用した。これら3つの遺伝子をHEK293細胞に導入し、酵素-RNA複合体を作成させたところ、変異EGFP mRNAが細胞内で修復され、緑色蛍光を発する細胞が観察された。RNAの変異修復は、RT-PCR後にRFLPとsequencingによって確認した。また、ADAR ファミリーのアイソフォームで脱アミノ化による修復効率の相違も調べた。 : To imitate RNA editing system for artificial and site-specific deamination of nucleic acid toward base-substitutions in vivo, we connected the active domain of ADAR1 with a guide RNA by using MS2 system. The nonsense mutated EGFP was used as a reporter gene. These 3 genes were transiently transfected into HEK293 cells at once. We observed green fluorescent cells in which parts of mutated EGFP mRNAs were restored in vivo. The genetic code restoration was confirmed by RT-PCR-RFLP and sequencing, respectively. We also compared efficiencies of RNA restorations by the site-specific deamination among ADAR family isoforms.
Description: 挑戦的萌芽研究
研究期間:2014~2015
課題番号:26670167
研究者番号:60207339
研究分野:生化学・分子生物学
Language: jpn
URI: http://hdl.handle.net/10119/13690
Appears in Collections:平成27年度 (FY 2015)

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