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  1. その他
  1. 部局別インデックス
  2. 医学部

ミニ染色体を用いた、遺伝子導入のための、ヒト人口染色体ベクターの構築と応用

http://hdl.handle.net/20.500.12000/13990
http://hdl.handle.net/20.500.12000/13990
8dfcc3ae-7df2-4527-87fb-a1d4e5eb8b1a
名前 / ファイル ライセンス アクション
15590293.pdf 15590293.pdf
Item type デフォルトアイテムタイプ(フル)(1)
公開日 2009-12-16
タイトル
タイトル ミニ染色体を用いた、遺伝子導入のための、ヒト人口染色体ベクターの構築と応用
言語 ja
作成者 要, 匡

× 要, 匡

ja 要, 匡

成富, 研二

× 成富, 研二

ja 成富, 研二

柳, 久美子

× 柳, 久美子

ja 柳, 久美子

Kaname, Tadashi

× Kaname, Tadashi

en Kaname, Tadashi

Naritomi, Kenji

× Naritomi, Kenji

en Naritomi, Kenji

Yanagi, Kumiko

× Yanagi, Kumiko

en Yanagi, Kumiko

アクセス権
アクセス権 open access
アクセス権URI http://purl.org/coar/access_right/c_abf2
主題
言語 ja
主題Scheme Other
主題 ヒト人工染色体
言語 ja
主題Scheme Other
主題 X染色体
言語 ja
主題Scheme Other
主題 部位特異的組換え
言語 ja
主題Scheme Other
主題 遺伝子治療
言語 ja
主題Scheme Other
主題 相同組換え
内容記述
内容記述タイプ Other
内容記述 科研費番号: 15590293
内容記述タイプ Other
内容記述 平成15年度~平成16年度科学研究費補助金(基盤研究(C)(2))研究成果報告書
内容記述タイプ Other
内容記述 研究概要:<ミニ染色体改変システムの構築,検証と応用> 1)BAC(細菌人工染色体)導入システムの構築と検証テロメア断片化ベクターにより作製された約2.7MbのヒトX染色体由来ミニ染色体に、さらに改変を加え、反応方向特異性をもつ部位特異的組換え系 (Cre/変異loxP)を用いたBAC導入システムを構築した。導入用BACには、作製済みベクターを用いた(Kaname and Huxley, BioTechniques 31:273(2001))。ヒトHPRT遺伝子を含む改変BAC(HPRTBAC66D2)をミニ染色体へ導入した場合の挿入効率は約75%であった。他のBAC(ヒトFactor IX遺伝子を含むF9BAC66D2)においても、効率は同程度(約80%)であった。一方、ネオマイシン耐性遺伝子発現ユニットのみをもつプラスミド (4.5kb)の挿入効率は、100%と非常に高率であった。ヒトHPRT遺伝子を挿入したミニ染色体は、これを保持しているニワトリpre-B細胞株 (DT40)において、遺伝子発現が認められた。よって、このシステムは、遺伝子全体をミニ染色体に挿入するシステムとして、有効であることが示された。(投稿中) 2)染色体ベクターとしての応用改変(挿入)を行ったミニ染色体の、宿主細胞であるDT40から他の細胞への移入を行った。ヒトHPRT遺伝子をもつミニ染色体を微小核融合法により、線維肉腫細胞亜株(HT1080,HPRT(-))へ移入した。G418耐性を指標にクローン (HT1080,HPRT(-))を検討したところ、ミニ染色体は、細胞あたりのコピー数に変動が見られる(1~8コピー)ものの、安定に存在していた。また、HPRT遺伝子発現も効率よく認められた。よって、このミニ染色体は、遺伝子導入のための染色体ベクターとして有効であると考えられた。
内容記述タイプ Other
内容記述 要約(欧文):We have established an efficient system for introducing a whole BAC clone including an intact gene unit onto a minichromosome based on the human X chromosome. The use of mutant loxP sites with Cre recombinase and direct selection for the retrofitting event led to a high efficiency of correctly retrofitted clones. A modified BAC containing the intact human HPRT gene was correctly retrofitted onto the minichromosome in DT40 cells in approximately 75% of analysed clones. Modification of BACs for retrofitting was used method previously reported (Kaname and Huxley, BioTechniques 31:273(2001)). For another BAC clone containing human factor IX gene, the efficiency of retrofitting was about 80%. For a plasmid (4.5kb) having a neoR expression unit, the retrofitting efficiency was 100%. The HPRT gene is expressed from the minichromosome at approximately the same level in both of these clones. This system allows one to introduce any gene or gene cluster which can be cloned as a BAC into the minichromosome for gene delivery. We also tried to transfer the modified minichromosomes into other cells (HT1080,HPRT(-)) by microcell-fusion method. It was efficient to isolate the transchromosomic cells by screening of G418 resistant clones. Of the resistant clones, copy number of the minichromosome per cell was in range of 1-8. Its expression was, however, efficient and stable. Thus, the minichromosome would be used as a chromosome vector for gene therapy.
内容記述タイプ Other
内容記述 未公開:P.9以降(別刷論文のため)
内容記述タイプ Other
内容記述 研究報告書
出版者
言語 ja
出版者 要匡
言語
言語 eng
資源タイプ
資源タイプ research report
資源タイプ識別子 http://purl.org/coar/resource_type/c_18ws
出版タイプ
出版タイプ VoR
出版タイプResource http://purl.org/coar/version/c_970fb48d4fbd8a85
識別子
識別子 http://hdl.handle.net/20.500.12000/13990
識別子タイプ HDL
収録物識別子
収録物識別子タイプ NCID
収録物識別子 BA74136818
書誌情報
発行日 2005-03
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エクスポート

OAI-PMH
  • OAI-PMH JPCOAR
  • OAI-PMH DublinCore
  • OAI-PMH DDI
Other Formats
  • JSON

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