{"created":"2022-01-28T01:03:15.234371+00:00","id":2005151,"links":{},"metadata":{"_buckets":{"deposit":"15cf41b7-4c77-46d4-b5a8-1c8159efbd22"},"_deposit":{"id":"2005151","owners":[1],"pid":{"revision_id":0,"type":"depid","value":"2005151"},"status":"published"},"_oai":{"id":"oai:u-ryukyu.repo.nii.ac.jp:02005151","sets":["1642838403123","1642838403551:1642838407795"]},"author_link":[],"item_1617186331708":{"attribute_name":"Title","attribute_value_mlt":[{"subitem_title":"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","subitem_title_language":"ja"},{"subitem_title":"Construction and application of human artificial chromosome vectors for gene introduction using minichromosomes.","subitem_title_language":"en"}]},"item_1617186419668":{"attribute_name":"Creator","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"要, 匡","creatorNameLang":"ja"}]},{"creatorNames":[{"creatorName":"成富, 研二","creatorNameLang":"ja"}]},{"creatorNames":[{"creatorName":"柳, 久美子","creatorNameLang":"ja"}]},{"creatorNames":[{"creatorName":"Kaname, Tadashi","creatorNameLang":"en"}]},{"creatorNames":[{"creatorName":"Naritomi, Kenji","creatorNameLang":"en"}]},{"creatorNames":[{"creatorName":"Yanagi, Kumiko","creatorNameLang":"en"}]}]},"item_1617186476635":{"attribute_name":"Access Rights","attribute_value_mlt":[{"subitem_access_right":"open access","subitem_access_right_uri":"http://purl.org/coar/access_right/c_abf2"}]},"item_1617186609386":{"attribute_name":"Subject","attribute_value_mlt":[{"subitem_subject":"ヒト人工染色体","subitem_subject_language":"ja","subitem_subject_scheme":"Other"},{"subitem_subject":"BAC","subitem_subject_language":"en","subitem_subject_scheme":"Other"},{"subitem_subject":"X染色体","subitem_subject_language":"ja","subitem_subject_scheme":"Other"},{"subitem_subject":"部位特異的組換え","subitem_subject_language":"ja","subitem_subject_scheme":"Other"},{"subitem_subject":"遺伝子治療","subitem_subject_language":"ja","subitem_subject_scheme":"Other"},{"subitem_subject":"DT40","subitem_subject_language":"en","subitem_subject_scheme":"Other"},{"subitem_subject":"相同組換え","subitem_subject_language":"ja","subitem_subject_scheme":"Other"},{"subitem_subject":"human artificial chromosomes","subitem_subject_language":"en","subitem_subject_scheme":"Other"},{"subitem_subject":"chromosome X","subitem_subject_language":"en","subitem_subject_scheme":"Other"},{"subitem_subject":"site specific recombination","subitem_subject_language":"en","subitem_subject_scheme":"Other"},{"subitem_subject":"gene therapy","subitem_subject_language":"en","subitem_subject_scheme":"Other"},{"subitem_subject":"homologous recombination","subitem_subject_language":"en","subitem_subject_scheme":"Other"}]},"item_1617186626617":{"attribute_name":"Description","attribute_value_mlt":[{"subitem_description":"科研費番号: 15590293","subitem_description_type":"Other"},{"subitem_description":"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","subitem_description_type":"Other"},{"subitem_description":"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。","subitem_description_type":"Other"},{"subitem_description":"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.","subitem_description_type":"Other"},{"subitem_description":"未公開：P.9以降(別刷論文のため)","subitem_description_type":"Other"},{"subitem_description":"研究報告書","subitem_description_type":"Other"}]},"item_1617186643794":{"attribute_name":"Publisher","attribute_value_mlt":[{"subitem_publisher":"要匡","subitem_publisher_language":"ja"}]},"item_1617186702042":{"attribute_name":"Language","attribute_value_mlt":[{"subitem_language":"eng"}]},"item_1617186783814":{"attribute_name":"Identifier","attribute_value_mlt":[{"subitem_identifier_type":"HDL","subitem_identifier_uri":"http://hdl.handle.net/20.500.12000/13990"}]},"item_1617186920753":{"attribute_name":"Source Identifier","attribute_value_mlt":[{"subitem_source_identifier":"BA74136818","subitem_source_identifier_type":"NCID"}]},"item_1617187056579":{"attribute_name":"Bibliographic Information","attribute_value_mlt":[{"bibliographicIssueDates":{"bibliographicIssueDate":"2005-03","bibliographicIssueDateType":"Issued"}}]},"item_1617258105262":{"attribute_name":"Resource Type","attribute_value_mlt":[{"resourcetype":"research report","resourceuri":"http://purl.org/coar/resource_type/c_18ws"}]},"item_1617265215918":{"attribute_name":"Version Type","attribute_value_mlt":[{"subitem_version_resource":"http://purl.org/coar/version/c_970fb48d4fbd8a85","subitem_version_type":"VoR"}]},"item_1617605131499":{"attribute_name":"File","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_access","filename":"15590293.pdf","mimetype":"application/pdf","url":{"objectType":"fulltext","url":"https://u-ryukyu.repo.nii.ac.jp/record/2005151/files/15590293.pdf"},"version_id":"0113b82d-7b8a-48d3-af2e-338772ee2e25"}]},"item_title":"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","item_type_id":"15","owner":"1","path":["1642838403123","1642838407795"],"pubdate":{"attribute_name":"PubDate","attribute_value":"2009-12-16"},"publish_date":"2009-12-16","publish_status":"0","recid":"2005151","relation_version_is_last":true,"title":["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"],"weko_creator_id":"1","weko_shared_id":-1},"updated":"2022-10-31T02:30:39.890939+00:00"}