{"created":"2022-01-28T01:03:16.903492+00:00","id":2005152,"links":{},"metadata":{"_buckets":{"deposit":"a9e4a49c-b433-41b3-a60b-704d9be12b78"},"_deposit":{"id":"2005152","owners":[1],"pid":{"revision_id":0,"type":"depid","value":"2005152"},"status":"published"},"_oai":{"id":"oai:u-ryukyu.repo.nii.ac.jp:02005152","sets":["1642838403123","1642838403551:1642838407795"]},"author_link":[],"item_1617186331708":{"attribute_name":"Title","attribute_value_mlt":[{"subitem_1551255647225":"安定した遺伝子発現の得られるヒト人工染色体の開発と応用","subitem_1551255648112":"ja"},{"subitem_1551255647225":"Construction and application of human artificial chromosome vectors for stable gene expression by minichromosome-modifying system.","subitem_1551255648112":"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_1522299639480":"open access","subitem_1600958577026":"http://purl.org/coar/access_right/c_abf2"}]},"item_1617186609386":{"attribute_name":"Subject","attribute_value_mlt":[{"subitem_1522299896455":"ja","subitem_1522300014469":"Other","subitem_1523261968819":"遺伝子"},{"subitem_1522299896455":"ja","subitem_1522300014469":"Other","subitem_1523261968819":"発現制御"},{"subitem_1522299896455":"ja","subitem_1522300014469":"Other","subitem_1523261968819":"ミニ染色体"},{"subitem_1522299896455":"ja","subitem_1522300014469":"Other","subitem_1523261968819":"遺伝子治療"},{"subitem_1522299896455":"en","subitem_1522300014469":"Other","subitem_1523261968819":"DT40"},{"subitem_1522299896455":"en","subitem_1522300014469":"Other","subitem_1523261968819":"Cre"},{"subitem_1522299896455":"en","subitem_1522300014469":"Other","subitem_1523261968819":"loxP"},{"subitem_1522299896455":"en","subitem_1522300014469":"Other","subitem_1523261968819":"position effect"},{"subitem_1522299896455":"en","subitem_1522300014469":"Other","subitem_1523261968819":"human artificial chromosomes"},{"subitem_1522299896455":"en","subitem_1522300014469":"Other","subitem_1523261968819":"BAC"},{"subitem_1522299896455":"en","subitem_1522300014469":"Other","subitem_1523261968819":"chromosome X"},{"subitem_1522299896455":"en","subitem_1522300014469":"Other","subitem_1523261968819":"site specific recombination"},{"subitem_1522299896455":"en","subitem_1522300014469":"Other","subitem_1523261968819":"gene therapy"}]},"item_1617186626617":{"attribute_name":"Description","attribute_value_mlt":[{"subitem_description":"科研費番号: 17590289","subitem_description_type":"Other"},{"subitem_description":"平成17年度～平成18年度科学研究費補助金(基盤研究(C))研究成果報告書","subitem_description_type":"Other"},{"subitem_description":"研究概要:＜多様なミニ染色体ベクターの作製＞ミニ染色体上のテロメア側、セントロメア内、中間領域での遺伝子発現および染色体の安定性を検討するため、それぞれの領域にゲノム遺伝子(BAC)を導入できるミニ染色体を個別に作製し、検討した。ミニ染色体の改変は、DT40細胞内で行った。1)ミニ染色体改変用プラスミドベクターの構築ヒトX染色体由来ミニ染色体(IKNFA3)の各部位へ、相同組換えにより変異型lox71を挿入するためのプラスミドベクターを作製した。変異型 lox71挿入用ベクターは、blasticidin R発現カセット(CAG lox71 bsr)を用いて作製した。また、ミニ染色体テロメア側の薬剤耐性遺伝子(neoR)をZeocinR遺伝子へ変更するためのtelomere- targeting vectorも新たに作製した。2)ミニ染色体の改変上記挿入用ベクターを用い、ミニ染色体の各部位へCAG lox71 bsrをそれぞれ挿入した改変ミニ染色体を作製した。同時に、テロメア側(短腕側)をZerocinR遺伝子へ変更した。3)改変ミニ染色体へのBAC導入上記改変ミニ染色体へ、遺伝子発現ユニットをもつBAC(HPRT-66, F9-66)をCre/変異lox系を用いて導入した。導入効率は、約70～75％であった。＜BAC導入ミニ染色体における遺伝子発現と安定性＞改変ミニ染色体のテロメア側、セントロメア側、中間領域のそれぞれの領域に挿入されたBAC内遺伝子(HPRT)の発現について検討した。定量PCRによる発現比較では、テロメア側にBACを挿入したクローンのヒトHPRT遺伝子発現を100とすると、セントロメア側挿入クローンでは83、中間領域挿入クローンでは122であった。細胞核あたりのコピー数は、1コピー(約70％)または2コピー(約30％)がほとんどであった。また、内在性Hprt遺伝子発現量に対し、ヒトHPRT遺伝子発現量は約15％であった。これは、宿主細胞の種の違いによると考えられた。長期培養による遺伝子発現は、テロメア側、中間領域挿入クローンで大きな変化はなかった。","subitem_description_type":"Other"},{"subitem_description":"要約(欧文):In order to know the position effect and stability of gene expression at various regions in human artificial minichromosomes, we have constructed four types of modified minichromosomes, which was introduced a BAC harboring a whole gene unit (HPRT, or Factor IX) into various sites such as near telomere, near centromere, and euchromatic region, and we investigated intensity and stability of the gene expression. Fist, we constructed plasmid vectors, which could introduce lox71 (mutant loxp) into the human minichromosome at various sites by homologous recombination or telomere targeting method. We also constructed a telomere-targeting vector, which could replace the neoR gene to the ZeoR gene located at the telomeric region in the minichromosome. Then, we modified the minichromosome using the vectors above, and introduced a BAC (HPRT-66 or F9-66) into the modified minichromosomes by the Cre/mutant lox recombination system. The efficiencies of BAC introduction into the minichromoscmes were approximately 75% and 70% for HPRT-66 BAC and F9-66 BAC, respectively. Relative gene expressions of human HPRT gene estimated by qPCR were 100, 83, and 122, when the gene was located near telomere near centromere, and euchromatic region, respectively. Copy number of the modified minichromosomes in DT40 cells was 1 copy/nucleus (approximately 70%) or 2 copies/nucleus (approximately 30%). Relative expression of the human HPRT gene was 15% of authentic Hprt gene expression in DT40. The gene expression was not significantly changed in any modified minichromosomes after 60 days culture.","subitem_description_type":"Other"},{"subitem_description":"未公開：P.14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_1522300295150":"ja","subitem_1522300316516":"要匡"}]},"item_1617186702042":{"attribute_name":"Language","attribute_value_mlt":[{"subitem_1551255818386":"eng"}]},"item_1617186783814":{"attribute_name":"Identifier","attribute_value_mlt":[{"subitem_identifier_type":"HDL","subitem_identifier_uri":"http://hdl.handle.net/20.500.12000/13993"}]},"item_1617186920753":{"attribute_name":"Source Identifier","attribute_value_mlt":[{"subitem_1522646500366":"NCID","subitem_1522646572813":"BA82750414"}]},"item_1617187056579":{"attribute_name":"Bibliographic Information","attribute_value_mlt":[{"bibliographicIssueDates":{"bibliographicIssueDate":"2007-03","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_1522305645492":"VoR","subitem_1600292170262":"http://purl.org/coar/version/c_970fb48d4fbd8a85"}]},"item_1617605131499":{"attribute_name":"File","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_access","filename":"17590289.pdf","mimetype":"application/pdf","url":{"objectType":"fulltext","url":"https://u-ryukyu.repo.nii.ac.jp/record/2005152/files/17590289.pdf"},"version_id":"b8eb3d5a-2c10-47b7-bc0f-f65b233316a0"}]},"item_title":"安定した遺伝子発現の得られるヒト人工染色体の開発と応用","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":"2005152","relation_version_is_last":true,"title":["安定した遺伝子発現の得られるヒト人工染色体の開発と応用"],"weko_creator_id":"1","weko_shared_id":-1},"updated":"2022-10-31T02:30:42.125648+00:00"}