{"created":"2023-05-15T14:52:30.736117+00:00","id":71695,"links":{},"metadata":{"_buckets":{"deposit":"2747df24-8374-47b7-87b0-86c2f450b04e"},"_deposit":{"created_by":1,"id":"71695","owners":[1],"pid":{"revision_id":0,"type":"depid","value":"71695"},"status":"published"},"_oai":{"id":"oai:repo.qst.go.jp:00071695","sets":["10:28"]},"author_link":["705486","705484","705480","705490","705479","705482","705477","705481","705487","705489","705485","705478","705483","705488"],"item_10005_date_7":{"attribute_name":"発表年月日","attribute_value_mlt":[{"subitem_date_issued_datetime":"2015-05-29","subitem_date_issued_type":"Issued"}]},"item_10005_description_5":{"attribute_name":"抄録","attribute_value_mlt":[{"subitem_description":"【目的】CrisprCas9を用いたゲノム編集技術によって格段にノックアウトマウスの作出は容易になった。本研究では人工の合成オリゴを用いることで、プラスミドを構築することなくノックアウトマウスを作出するための検討を行った。【方法】全身性に赤色蛍光タンパク質（DsRed）を発現するトランスジェニックマウスのDsRed遺伝子の２箇所をgRNAの標的配列として選定した。これらの配列を含んだgRNAの全長（130bp）をT7プロモーターから転写できるようにオリゴDNA（gBlocks）として人工的に合成しin vitro転写によってgRNAを生成した。一方、Cas9は、Cas9タンパク質をコードするDNAを鋳型にT7プロモーターを付加したプライマーで必要領域を増幅し、これを鋳型にmRNAを生成した。次にDsRed雄マウスと野生型（C57BL/B6）雌マウスのそれぞれから精子と卵子を採取し体外受精を行い、受精４－５時間後の１細胞の細胞質に生成\nした２つのgRNAを別々にあるいは混合したものにCas9のmRNAを混ぜて顕微注入した。２つのgRNAを別々に顕微注入した受精卵は培養して胚盤胞を用いて変異導入の有無を検討した。一方で、２つのgRNAを混ぜて顕微注入した受精卵は、翌日に２細胞へ卵割したものを仮親へ移植して得られた産仔を用いて同様の解析を行った。【結果および考察】上記の方法で得られた胚盤胞（４０個）と産仔（２０匹）を蛍光顕微鏡下で観察したところ、すべてにおいて赤色蛍光は観察されなかった。このことから標的遺伝子であるDsRedに高効率で変異が導入されていることが示唆された。実際に変異型DsRedをゲノムに持つマウス３匹の標的配列周辺を調べたところ、２箇所の両方で１塩基から数塩基の欠損とgRNA認識部位で挟まれた領域（およそ150bp）が欠損していることが明らかとなった。また、調べた限りにおいてはオフターゲット効果は２０匹の中に認められなかった。その後に本法を用いてマウスの内在性遺伝子Rhot1を標的にし\nたところ、得られた産仔２０匹のすべてに変異が導入され、すでに報告されていたRhot1全身ノックアウトマウスの表現型（出生直後の致死）と一致することも確認している。クローニングが不要で簡便な本法のメリットを最近の実施例と共に紹介する。\n抄録本文（英語）:\nCRISPR/Cas9 system is a new way of generating knockout mouse models. Here, w e optimized the gBlocks-based CRISPR/Cas9 system, in which plasmid construct ion is not a requirement. We designed two distinct gRNAs targeting DsRed2 in  a transgenic mouse that systemically expresses DsRed protein. A DNA fragmen t (130bp, gBlocks), required for the gRNA function, was produced artificiall y and used as a template for in vitro transcription. gRNAs and Cas9 mRNA wer e produced by in vitro transcription, and a mixture of or individual gRNAs, combined with Cas9 mRNA, were microinjected into fertilized 1-cell embryos, which were collected at 4-5 h after in vitro fertilization between DsRed TG males and C57BL/6 females. These embryos were cultured further until the bla stocyst stage or were transferred to a foster mother, and the resulting blas tocysts and pups, respectively, were analyzed. Although these experiments yi elded 40 blastocysts and 20 pups, no red fluorescent signal was observed und er fluorescent microscopy, suggesting that DsRed2 was successfully mutageniz ed. Indeed, sequencing analysis revealed that 3 pups possessed a disrupted D\nsRed2 gene. In summary, the gBlocks-based CRISPR/Cas9 system is an effective  way to generate knockout mice without plasmid construction.","subitem_description_type":"Abstract"}]},"item_10005_description_6":{"attribute_name":"会議概要（会議名, 開催地, 会期, 主催者等）","attribute_value_mlt":[{"subitem_description":"第62回日本実験動物学会総会","subitem_description_type":"Other"}]},"item_access_right":{"attribute_name":"アクセス権","attribute_value_mlt":[{"subitem_access_right":"metadata only access","subitem_access_right_uri":"http://purl.org/coar/access_right/c_14cb"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"塚本, 智史"}],"nameIdentifiers":[{"nameIdentifier":"705477","nameIdentifierScheme":"WEKO"}]},{"creatorNames":[{"creatorName":"伊林, 恵美"}],"nameIdentifiers":[{"nameIdentifier":"705478","nameIdentifierScheme":"WEKO"}]},{"creatorNames":[{"creatorName":"和田, 彩子"}],"nameIdentifiers":[{"nameIdentifier":"705479","nameIdentifierScheme":"WEKO"}]},{"creatorNames":[{"creatorName":"道川, 祐市"}],"nameIdentifiers":[{"nameIdentifier":"705480","nameIdentifierScheme":"WEKO"}]},{"creatorNames":[{"creatorName":"安井, 孝彰"}],"nameIdentifiers":[{"nameIdentifier":"705481","nameIdentifierScheme":"WEKO"}]},{"creatorNames":[{"creatorName":"矢野, 実"}],"nameIdentifiers":[{"nameIdentifier":"705482","nameIdentifierScheme":"WEKO"}]},{"creatorNames":[{"creatorName":"鬼頭, 靖司"}],"nameIdentifiers":[{"nameIdentifier":"705483","nameIdentifierScheme":"WEKO"}]},{"creatorNames":[{"creatorName":"小久保, 年章"}],"nameIdentifiers":[{"nameIdentifier":"705484","nameIdentifierScheme":"WEKO"}]},{"creatorNames":[{"creatorName":"塚本 智史","creatorNameLang":"en"}],"nameIdentifiers":[{"nameIdentifier":"705485","nameIdentifierScheme":"WEKO"}]},{"creatorNames":[{"creatorName":"伊林 恵美","creatorNameLang":"en"}],"nameIdentifiers":[{"nameIdentifier":"705486","nameIdentifierScheme":"WEKO"}]},{"creatorNames":[{"creatorName":"和田 彩子","creatorNameLang":"en"}],"nameIdentifiers":[{"nameIdentifier":"705487","nameIdentifierScheme":"WEKO"}]},{"creatorNames":[{"creatorName":"道川 祐市","creatorNameLang":"en"}],"nameIdentifiers":[{"nameIdentifier":"705488","nameIdentifierScheme":"WEKO"}]},{"creatorNames":[{"creatorName":"鬼頭 靖司","creatorNameLang":"en"}],"nameIdentifiers":[{"nameIdentifier":"705489","nameIdentifierScheme":"WEKO"}]},{"creatorNames":[{"creatorName":"小久保 年章","creatorNameLang":"en"}],"nameIdentifiers":[{"nameIdentifier":"705490","nameIdentifierScheme":"WEKO"}]}]},"item_language":{"attribute_name":"言語","attribute_value_mlt":[{"subitem_language":"jpn"}]},"item_resource_type":{"attribute_name":"資源タイプ","attribute_value_mlt":[{"resourcetype":"conference object","resourceuri":"http://purl.org/coar/resource_type/c_c94f"}]},"item_title":"gBlocks-based CrisprCas9システムによるノックアウトマウス作出条件の検討","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"gBlocks-based CrisprCas9システムによるノックアウトマウス作出条件の検討"}]},"item_type_id":"10005","owner":"1","path":["28"],"pubdate":{"attribute_name":"公開日","attribute_value":"2015-06-03"},"publish_date":"2015-06-03","publish_status":"0","recid":"71695","relation_version_is_last":true,"title":["gBlocks-based CrisprCas9システムによるノックアウトマウス作出条件の検討"],"weko_creator_id":"1","weko_shared_id":-1},"updated":"2023-05-15T19:48:25.284679+00:00"}