JPWO2009075119A1 - 効率的な核初期化方法 - Google Patents
効率的な核初期化方法 Download PDFInfo
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Abstract
Description
本発明の好ましい態様によれば、該miRNAが、(a)胚性幹細胞において体細胞よりも高度に発現しており、かつ(b)該miRNAの存在下において該miRNAの非存在下の場合よりも高い核初期化効率を与える性質を有するmiRNAである上記の方法が提供される。
さらに本発明により、上記の方法により得られた人工多能性幹細胞を分化誘導して得られる各種細胞を用いて、化合物、薬剤、毒物などの生理作用や毒性を評価する方法が提供される。
組換えベクターを体細胞に導入する方法も特に限定されず、当業者に公知の方法で行うことができる。一時的トランスフェクション、微細注射、リン酸カルシウム沈澱法、リポソーム媒介トランスフェクション、DEAEデキストラン媒介トランスフェクション、エレクトロポレーション、遺伝子銃による方法などを用いることができる。
(a)Octファミリー遺伝子及びSoxファミリー遺伝子からなる2種の遺伝子の組み合わせ
(b)Octファミリー遺伝子、Klfファミリー遺伝子、及びSoxファミリー遺伝子からなる3種の遺伝子の組み合わせ;
(c)Octファミリー遺伝子、Soxファミリー遺伝子、Linファミリー遺伝子、及びNanog遺伝子からなる4種の遺伝子の組み合わせ;
などを挙げることができるが、これらに限定されることはない。
好ましい組み合わせとして、例えば、
(e)Octファミリー遺伝子、Klfファミリー遺伝子、Soxファミリー遺伝子、及びTERT遺伝子からなる4種の遺伝子の組み合わせ;
(f)Octファミリー遺伝子、Klfファミリー遺伝子、Soxファミリー遺伝子、及びSV40 Large T antigen遺伝子からなる4種の遺伝子の組み合わせ;
(g)Octファミリー遺伝子、Klfファミリー遺伝子、Soxファミリー遺伝子、TERT遺伝子、及びSV40 Large T antigen遺伝子からなる5種の遺伝子の組み合わせを挙げることができる。
必要に応じて、上記の組み合わせからKlfファミリー遺伝子を除くことができる場合もある。
(1)Oct3/4及びSox2からなる2種の遺伝子の組み合わせ;
(2)Oct3/4、Klf4、及びSox2からなる3種の遺伝子の組み合わせ;
(3)Oct3/4、Sox2、Lin28、及びNanogからなる4種の遺伝子の組み合わせ;
(4)Oct3/4、Sox2、TERT、及びSV40 Large T antigenからなる4種の遺伝子の組み合わせ;
(5)Oct3/4、Klf4、Sox2、TERT、及びSV40 Large T antigenからなる5種の遺伝子の組み合わせ
などであるが、これらに限定されることはない。
例1:マウス胎児線維芽細胞の核初期化による人工多能性幹細胞の調製
マウス由来Oct3/4、Sox2、及びKlf4の3種の遺伝子、コントロール用のDsRed、及び18種類のmiRNAのそれぞれをコードするpMXs-ベースのレトロウイルスベクターをPLAT-E細胞にFugene6 (Roche)を用いてトランスフェクトした。翌日、Nanog遺伝子のプロモーター領域の下流にEGFPとpuromycinの耐性遺伝子をコードした配列をつないだトランスジェニックマウス由来の胎児線維芽細胞(Nanog GFP MEF、WO2007/69666)を6 well plateに1×105個ずつまいた。翌日、この細胞にOct3/4、Sox2、及びKlf4、並びに18種類のmiRNAから選ばれる各1種類のmiRNAを発現するレトロウイルスを3因子の発現ウイルスの混合液1 ml+miRNA発現ウイルス液1 mlの割合で感染させ核初期化により人工多能性幹細胞の調製を行った。
マウス由来Oct3/4、Sox2、及びKlf4の3種の遺伝子、DsRed(コントロール)、mmu-miR-295のそれぞれをコードするpMXs-ベースのレトロウイルスベクターをPLAT-E細胞にFugene6 (Roche)を用いてトランスフェクトした。翌日、Nanog遺伝子のプロモーター領域の下流にEGFPとpuromycinの耐性遺伝子をコードした配列をつないだトランスジェニックマウス由来の尻尾線維芽細胞(Nanog GFP tailtip fibroblasts)を6 well plateに1×105個ずつまいた。翌日、この細胞にOct3/4、Sox2、及びKlf4の3因子とDsRedあるいはmmu-miR-295を発現するレトロウイルスを(1:1:1:1)の割合で感染させ核初期化により人工多能性幹細胞の調製を行った。
ヒト由来Oct3/4、Sox2、及びKlf4の3種の遺伝子に加え、コントロール用のDsRedと23種類のmiRNA又はmiRNAクラスターをコードするpMXs-ベースのレトロウイルスベクターをPLAT-E細胞にFugene6 (Roche)を用いてトランスフェクトした。翌日、げっ歯類のみに感染するウイルスのレセプターであるSlc7a1を発現するようにしたヒト成人皮膚細胞(adult human dermal fibroblasts)を6cm dishに3×105個ずつまいた。翌日、この細胞にOct3/4、Sox2、及びKlf4の3種の遺伝子と各種miRNAを発現するレトロウイルスを1:1:1:1の割合で感染させ核初期化による人工多能性幹細胞の生成を試みた。
Claims (7)
- 人工多能性幹細胞の製造方法であって、miRNAの存在下で核初期化因子により核初期化する工程を含み、該miRNAが該miRNAの存在下において該miRNAの非存在下の場合よりも高い核初期化効率を与える性質を有するmiRNAである方法。
- 該miRNAが胚性幹細胞において体細胞よりも高度に発現しているmiRNAである請求項1に記載の方法。
- 核初期化因子がOct3/4、Klf4、及びSox2からなる3種の遺伝子の遺伝子産物である請求項1又は2に記載の方法。
- 核初期化因子による核初期化を体細胞への上記遺伝子の導入により行う請求項1ないし3のいずれか1項に記載の方法。
- miRNAが下記の群:hsa-miR-372、hsa-miR-373、hsa-miR-302b、hsa-miR-302c、hsa-miR-302a、hsa-miR-302d、hsa-miR-367、hsa-miR-520c、mmu-miR-291a、mmu-miR-294、及びmmu-miR-295からなる群から選ばれる1又は2以上のRNA(記号はmiRBaseデータベースの登録名を示す)に含まれる1又は2以上のmiRNAである請求項1ないし4のいずれか1項に記載の方法。
- miRNAがmiRBaseデータベースの登録名hsa-miR-302b-367で特定されるRNA配列に含まれる1又は2以上のmiRNAである請求項1ないし4のいずれか1項に記載の方法。
- miRNAが配列表の配列番号1ないし12から選択される1又は2以上のRNA配列に含まれる1又は2以上のmiRNAである請求項1ないし4のいずれか1項に記載の方法。
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US8791248B2 (en) | 2014-07-29 |
US20130102768A1 (en) | 2013-04-25 |
KR101532442B1 (ko) | 2015-06-29 |
WO2009075119A1 (ja) | 2009-06-18 |
AU2008286249A1 (en) | 2009-06-25 |
US20100075421A1 (en) | 2010-03-25 |
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