JP5557288B2 - 細胞のリプログラミングに用いられる複数遺伝子の発現制御システム - Google Patents
細胞のリプログラミングに用いられる複数遺伝子の発現制御システム Download PDFInfo
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Description
本発明は、細胞再生医療分野における複数の転写因子の発現制御による治療用細胞・組織の分化誘導および製造に関する。より具体的には、本発明は、複数の転写因子の制御された発現を可能とする発現カセット、ならびに該発現カセットを用いた体細胞リプログラミングおよび分化多能性幹細胞(iPS細胞など)の誘導に関する。
iPS細胞(induced pluripotent stem cell)とは、生体の組織・臓器を構成する分化した体細胞を、Oct4、Sox2、cMyc、Klf4などの転写因子により人工的に初期化して得られる、三胚葉分化能および個体発生能をもつ胚性幹細胞(ES細胞)様の分化多能性幹細胞である。
(1)Oct4、Sox2、cMycおよびKlf4の遺伝子の連結
図1に示す7種類の4因子連結カセット:SKtMO、MKtSO、MKiresSO、SOiresMK、SKiresMO、MOiresSK、およびMKtOSを以下の手順で作製した。
実施例1(1)で作製した4因子連結カセットベクターをBamHIおよびNotIで消化することにより、4因子連結カセットを調製し、pMXsベクター(東京大学北村俊雄教授より供与;Takahashi et al., Cell, 2006, 126: 663-676)のBamHI−NotI部位間へ挿入した。得られたベクターを、それぞれpMXs−SKtMO、pMXs−SKiresMO、pMXs−MOiresSK、pMXs−MKtSO、pMXs−MKiresSO、pMXs−SOiresMK、およびpMXs−MKtOSと命名した。
(1)連結4因子SKtMOによるmBMMNCのリプログラミング
細胞のリプログラミングは、実施例1(2)で作製した7種の連結4因子導入pMXsベクターのうち、pMXs−SKtMOを導入した場合にのみ成功した。この実験の詳細を以下に示す。
遺伝子導入の約48時間後に培養上清を回収し、0.45μmフィルターでろ過後、mBMMNCへのRVベクター1感染反応あたり培養上清の3分の2量を分注し、5800×g、4℃で4時間遠心した。コントロールは、各因子4種の培養上清を分注する際に混合し、遠心調製した。ペレットをRVベクター1感染反応あたり0.75mlのマウスES細胞用培地で再懸濁し、これをRVベクター濃縮サンプルとした。マウスES細胞用培地としては、20%FBS入りDMEM培地に1/100容の2-メルカプトエタノール(SIGMA社)、1000 IU/mlマウスLIF(1/10000容のESGRO;フナコシ社)を添加したものを用いた。
pMXs−SKtMOベクターに加えて、さらに1遺伝子ORFのpMXsベクターを用いた遺伝子導入による細胞リプログラムを試みた。方法は、実施例2(1)に従い、独立に3セットの実験を行った。その結果、全ての実験回において、pMXs−SKtMOベクターとpMXs−Oct4ベクターとの組み合わせを用いることにより、Oct4−GFP陽性細胞はRVベクター感染後3日目から出現し、増殖した。さらに、感染後16日目からES様コロニー形成が確認された。pMXs−SKtMOベクターのみを用いた場合は、感染後4日目においてもOct4−GFP陽性細胞を殆ど検出できなかった。感染後4日目の写真を図8に示す。
連結4因子の発現プロセシングを確認するために、実施例1(1)で作製した連結4因子ベクター:pmSKtMO、pmMKtSO、pmMKiresSO、pmSOiresMK、pmSKiresMOおよびpmMOiresSKを、CHO細胞で一過的に発現させ、ウェスタンブロット解析を行った。
Claims (10)
- 複数の核初期化転写因子のそれぞれをコードする複数のDNAを、細胞中の内在酵素によって切断されるペプチドをコードする介在DNA配列を介してポリシストロニックに連結してなる発現カセットであって、
介在DNA配列が2AペプチドをコードするDNA配列を含むものであり、
複数の核初期化転写因子が、Sox2、Klf4、cMyc、およびOct4であり、
Sox2をコードするDNA、Klf4をコードするDNA、cMycをコードするDNA、およびOct4をコードするDNAを、5’末端から3’末端に向けてこの順番で含んでなる、発現カセット。 - 請求項1に記載の発現カセットを作動可能な形で含んでなる、発現ベクター。
- レトロウイルスベクターである、請求項2に記載の発現ベクター。
- 請求項2または3に記載の発現ベクターを含んでなる、細胞。
- 細胞をリプログラミングする方法であって、該細胞に、請求項1に記載の発現カセットまたは請求項2もしくは3に記載の発現ベクターを導入する工程を含んでなる、方法。
- 前記細胞に、1種の核初期化転写因子をコードするDNAを含む第二の発現カセットまたは該発現カセットを作動可能な形で含む第二の発現ベクターを導入する工程をさらに含んでなる、請求項5に記載の方法。
- 第二の発現カセットまたは第二の発現ベクターに含まれる前記DNAが、Oct4をコードするものである、請求項6に記載の方法。
- 前記細胞が骨髄由来単核球細胞である、請求項5に記載の方法。
- iPS細胞が得られる、請求項5に記載の方法。
- リプログラミングされた細胞の製造方法である、請求項5〜9のいずれか一項に記載の方法。
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US9365866B2 (en) | 2009-06-03 | 2016-06-14 | National Institute Of Advanced Industrial Science And Technology | Vectors for generating pluripotent stem cells and methods of producing pluripotent stem cells using the same |
WO2011145615A1 (ja) * | 2010-05-18 | 2011-11-24 | タカラバイオ株式会社 | 多能性幹細胞の製造のための核酸 |
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EP2639297B1 (en) * | 2010-11-09 | 2018-01-03 | National Institute of Advanced Industrial Science And Technology | Method for producing peripheral blood monocyte-derived pluripotent stem cells |
CN106148286B (zh) * | 2016-06-29 | 2019-10-29 | 牛刚 | 一种用于检测热原的细胞模型的构建方法和细胞模型及热原检测试剂盒 |
CN116529361B (zh) * | 2019-10-18 | 2024-01-26 | 北赛泓升(北京)生物科技有限公司 | 使用多顺反子sox2、klf4和任选的c-myc产生诱导多能干细胞 |
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