JP4340736B2 - レポーター遺伝子を組み込んだベクター - Google Patents
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- JP4340736B2 JP4340736B2 JP2004007549A JP2004007549A JP4340736B2 JP 4340736 B2 JP4340736 B2 JP 4340736B2 JP 2004007549 A JP2004007549 A JP 2004007549A JP 2004007549 A JP2004007549 A JP 2004007549A JP 4340736 B2 JP4340736 B2 JP 4340736B2
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Description
Biochem. Pharmacol. 1999 Sep 1;58(5):749-57 Reporter gene technology:The future looks bright. Naylor LH. Annu. Rev. Biochem. 1998;67:509-44 The green fluorescent protein. Tsien RY.
作製したプラスミドやウィルスは、プラスミドのばあいは大腸菌を宿主として増幅して十分な量を生産し、また、ウィルスベクターの場合は宿主細胞をもちいて増幅して十分な量を生産したのち、精製して、培養細胞や動物に適用する。
MERを含むcDNA配列を、pAN-Mer-Cre-Merを鋳型としてPCRにより5'端にSpe1、3'端にEcoR1の制限酵素認識部位を付加して増幅し、pGEM-T easy(プロメガ社)に導入してDNA配列に間違いのないことを確認した。このプラスミドをSpe1とEcoE1で消化して得られるフラグメントを、誘導型哺乳類発現用プラスミドpGene/V5-His(インビトロゲン社)のGAL4-UASを含むプロモーターの下流にあるマルチクローニングサイトに導入しpGene/Merを作製した。pIRES-hrGFP-1a(ストラタジーン社)を鋳型として、PCRにより、5'端にBamH1、3'端にMfe1の制限酵素認識部位を付加してIRESの遺伝子配列を増幅し、また、5'端にMfe1、3'端にSpe1の制限酵素認識部位を付加してGFPをコードする配列を増幅し、両者をpGEM-T easy(プロメガ社)に導入してDNA配列に間違いのないことを確認した。GFPをコードする配列を導入したpGEMをMfe1とSpe1で消化し、ここにIRESを導入したpGEMをMfe1とSpe1で消化して得られたフラグメントを挿入してプラスミドpGEM/GIを作製した。pGene/MerをBamH1とMfe1で消化し、ここにpGEM/GIをBamH1とMfe1で消化してえられたフラグメントを挿入して、GAL4-UASを含むプロモーターの下流にGFPをコードする遺伝子配列、IRES、MERをコードする遺伝子配列が順にならぶプラスミド、pGene/GIMを作製した。(図1a)
ヒトTPをコードするcDNAの開始コドンからストップコドンまでを含む領域を、福井医科大学外科学Dr.Riより譲り受けたプラスミド(pCIhTP)を鋳型に、PCRにより5'端EcoR1,3'端にMlu1の制限酵素認識部位をつけて増幅、pGEM-T easy(プロメガ社)に導入してDNA配列に間違いのないことを確認した後、EcoR1で消化し、得られたフラグメントをほ乳類発現用プラスミドpCI(プロメガ社)のCMVプロモーターの下流にあるマルチクローニングサイトに挿入した。実施例1で作製したpGene/GIMを鋳型として、PCRにより、5'端にMlu1の制限酵素認識部位が付加されるようIRESとMERをコードする配列を含むフラグメントを増幅し、pGEM-T easy(プロメガ社)に導入してDNA配列に間違いのないことを確認した後、Mlu1とNot1で消化してIRESとMERをコードする配列を含むフラグメントを得た。このフラグメントを、上記のTPを導入したプラスミドをMlu1とNot1で消化した位置に挿入し、TPをコードする配列の下流に導入し、CMVプロモーターの下流に、TPをコードする配列、IRES、MERをコードする配列がこの順にならぶプラスミド、pTIMを作製した。(図1b)
〔試験例1〕
〔試験例2〕
〔試験例3〕
Claims (5)
- 動物細胞で機能可能なプロモーター/エンハンサーの下流に接続される治療遺伝子に、マウスエストロゲン・レセプターの525番目のアミノ酸がGlyからArgへの1アミノ酸変異を持ち、天然のエストロゲンとの結合能を失っているがタモキシフェンとの選択的結合能を有するマウス変異型エストロゲンレセプターリガンド結合領域をコードする遺伝子(Mer遺伝子)をレポーター遺伝子として接続したベクター。
- 動物細胞で機能可能なプロモーターが、CMVプロモーターである請求項1記載のベクター。
- 動物細胞で機能可能なプロモーターが、GAL4−UASプロモーターである請求項1記載のベクター。
- 治療遺伝子にエストロゲンレセプターリガンド結合領域をIRES(Internal Ribosomal Entry Site)配列で接続した請求項1記載のベクター。
- ベクターが、プラスミドまたはウィルスベクターである請求項1記載のベクター。
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JP2008307007A (ja) | 2007-06-15 | 2008-12-25 | Bayer Schering Pharma Ag | 出生後のヒト組織由来未分化幹細胞から誘導したヒト多能性幹細胞 |
EP2268809B1 (en) * | 2008-05-02 | 2019-02-06 | Kyoto University | Method of nuclear reprogramming |
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