1999 Fiscal Year Annual Research Report

細胞制御遺伝子を用いた造血幹細胞改変技術の開発と応用

Research Project

Project/Area Number	11557075
Research Category	Grant-in-Aid for Scientific Research (B)
Research Institution	Jichi Medical University
Principal Investigator	小澤敬也自治医科大学, 医学部, 教授 (30137707)
Co-Investigator(Kenkyū-buntansha)	長谷川護ディナベック研究所, 所長(研究職) 久米晃啓自治医学大学, 医学部, 講師 (10264293)
Keywords	造血幹細胞 / 体内増幅 / 体外増幅 / 遺伝子導入 / 分化制御遺伝子 / 選択的増幅遺伝子
Research Abstract	1.造血幹細胞の体外増幅のための分化制御遺伝子の開発とその解析:分化抑制活性を持つと考えられるドミナントネガティブレチノイン酸受容体α遺伝子(RAR403遺伝子あるいはRAR-E遺伝子)とネオマイシン耐性遺伝子(neo)をIRESで連結し、loxP配列で挟んだ後、レトロウイルスベクターに搭載した(pMXL403LneoあるいはpMXLELneo)。次に、分化制御遺伝子の活性を、分化能を有するIL-3依存性マウス骨髄細胞株(FDCPmixあるいは32Dcl3)への遺伝子導入実験により検討したところ、コントロールが分化する条件でもこれらの遺伝子を導入した細胞は未分化状態で増殖反応を示した。導入遺伝子の除去法については、Creリコンビナーゼを作用させてloxP間の組換え反応を起こさせ、その間に挟まれた分化制御遺伝子を取り外すというストラテジーの有効性を調べるため、制御可能なCreリコンビナーゼの発見ユニットをさらにはめ込んだベクターの構築を行った。現在、そのシステムの解析を行っている。 2.遺伝子導入造血幹細胞の体内増幅のための選択的増幅遺伝子の開発とその解析:新規選択的増幅遺伝子として、Mpl遺伝子(TPO結合領域を除いた部分)とエストロゲン受容体(あるいはタモキシフェン受容体)のリガンド結合領域とのキメラ遺伝子(Mpl-ERあるいはMpl-TmR)の発現ベクターを構築した。その増殖刺激活性について、IL-3依存性細胞株(Ba/F3)を用いて検討し、狙い通り機能することを確認した。選択的増幅遺伝子を用いた遺伝子導入造血幹細胞の体内増幅効率については、致死量放射線照射マウスの造血系再構築実験にて検討を進めている。

Research Products

(12 results)

All Other

All Publications (12 results)

[Publications] Matuda,K.M.: "A novel strategy for the tumor angiogenesis-targeted gene therapy : Generation of angiostatin endogenous plasminogen by protease gene transfer"Cancer Gene Ther.. (in press).
[Publications] Urabe,M.: "DNA/calcium phosphate presipitates mixed with medium are stabel and maintain high transduction efficiency"Anal.Biochem.. 278. 91-92 (2000)
[Publications] Maeda,Y.: "Endogenously generated nitric oxide by nitric-oxide synthase gene transfer inhibits cellular proliferation"J.Phamacol.Exp.Therapeut.. 292. 387-393 (2000)
[Publications] Ogasawara,Y.: "Potential application of dominant negative retinoic acid receptor genes for ex vivo expansion of hematopoietic stem cells"Gene Ther. Mol. Biol.. 3. 293-300 (1999)
[Publications] Ogasawara,Y.: "Highly-regulated expression of adeno-associated virus large Rep proteins in stable 293 cell lines using the Cre-loxP switching system"J. Gen. Virol.. 80. 2477-2480 (1999)
[Publications] Xu,R.: "A selective amplifier gene for tamoxifen-inducible expansion of hematopoietic cells"J. Gene Med.. 1. 236-244 (1999)
[Publications] Matsuda,K.M.: "Development of a modified selective a mplifier gene for hematopoietic stem cell gene therapy"Gene Ther.. 6. 1038-1044 (1999)
[Publications] Kume,A.: "Green fluorescent protein as a selectable marker of retrovirally transduced hematopoietic progenitors"Stem Cells. 17. 226-232 (1999)
[Publications] Kume,A.: "A G-CSF receptor-gyrase B fusion gene : a new type of molecular switch for expansion of genetically modified hematopoietic cells"Biochem.Biophys.Res.Commun.. 260. 9-12 (1999)
[Publications] Ozawa,K.: "Strategies for gene therapy of Parkinson's disease using adeno-associated virus (AAV) vectors"Biogenic Amines. 15. 21-37 (1999)
[Publications] Ogasawara,Y.: "Efficient production of adeno-associated virus vectors using split-type helper plasmids"Jpn. J. Cancer Res.. 90. 476-483 (1999)
[Publications] Urabe,M.: "Charged-to-alanine scanning mutagenesis of the N-terminal half of adeno-associated virus type 2 Rep78 protein"J. Virol.. 73. 2682-2693 (1999)

1999 Fiscal Year Annual Research Report

細胞制御遺伝子を用いた造血幹細胞改変技術の開発と応用

Principal Investigator

小澤 敬也 自治医科大学, 医学部, 教授 (30137707)

Research Products

[Publications] Matuda,K.M.: "A novel strategy for the tumor angiogenesis-targeted gene therapy : Generation of angiostatin endogenous plasminogen by protease gene transfer"Cancer Gene Ther.. (in press).

[Publications] Urabe,M.: "DNA/calcium phosphate presipitates mixed with medium are stabel and maintain high transduction efficiency"Anal.Biochem.. 278. 91-92 (2000)

[Publications] Maeda,Y.: "Endogenously generated nitric oxide by nitric-oxide synthase gene transfer inhibits cellular proliferation"J.Phamacol.Exp.Therapeut.. 292. 387-393 (2000)

[Publications] Ogasawara,Y.: "Potential application of dominant negative retinoic acid receptor genes for ex vivo expansion of hematopoietic stem cells"Gene Ther. Mol. Biol.. 3. 293-300 (1999)

[Publications] Ogasawara,Y.: "Highly-regulated expression of adeno-associated virus large Rep proteins in stable 293 cell lines using the Cre-loxP switching system"J. Gen. Virol.. 80. 2477-2480 (1999)

[Publications] Xu,R.: "A selective amplifier gene for tamoxifen-inducible expansion of hematopoietic cells"J. Gene Med.. 1. 236-244 (1999)

[Publications] Matsuda,K.M.: "Development of a modified selective a mplifier gene for hematopoietic stem cell gene therapy"Gene Ther.. 6. 1038-1044 (1999)

[Publications] Kume,A.: "Green fluorescent protein as a selectable marker of retrovirally transduced hematopoietic progenitors"Stem Cells. 17. 226-232 (1999)

[Publications] Kume,A.: "A G-CSF receptor-gyrase B fusion gene : a new type of molecular switch for expansion of genetically modified hematopoietic cells"Biochem.Biophys.Res.Commun.. 260. 9-12 (1999)

[Publications] Ozawa,K.: "Strategies for gene therapy of Parkinson's disease using adeno-associated virus (AAV) vectors"Biogenic Amines. 15. 21-37 (1999)

[Publications] Ogasawara,Y.: "Efficient production of adeno-associated virus vectors using split-type helper plasmids"Jpn. J. Cancer Res.. 90. 476-483 (1999)

[Publications] Urabe,M.: "Charged-to-alanine scanning mutagenesis of the N-terminal half of adeno-associated virus type 2 Rep78 protein"J. Virol.. 73. 2682-2693 (1999)

小澤敬也自治医科大学, 医学部, 教授 (30137707)