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Antisense Display-新しい機能性遺伝子スクリーニング法開発の試み

Research Project

Project/Area Number 08283208
Research Category

Grant-in-Aid for Scientific Research on Priority Areas

Allocation TypeSingle-year Grants
Research InstitutionKanazawa University

Principal Investigator

山本 博  金沢大学, 医学部, 教授 (00115198)

Co-Investigator(Kenkyū-buntansha) 米倉 秀人  金沢大学, 医学部, 助教授 (80240373)
Project Period (FY) 1996
Project Status Completed (Fiscal Year 1996)
Budget Amount *help
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1996: ¥2,000,000 (Direct Cost: ¥2,000,000)
Keywordsアンチセンス / Antisense Display / mRNA / 遺伝子機能 / 血管新生
Research Abstract

本研究は、新しい遺伝子機能解析手段としての"Antisense Display"法の創出を目的とする。本法は、(1)mRNAの5'領域に対し全mRNA分子種をカバーするアンチセンスDNAレパトリ-の作製→(2)所定の細胞形質変化を来すアンチセンス分子種のスクリーニング→(3)5'センスプライマーと3'アンカー型プライマーを用いた翻訳枠全長のクローニング→(4)構造決定→(5)候補遺伝子の機能検定の5ステップからなる。
本年度は、ステップ(1)、(2)の検証を行った。
1.Kozakのコンセンサス配列に対応する鎖長6〜12塩基のホスホロチオエ-ト型アンチセンスDNAレパートリー化学合成した。
2.当該レパートリーの翻訳阻害活性を無細胞蛋白合成系で検証した。その結果、(1)ウサギ網状赤血球ライゼ-トでの翻訳反応はアンチセンスDNAの鎖長に依存して阻害され、10mer以上のレパトリ-ではほぼ完全な阻害が得られた。この蛋白合成阻害は、(2)RNaseHで増強され、(3)鋳型RNAの熱変形なしに達成された。
3.以上の検証に基づき、鎖長10merのアンチセンスレパートリーを計128のサブグループに分けて大量合成し、2段階のHPLCで精製した。
4.本法は、アンチセンス効果がgain-of-functionとなる抑制性因子の探索により大きな適応があると考えられたので、まず血管新生抑制遺伝子を候補遺伝子として細胞でのスクリーニングを開始した。すなわち、ヒト微小血管内皮細胞を96穴プレートに播種後、アンチセンスサブグループあるいは対照の10merオリゴd(A)の存在下に培養し、内皮細胞の(1)増殖促進と(2)マトリゲル上での管腔形成促進を指標として全サブグループの一次スクリーニングを完了した。この結果、数種の候補サブグループを得、現在二次スクリーニングを進めている。

Report

(1 results)
  • 1996 Annual Research Report
  • Research Products

    (30 results)

All Other

All Publications (30 results)

  • [Publications] Sho-ichi Yamagishi: "Advanced glycation endproducts-driven angiogenesis in vitro." J.Biol.Chem.272. 8723-7830 (1997)

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      1996 Annual Research Report
  • [Publications] Yasuhiko Hayashi: "Induction of various blood-brain barrier properties in non-neural endothelial cells by close apposition to co-cultured astrocytes." Glia. 19. 13-26 (1997)

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      1996 Annual Research Report
  • [Publications] Masahide Kaji: "Participation of c-met in the progression of human gastric cancers ; anti-c-met oligonucleotides inhibit proliferation or invasiveness of gastric cancer cells." Cancer Gene Therapy. 3. 393-404 (1996)

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      1996 Annual Research Report
  • [Publications] Sho-ichi Yamagishi: "Advanced glycosylation end products stimulate the growth but inhibit the prostacyclinproducing ability of endothelial cells through interactions with their receptors." FEBS Letters. 384. 103-106 (1996)

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      1996 Annual Research Report
  • [Publications] Yasuhiko Yamamoto: "Advanced glycation endproducts-receptor interactions stimulate the growth of human pancreatic cancer cells through the induction of platelet-derived growth factor-B." Biochem.Biophys.Res.Commun.222. 700-705 (1996)

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      1996 Annual Research Report
  • [Publications] Yutaka Yonemura: "Correlation between overexpression of c-met gene and the progression of gastric cancer." Int.J.Oncology. 8. 555-560 (1996)

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      1996 Annual Research Report
  • [Publications] Hiroshi Yamamoto: "Vascular VEGF : The expression and roles in hypoxia-driven angiogenesis." Journal of Vascular Research. 33. 110 (1996)

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      1996 Annual Research Report
  • [Publications] Shoichi Yamagishi: "Advanced glycation endproducts can promote angiogenesis through autocrine vascular endothelial growth factors." Journal of Vascular Research. 33. 110 (1996)

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      1996 Annual Research Report
  • [Publications] Yasuhiko Hayashi: "Astrocytes can transdifferentiate non-neural endothelial cells into the neural type through endfeet-mediated contacts." Journal of Vascular Research. 33. 33 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Haruhiko Hatakeyama: "The expression of steroidogenic enzyme genes in human vascular cells." Biochem.Mol.Biol.Int.40. 639-645 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Yasuo Hirono: "Expression of autocrine motility factor receptor correlates with disease progression in human gastric cancer." British Journal of Cancer. 74. 2003-2007 (1996)

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      1996 Annual Research Report
  • [Publications] Ronan,P.: "Effect of low frequency magnetic fields and induced current on DNA digestion." Proc.of Australian university Power Engineering Conference. 259-262 (1996)

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      1996 Annual Research Report
  • [Publications] Nafalshki,A.: "Measurement of magnetic fields in urban environment." Proc.of Australian University Power Engineering Conference. 247-252 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] 山本博: "アンチセンスDNA" 生化学. 68. 155 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] 山本博: "血管系細胞の"co-culture"と"hypoxic culture"" 口腔組織培養研究会誌. 5. 1-7 (1996)

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      1996 Annual Research Report
  • [Publications] 山岸昌一: "後期糖化反応生成物(AGE)による血管内皮細胞・周皮細胞障害とその分子機構" 分子糖尿病学. 7. 29-34 (1996)

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      1996 Annual Research Report
  • [Publications] 三浦順之助: "長期罹病IDDM患者の末梢血単球におけるAGEレセプター遺伝子の発現" 分子糖尿病学. 7. 35-39 (1996)

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      1996 Annual Research Report
  • [Publications] Narushima,Y.,et al.: "Structure,chromosomal localization,and expression of mouse genes encoding type III Reg RegIIIα,RegIIIβ,RegIIIγ." Gene. (in press). (1997)

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      1996 Annual Research Report
  • [Publications] Noguchi,N.,et al.: "Cyclic ADP-ribose binds to FK506-binding protein 12.6 to release Ca2+from islet microsome." Journal of Biological Chemistry. 272. 3133-3136 (1997)

    • Related Report
      1996 Annual Research Report
  • [Publications] Yonekura,H.,et al.: "Identification of the five essential histidine residues for peptidylglycine monooxygenase." Biochemical and Biophysical Research Communication. 218. 495-499 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] 山岸昌一他: "Advanced glycation endproducts の血管新生活性について" 分子糖尿病学. 7(印刷中). (1997)

    • Related Report
      1996 Annual Research Report
  • [Publications] 山本靖彦他: "糖尿病-膵癌合併の分子メカニズム" 分子糖尿病学. 7(印刷中). (1997)

    • Related Report
      1996 Annual Research Report
  • [Publications] 山本博: "「医学のための基礎分子細胞生物学」細胞の基本構造、老化、遺伝病(1)、遺伝病(2)、血管新生" 南山堂(印刷中), (1997)

    • Related Report
      1996 Annual Research Report
  • [Publications] 山本博: "VEGF、動脈硬化+高脂血症研究ストラテジー" 秀潤社, 464 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] 山岸昌一: "周皮細胞、「血管新生のメカニズムと疾患」" 医薬ジャーナル社, 287 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] 山本博: "KEY WORD 1996-'97『炎症・免疫系』" 先端医学社, 191 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Hiroshi Yamamoto: "Contemporary approaches toward cure of gastric cancer" Maeda Shoten Co.,Ltd., 197 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Yutaka Yonemura: "腹膜播種(Peritoneal Dissemination)" へるす出版, 134 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] 米倉秀人: "「医学のための基礎分子細胞生物学」25章"インスリン、グルカゴン"" 平賀紘一、伊達孝保、山本博、野口民夫編(南山堂)(印刷中), (1997)

    • Related Report
      1996 Annual Research Report
  • [Publications] 米倉秀人: "「医学のための基礎分子細胞生物学」43章"血管作動性物質"" 平賀紘一、伊達孝保、山本博、野口民夫編(南山堂)(印刷中), (1997)

    • Related Report
      1996 Annual Research Report

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Published: 1996-04-01   Modified: 2016-04-21  

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