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レンチウイルスベクターとsh・RNAを用いたノックダウンマウスの作製

Research Project

Project/Area Number 18659325
Research Category

Grant-in-Aid for Exploratory Research

Allocation TypeSingle-year Grants
Research Field Dermatology
Research InstitutionEhime University

Principal Investigator

橋本 公二  Ehime University, 大学院・医学系研究科, 教授 (00110784)

Co-Investigator(Kenkyū-buntansha) 花川 靖  愛媛大学, 大学院・医学系研究科, 助教 (90284398)
Project Period (FY) 2006 – 2007
Project Status Completed (Fiscal Year 2007)
Budget Amount *help
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2007: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 2006: ¥1,700,000 (Direct Cost: ¥1,700,000)
KeywordsToll-like receptor / sh-RNA / レンチウィルスベクター / ノックダウン / トランスジェニックマウス / TLR2 / TLR3 / 角化細胞
Research Abstract

本研究の目的は、sh-RNA法とレンチウイルスベクターの技術を融合したノックダウンマウスの作製法を樹立することである。この技術が確立されれば低コストで短時間に多くの遺伝子の機能が抑制されたマウスを作製することが可能となり、皮膚科領域のみならず、生物、医学研究の発展に大きな貢献ができるのではないかと考えられる。具体的には、プロトタイプモデルとしてToll-like recptor(TLR)アイソタイプを同時に抑制するトランスジェニックマウスを作成する。TLR2,TLR3の発現を効果的に抑制できるsh-RNAを作製し、レンチウィルスベクターに組み込んだ。このウィルスベクターを角化細胞に導入し、抑制効果を確認した。生後1日の新生児マウス背部皮膚を剥離切除しディスパーゼ処理、続いてトリプシン処理を行い、低カルシウム無血清培地をもちいて表皮細胞を培養した。レンチウイルスベクターをMOI20にて感染し48h後にtotal RNAを回収した。標的配列を持つウイルスの効果をコントロールスクランブル配列と比較してABI prismを用いてreal-time PCRを行った。TLR2,TLR3のリガンド刺激においてシグナル伝達は抑制されていた。レンチウイルスベクターのpLe-U6iのU6 promoterとsh-RNA配列の間にバクテリオファージ由来のloxP配列、終止配列(TTTTTT)、loxP配列をタンデムにつないだスタッファーを挿入しsilent-lentivirus vector(U6LSL-shRNA,LSL=loxP-stop-loxP)を作製した。このベクターを用いてトランスジェニックマウスを作製する準備までは完了したが、実際のマウス作製には至らなかった。

Report

(2 results)
  • 2007 Annual Research Report
  • 2006 Annual Research Report
  • Research Products

    (21 results)

All 2008 2007 2006

All Journal Article (15 results) (of which Peer Reviewed: 9 results) Presentation (6 results)

  • [Journal Article] An intermediary role of proHB-EGF shedding in growth factor-induced c-Myc gene expression2008

    • Author(s)
      Nanba D, Inoue H, Shigemi Y, Shirakata Y, Hashimoto K, Higashiyama S
    • Journal Title

      J Cell Physiol 214

      Pages: 465-73

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] PPARgamma is an important transcription factor in lalpha, 25-dihydroxyvitamin D3-induced in volucrin expression2008

    • Author(s)
      Dai X, Sayama K, Shirakata Y, Tokumaru S, Yang L, Tohyama M, Hirakawa S, Hanakawa Y, Hashimoto K
    • Journal Title

      J Dermatol Sci 50

      Pages: 53-60

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Human corneal epithelial cell proliferation by epiregulin and its cross-induction by other EGF family members2007

    • Author(s)
      Morita S, Shirakata Y, Shiraishi A, I Kadota Y, Hashimoto K, Higashiyama S, Ohashi Y
    • Journal Title

      Molecular Vision 13

      Pages: 2119-2128

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Epiregulin, a member of the EGF family, is over-expressed in psoriatic epidermis2007

    • Author(s)
      Shirakata Y, Kishimoto J, Tokumaru S, Yamasaki K, Hanakawa Y, Tohyama M, Sayama K, Hashimoto K
    • Journal Title

      J Dermatol Sci 45

      Pages: 69-72

    • NAID

      10019994988

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Hair follicle regeneration using grafted rodent and human cells2007

    • Author(s)
      Ehama R, Ishimatsu-Tsuji Y, Iriyama S, Ideta R, Soma T, Yano K, Kawasaki C, Suzuki S, Shirakata Y, Hashimoto K, Kishimoto J
    • Journal Title

      J Invest Dermatol 127

      Pages: 2106-15

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] STAT5a/PPARgamma Pathway Regulates Involucrin Expression in Keratinocyte Differentiation2007

    • Author(s)
      Dai X, Sayama K, Shirakata Y, Hanakawa Y, Yamasaki K, Tokumaru S, Yang L, Wang X, Hirakawa S, Tohyama M, Yamauchi T, Takashi K, Kagechika H, Hashimoto K
    • Journal Title

      J Invest Dermatol 127

      Pages: 1728-35

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Suppressor of cytokine signaling 3 negative regulation of signal transducer and activator of transcription 3 in platelet-derived growth factor-induced fibroblast migration2007

    • Author(s)
      Nagai H, Tokumaru S, Sayama K, Shirakata Y, Hanakawa Y, Hirakawa S, Dai X, Tohyama M, Yang L, Hashimoto K
    • Journal Title

      J Dermatol 34

      Pages: 523-530

    • NAID

      10021306180

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] CXCL16 is a novel mediator of the innate immunity of epidermal keratinocytes2007

    • Author(s)
      Tohyma M, Sayama K, Komatsuzawa H, Hanakawa Y, Shirakata Y, Dai X, Yang L, Tokumaru S, Nagai H, Hirakawa S, Sugai M, Hashimoto K
    • Journal Title

      Int Immunol 19

      Pages: 1095-1102

    • NAID

      10020108093

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Association of human herpesvirus 6 reactivation with the flaring and severity of drug-induced hypersensitivity syndrome2007

    • Author(s)
      Tohyama M, Hashimoto K, Yasukawa M, Kimura H, Horikawa T, Nakajima K, Urano Y, Matsumoto K, Iijima M, Shear NH
    • Journal Title

      Br J Dermatol 157

      Pages: 934-40

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Epiregulin, a member of the EGF family, is over-expressed in psoriatic epidermis.2007

    • Author(s)
      Shirakata Y, Kishimoto J, et al.
    • Journal Title

      The Journal of Dermatological Science 45

      Pages: 69-72

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Pre-B-cell leukemia transcription factor 1 is a major target of promyelocytic leukemia zinc-finger-mediated melanoma cell growth suppression.2007

    • Author(s)
      Shiraishi K, Yamasaki K, Shirakata Y, et al.
    • Journal Title

      Oncogene 26

      Pages: 339-348

    • Related Report
      2006 Annual Research Report
  • [Journal Article] TAK1 is essential for differentiation and the prevention of apoptosis in epidermis.2006

    • Author(s)
      Sayama K, Hanakawa Y, Nagai H, Shirakata Y, et al.
    • Journal Title

      The Journal of Biological Chemistry 281

      Pages: 22013-22020

    • Related Report
      2006 Annual Research Report
  • [Journal Article] New skin-equivalent model from de-epithelialized amnion membrane.2006

    • Author(s)
      Yang L, Shirakata Y, et al.
    • Journal Title

      Cell and Tissue Research 326

      Pages: 69-77

    • Related Report
      2006 Annual Research Report
  • [Journal Article] A novel function of angiotensin II in skin wound healing.2006

    • Author(s)
      Yahata Y, Shirakata Y, et al.
    • Journal Title

      The Journal of Biological Chemistry 281

      Pages: 13209-13216

    • Related Report
      2006 Annual Research Report
  • [Journal Article] SOCSI negative feedback of STAT1 activation is a key pathway in the dsRNA-induced innate immune response of human keratinocytes.2006

    • Author(s)
      Dai X, Sayama K, Yamasaki K, Tohyama M, Shirakata Y, et al.
    • Journal Title

      The Journal of Investigative Dermatology 126

      Pages: 1574-1581

    • Related Report
      2006 Annual Research Report
  • [Presentation] A new skin equivalent using de-epithelialized amnion membrane2007

    • Author(s)
      Shirakata Y, Yang L, Hashimoto K
    • Organizer
      8^<th> Congress of the German-Japanese Society of Dermatology
    • Place of Presentation
      Yokohama
    • Related Report
      2007 Annual Research Report
  • [Presentation] PRAR-gamma is involved in 1.25-dihydroxyvitamin D3-induced beta-defensin-3 and cathelicidin expression in normal keratinocyte2007

    • Author(s)
      Dai X, Shirakata Y, Sayama K, Tokumaru S, Hirakawa S, Tohyama M, Yang L, Hashimoto K
    • Organizer
      68^<th> Annual Meeting of the Society for Investigative Dermatology
    • Place of Presentation
      Los Angeles
    • Related Report
      2007 Annual Research Report
  • [Presentation] Development of a new skin equivalent model using de-epithelialized amnion membrane2007

    • Author(s)
      Yang L, Shirakata Y, Dai X, Tokumaru S, Hirakawa S, Sayama K, Hashimoto K
    • Organizer
      68^<th> Annual Meeting of the Society for Investigative Dermatology
    • Place of Presentation
      Los Angeles
    • Related Report
      2007 Annual Research Report
  • [Presentation] Targeted overexpression of VEGF-A of -C in the skin promotes lymph node lymphangiogenesis and metastasis2007

    • Author(s)
      Hirakawa S, Kodama S, Brown LF, Paavonen K, Hashimoto K, Alitalo K, Detmar M
    • Organizer
      2^<nd> Intemational symposium on cancer metastasis and the lymphovascular system
    • Place of Presentation
      Sanfrancisco
    • Related Report
      2007 Annual Research Report
  • [Presentation] Ablation of TAKl in mouse keratinocytes disturbed hair follicle development2007

    • Author(s)
      Sayama K, Kajiya K, Shirakata Y, Hanakawa Y, Hirakawa S, Dai X, Nagai H, Akira S, Kishimoto J, Hashimoto K
    • Organizer
      68^<th> Annual Meeting of the Society for Investigative Dermatology
    • Place of Presentation
      Los Angeles
    • Related Report
      2007 Annual Research Report
  • [Presentation] Milroy's disease:a case report of the congenital lymphedema pedigree2007

    • Author(s)
      Hirakawa S, Murakaami S, Hashimoto K
    • Organizer
      The 15^<th> Korea-Japan Joint Meeting of Dermatology
    • Place of Presentation
      Jeju
    • Related Report
      2007 Annual Research Report

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Published: 2006-04-01   Modified: 2016-04-21  

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