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

• Project/Area Number: 18659325
• Research Category: Grant-in-Aid for Exploratory Research
• Allocation Type: Single-year Grants
• Research Field: Dermatology
• Research Institution: Ehime 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: ¥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)
• Keywords: Toll-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。

Research Products

• [Journal Article] An intermediary role of proHB-EGF shedding in growth factor-induced c-Myc gene expression (2008)
  • Author(s): Nanba D, Inoue H, Shigemi Y, Shirakata Y, Hashimoto K, Higashiyama S
  • Journal Title: J Cell Physiol 214 Pages: 465-73
  • Peer Reviewed
• [Journal Article] PPARgamma is an important transcription factor in lalpha, 25-dihydroxyvitamin D3-induced in volucrin expression (2008)
  • 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
  • Peer Reviewed
• [Journal Article] Human corneal epithelial cell proliferation by epiregulin and its cross-induction by other EGF family members (2007)
  • 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
  • Peer Reviewed
• [Journal Article] Epiregulin, a member of the EGF family, is over-expressed in psoriatic epidermis (2007)
  • 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
  • Peer Reviewed
• [Journal Article] Hair follicle regeneration using grafted rodent and human cells (2007)
  • 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
  • Peer Reviewed
• [Journal Article] STAT5a/PPARgamma Pathway Regulates Involucrin Expression in Keratinocyte Differentiation (2007)
  • 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
  • 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 migration (2007)
  • 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
  • Peer Reviewed
• [Journal Article] CXCL16 is a novel mediator of the innate immunity of epidermal keratinocytes (2007)
  • 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
  • Peer Reviewed
• [Journal Article] Association of human herpesvirus 6 reactivation with the flaring and severity of drug-induced hypersensitivity syndrome (2007)
  • 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
  • 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
• [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
• [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
• [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
• [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
• [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
• [Presentation] A new skin equivalent using de-epithelialized amnion membrane (2007)
  • Author(s): Shirakata Y, Yang L, Hashimoto K
  • Organizer: 8^<th> Congress of the German-Japanese Society of Dermatology
  • Place of Presentation: Yokohama
• [Presentation] PRAR-gamma is involved in 1.25-dihydroxyvitamin D3-induced beta-defensin-3 and cathelicidin expression in normal keratinocyte (2007)
  • 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
• [Presentation] Development of a new skin equivalent model using de-epithelialized amnion membrane (2007)
  • 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
• [Presentation] Targeted overexpression of VEGF-A of -C in the skin promotes lymph node lymphangiogenesis and metastasis (2007)
  • 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
• [Presentation] Ablation of TAKl in mouse keratinocytes disturbed hair follicle development (2007)
  • 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
• [Presentation] Milroy's disease:a case report of the congenital lymphedema pedigree (2007)
  • Author(s): Hirakawa S, Murakaami S, Hashimoto K
  • Organizer: The 15^<th> Korea-Japan Joint Meeting of Dermatology
  • Place of Presentation: Jeju