Development and its application of transgene integration system targeted to the genome of liver cells.

• Project/Area Number: 25850226
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Integrative animal science
• Research Institution: National Defense Medical College
• Principal Investigator: NAKAMURA Shingo 防衛医科大学校(医学教育部医学科進学課程及び専門課程、動物実験施設、共同利用研究, 防衛医学研究センター, 講師 (00505323)
• Project Period (FY): 2013-04-01 – 2016-03-31
• Project Status: Completed (Fiscal Year 2015)
• Budget Amount: ¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000); Fiscal Year 2015: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2014: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000); Fiscal Year 2013: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
• Keywords: 生体内遺伝子導入 / 遺伝子持続発現 / 非ウイルスベクター / 遺伝子発現切り替え / バイオマテリアル / 遺伝子発現制御 / 遺伝子長期発現 / 生体材料 / 肝疾患モデル / 遺伝子導入

Outline of Final Research Achievements

In this study, we attempted to develop easy and novel technique for gene-based manipulation in murine liver that is achieved by hydrodynamics-based gene delivery system and Cre-loxP-mediated gene switching system containing piggyBac transposon system to continuously express gene of interest. With this technique, we demonstrated to create liver disease model mouse and investigate whether the mouse is recovered by administration of our original hepatocyte growth factor-loaded biomaterial. In conclusion, we succeeded to show possibility of the easy and novel technique for gene-based manipulation in murine liver.

Research Products

• [Journal Article] Direct injection of CRISPR/Cas9-related mRNA into cytoplasm of parthenogenetically activated porcine oocytes causes frequent mosaicism for indel mutations. (2015)
  • Author(s): Sato M, Koriyama M, Watanabe S, Ohtsuka M, Sakurai T, Inada E, Saitoh I, Nakamura S, Miyoshi K
  • Journal Title: International Journal of Molecular Sciences, Volume: 16 Issue: 8 Pages: 17838-17856
  • DOI: 10.3390/ijms160817838
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Generation of α-1,3-galactosyltransferase-deficient porcine embryonic fibroblasts by CRISPR/Cas9-mediated knock-in of a small mutated sequence and a targeted toxin-based selection system. (2015)
  • Author(s): Sato M, Kagoshima A, Saitoh I, Inada E, Miyoshi K, Ohtsuka M, Nakamura S, Sakurai T, Watanabe S
  • Journal Title: Domestic Animal Reproduction Volume: 50 Issue: 5 Pages: 872-880
  • DOI: 10.1111/rda.12565
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Improved angiogenesis and healing in crush syndrome by fibroblast growth factor-2–containing low-molecular-weight heparin (Fragmin)/protamine nanoparticles (2015)
  • Author(s): Makoto Takikawa,Shingo Nakamura,Masayuki Ishihara,Yuki Takabayashi,Masanori Fujita, Hidemi Hattori,Toshihiro Kushibiki,Miya Ishihara
  • Journal Title: Journal of Surgical Research Volume: 印刷中 Issue: 2 Pages: 247-257
  • DOI: 10.1016/j.jss.2015.03.022
  • Peer Reviewed / Open Access
• [Journal Article] Enhanced healing of mitomycin C-treated healing-impaired wounds in rats with PRP-containing fragmin/protamine microparticles (PRP&F/P MPs). (2015)
  • Author(s): Megumi Takikawa, Masayuki Ishihara, Yuki Takabayashi, Yuki Sumi, Makoto Takikawa, Ryuichi Yoshida, Shingo Nakamura, Hidemi Hattori, Satoshi Yanagibayashi, Naoto Yamamoto, Tomoharu Kiyosawa.
  • Journal Title: Journal of Plastic Surgery and Hand Surgery Volume: 49 Issue: 5 Pages: 268-274
  • DOI: 10.3109/2000656x.2015.1034723
  • Peer Reviewed
• [Journal Article] Liver lobe and strain difference in gene expression after hydrodynamics-based gene delivery in mice. (2015)
  • Author(s): Nakamura Shingo, Maehara Tadaaki, Watanabe Satoshi, Ishihara Masayuki, Sato Masahiro
  • Journal Title: Animal Biotechnology Volume: 26 Pages: 51-57
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] A combination of targeted toxin technology and the piggyBacmediated gene transfer system enables efficient isolation of stable transfectants in nonhuman mammalian cells. (2015)
  • Author(s): Masahiro Sato, Emi Inada, Issei Saitoh, Yuko Matsumoto, Masato Ohtsuka, Hiromi Miura, Shingo Nakamura, Takayuki Sakurai, Satoshi Watanabe
  • Journal Title: Biotechnology Journal Volume: 10 Pages: 143-153
  • Peer Reviewed
• [Journal Article] Therapeutic Angiogenesis for Limb Ischemia Using Angiogenic Growth Factors and Carriers. (2015)
  • Author(s): Shingo Nakamura, Masahiro Sato, Hidemi Hattori, Masafumi Shimizu, Masanori Fujita, Tadaaki Maehara and Masayuki Ishihara
  • Journal Title: Current Tissue Engineering Volume: 4 Pages: 1-7
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Novel Cancer Vaccination System Based on Human Endo-Β-N-Acetyl Glucosaminidase Gene Delivery. (2014)
  • Author(s): Satoshi Watanabe, Seiki Haraguchi, Shingo Nakamura, Takayuki Sakurai, Shin-ichiro Mugikura, Kagemasa Kajiwara, Minoiru Kimura, Masahiro Sato
  • Journal Title: Journal of Glycobiolog Volume: 3 Pages: 1-8
  • Peer Reviewed
• [Journal Article] The combinational use of CRISPR/Cas9-based gene editing and targeted toxin technology enables efficient biallelic knockout of the α-1,3-galactosyltransferase gene in porcine embryonic fibroblasts. (2014)
  • Author(s): Masahiro Sato, Kazuchika Miyoshi, Yozo Nagao, Yohei Nishi, Masato Ohtsuka, Shingo Nakamura, Takayuki Sakurai, Satoshi Watanabe
  • Journal Title: Xenotransplantation Volume: 21 Pages: 291-300
  • Peer Reviewed
• [Journal Article] Effective wound healing in streptozotocin-induced diabetic rats by adipose-derived stromal cell transplantation in plasma-gel containing fragmin/protamine microparticles. (2014)
  • Author(s): Sumi Y, Ishihara M, Kishimoto S, Takikawa M, Hattori H, Takikawa M, Azuma R, Nakamura S, Fujita M, Kiyosawa T.
  • Journal Title: Annals of Plastic Surgery Volume: 72 Issue: 1 Pages: 113-120
  • DOI: 10.1097/sap.0000000000000014
  • Peer Reviewed
• [Journal Article] Interaction of silver nanoparticles and chitin powder with different sizes and surface structures: The correlation with antimicrobial activities. (2013)
  • Author(s): VQ Nguyen, M Ishihara, S Nakamura, H Hattori, T Ono, Y Miyahira, T Matsui.
  • Journal Title: Journal of Nanomaterials Volume: 467534 Issue: 1 Pages: 1-9
  • DOI: 10.1155/2013/467534
  • Peer Reviewed
• [Journal Article] Site-targeted non-viral gene delivery by direct DNA injection into the pancreatic parenchyma and subsequent in vivo electroporation in mice. (2013)
  • Author(s): M Sato, E Inada, I Saitoh, M Ohtsuka, S Nakamura, T Sakurai, S Watanabe.
  • Journal Title: Biotechnology Journal Volume: 8 Pages: 1355-1361
  • Peer Reviewed
• [Presentation] zygote injectionに依らない生殖細胞、胚を標的とした遺伝子導入によるCRISPR/Cas9 genome editingの可能性. (2015)
  • Author(s): 佐藤正宏, 大塚正人, 中村伸吾, 桜井敬之, 稲田絵美, 齋藤一誠, 渡部 聡.
  • Organizer: BMB2015（第38回日本分子生物学会年会、第88回日本生化学会大会 合同大会）
  • Place of Presentation: 神戸国際会議場
  • Year and Date: 2015-12-01
• [Presentation] ブタ単為発生卵細胞質へのCRISPR/Cas9系mRNAの直接顕微注入によるゲノム編集は多様な標的遺伝子変異を持つ細胞を生む. (2015)
  • Author(s): 佐藤正宏, 郡山実優, 渡部 聡, 大塚正人, 桜井敬之, 稲田絵美, 齋藤一誠, 中村伸吾, 三好和睦.
  • Organizer: 第108回日本繁殖生物学会大会
  • Place of Presentation: 宮崎大学および宮崎市民プラザ
  • Year and Date: 2015-09-12
• [Presentation] GAG/プロタミンナノ粒子の医療応用 (2015)
  • Author(s): 中村伸吾
  • Organizer: 第23回プロテオグライカンフォーラム
  • Place of Presentation: 東京医科歯科大学 （東京都・文京区）
  • Year and Date: 2015-01-24
  • Invited
• [Presentation] EndoGalCとtargeted toxin法の組み合わせを用いたCRISPR系でノックアウトされた遺伝子改変細胞濃縮法の開発. (2014)
  • Author(s): 渡部聡, 桜井敬之, 中村伸吾, 梶原景正, 木村穣, 佐藤正宏
  • Organizer: 第37回日本分子生物学会
  • Place of Presentation: パシフィコ横浜 （神奈川県・横浜市）
  • Year and Date: 2014-11-25 – 2014-11-27
• [Presentation] 標的細胞破壊法とpiggyBacの組み合わせは遺伝子導入安定株の効率的取得を可能とする. (2014)
  • Author(s): 佐藤正宏, 稲田絵美, 齋藤一誠, 松本祐子, 大塚正人, 三浦浩美, 中村伸吾, 桜井敬之, 渡部聡
  • Organizer: 第37回日本分子生物学会
  • Place of Presentation: パシフィコ横浜 （神奈川県・横浜市）
  • Year and Date: 2014-11-25 – 2014-11-27
• [Presentation] ブタにおけるCRISPR/Cas9, targeted toxin法を用いたmultiple constructsの標的遺伝子導入システムの開発. (2014)
  • Author(s): 佐藤正宏, 郡山実優, 三好和睦, 大塚正人, 中村伸吾, 桜井敬之, 渡部聡
  • Organizer: 第107回日本繁殖生物学会大会
  • Place of Presentation: 帯広畜産大学 （北海道・帯広市）
  • Year and Date: 2014-08-21 – 2014-08-24
• [Presentation] 多糖類で簡便に作成したナノサイズドラッグデリバリーキャリアの安定性. (2014)
  • Author(s): 中村伸吾, 石原雅之
  • Organizer: 第33回日本糖質学会
  • Place of Presentation: 名古屋大学 （愛知県・名古屋市）
  • Year and Date: 2014-08-10 – 2014-08-12
• [Presentation] 増殖因子キャリアとしてのフラグミン/プロタミン/ナノ粒子(F/P M/NPs). (2014)
  • Author(s): 石原雅之, 中村伸吾, 瀧川真人, 服部秀美, 藤田真敬.
  • Organizer: 第13回日本再生医療学会総会
  • Place of Presentation: 国立京都国際会館
• [Presentation] ハイドロダイナミクスに基づく生体内遺伝子導入における外来遺伝子発現のマウス系統差および肝臓ローブ間の差. (2013)
  • Author(s): 中村伸吾, 前原正明, 渡部聡, 石原雅之, 佐藤正宏.
  • Organizer: 第36回日本分子生物学会年会
  • Place of Presentation: 神戸国際会議場
• [Presentation] CRISPR/Cas9 による遺伝子編集と標的毒素法との組み合わは、alpha-1,3- galactosyltransferase 遺伝子を完全にKOしたブタ胎仔性線維芽細胞の効率的作製に有効である. (2013)
  • Author(s): 佐藤正宏, 三好和睦, 長尾 洋三, 西 洋平, 大塚正人, 中村伸吾, 桜井敬之, 渡部聡.
  • Organizer: 第36回日本分子生物学会年会
  • Place of Presentation: 神戸国際会議場
• [Presentation] 膜結合型C-type レクチンであるDgcr2 タンパクはI 型BMP 受容体と結合しシグナル伝達制御に関与する. (2013)
  • Author(s): 渡部聡, 梶原景正, 麦倉信一郎, 桜井敬之, 中村伸吾, 木村穣, 佐藤正宏.
  • Organizer: 第36回日本分子生物学会年会
  • Place of Presentation: 神戸国際会議場
• [Presentation] PRPキャリアとしての低分子化ヘパリン/プロタミン粒子の適用. (2013)
  • Author(s): 石原雅之, 服部秀美, 藤田真敬, 中村伸吾.
  • Organizer: 第51回日本人工臓器学会大会
  • Place of Presentation: パシフィコ横浜
• [Presentation] 多糖類を用いたマウス生体組織へのタンパク質・遺伝子運搬システムの検討. (2013)
  • Author(s): 中村伸吾, 石原雅之, 磯田晋, 木村民蔵, 山中望, 西村健二, 藤田真敬, 前原正明.
  • Organizer: 第32回日本糖質学会年会
  • Place of Presentation: 大阪国際交流センター
• [Presentation] 分子量の異なるキトサンの血液凝固効果. (2013)
  • Author(s): 石原雅之, 服部秀美, 中村伸吾.
  • Organizer: 第32回日本糖質学会年会
  • Place of Presentation: 大阪国際交流センター
• [Book] Nanomedicine (2014)
  • Author(s): Masayuki Ishihara, Makoto Takikawa, Hidemi Hattori, Masanori Fijita, Miya Ishihara, Shingo Nakamura
  • Total Pages: 528
  • Publisher: One Central Press