The production of the organ deficient model using rat pluripotent stem cells, and organ regeneration by blastocyst complementation procedure

• Project/Area Number: 25290037
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Partial Multi-year Fund
• Section: 一般
• Research Field: Laboratory animal science
• Research Institution: National Institute for Physiological Sciences
• Principal Investigator: HIRABAYASHI MASUMI 生理学研究所, 行動・代謝分子解析センター, 准教授 (20353435)
• Co-Investigator(Renkei-kenkyūsha): HARA HIROMASA 自治医科大学, 分子病態治療研究センター, 助教 (50751244)
• Project Period (FY): 2013-04-01 – 2016-03-31
• Project Status: Completed (Fiscal Year 2015)
• Budget Amount: ¥17,030,000 (Direct Cost: ¥13,100,000、Indirect Cost: ¥3,930,000); Fiscal Year 2015: ¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000); Fiscal Year 2014: ¥5,460,000 (Direct Cost: ¥4,200,000、Indirect Cost: ¥1,260,000); Fiscal Year 2013: ¥6,890,000 (Direct Cost: ¥5,300,000、Indirect Cost: ¥1,590,000)
• Keywords: 胚性幹細胞 / 異種キメララット / ノックアウトラット / 胚盤胞補完法 / 臓器欠損 / 人工ヌクレアーゼ / CRISPR/Cas9 / CRISPR/Cas9法 / Foxn1遺伝子 / マウスES細胞 / 胸腺 / GFP / 末梢血T細胞 / 遺伝子ターゲティングベクター / CRISPR/Cas法 / FoxN1遺伝子 / ラットES細胞 / 単為発生胚 / 生殖寄与能 / Hex遺伝子 / Prdm14遺伝子 / フォルスコリン

Outline of Final Research Achievements

Pancreas- and kidney-deficient rats were prepared by knocking-out (KO) the Pdx1 gene and the Sall1 gene, respectively. By applying blastocyst complementation, both organs could be regenerated from rat ES cells in homozygous KO rat individuals. Knocking-out the Foxn1 gene resulted in mutant rats lacking thymus and T-cell population. Interspecies blastocyst complementation with mouse ES cells gave birth to viable rat offspring carrying mouse thymus. Thus, allogenic or xenogenic stem cells successfully contributed to regeneration of target organs in the KO models.

Research Products

• [Journal Article] Hypomorphic phenotype of Foxn1 gene-modified rats by CRISPR/Cas9 system. (2016)
  • Author(s): Goto T, Hara H, Nakauchi H, Hochi S, Hirabayashi M.
  • Journal Title: Transgenic Research Volume: 印刷中 Issue: 4 Pages: 533-544
  • DOI: 10.1007/s11248-016-9941-9
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Transgenic rat model of childhood-onset dermatitis by overexpressing telomerase reverse transcriptase (TERT) (2016)
  • Author(s): Kaneko R, Sato A, Hamada S, Yagi T, Ohsawa I, Ohtsuki M, Kobayashi E, Hirabayashi M, and Murakami T
  • Journal Title: Transgenic Research Volume: 17 Feb Issue: 4 Pages: 1-12
  • DOI: 10.1007/s11248-016-9939-3
  • Peer Reviewed / Open Access
• [Journal Article] Rat blastocysts from nuclear injection and time-lagged enucleation and their commitment to embryonic stem cells. (2016)
  • Author(s): Hara H, Goto T, Takizawa A, Sanbo M, Jacob HJ, Kobayashi T, Nakauchi H, Hochi S, Hirabayashi M
  • Journal Title: Cell. Reprogram. Volume: 印刷中 Issue: 2 Pages: 108-115
  • DOI: 10.1089/cell.2015.0084
  • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Lack of pulse and surge modes and glutamatergic stimulation of LH release in Kiss1 knockout rats. (2015)
  • Author(s): Uenoyama, Y., Nakamura, S., Hayakawa, Y., Ikegami, K., Watanabe, Y., Deura, C., Minabe, S., Tomikawa, J., Goto, T., Ieda, N., Inoue, N., Sanbo, M., Tamura, C., Hirabayashi, M., Maeda, K., Tsukamura, H.
  • Journal Title: J Neuroendocrinol Volume: 27 Issue: 3 Pages: 187-197
  • DOI: 10.1111/jne.12257
  • Peer Reviewed / Open Access
• [Journal Article] Knock-in of a histone H2B-tdTomato reporter into the Rosa26 locus allows visualization of cell nuclei in rats. (2015)
  • Author(s): GotoT, Kobayashi T, Hara H, Sanbo M, Hochi S, Nakauchi H, Hirabayashi M
  • Journal Title: Mol. Reprod. Dev. Volume: 82 Issue: 12 Pages: 916-917
  • DOI: 10.1002/mrd.22584
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Derivation of embryonic stem cell lines from parthenogenetically developing rat blastocysts. (2014)
  • Author(s): Hirabayashi M, Goto T, Tamura C, Sanbo M, Hara H, Kato-Itoh M, Sato H, Kobayashi T, Nakaushi H, Hochi S.
  • Journal Title: Stem Cells and Development Volume: VOL.23 Issue: 2 Pages: 107-114
  • DOI: 10.1089/scd.2013.0200
  • Peer Reviewed
• [Journal Article] Effect of Leukemia Inhibitory Factor and Forskolin on Establishment of Rat Embryonic Stem Cell Lines (2014)
  • Author(s): Hirabayashi M, Goto T, Tamura C, Sanbo M, Hara H, Hochi S.
  • Journal Title: Journal of Reproduction and Development Volume: 60 Issue: 1 Pages: 78-82
  • DOI: 10.1262/jrd.2013-109
  • NAID: 130003381862
  • ISSN: 0916-8818, 1348-4400
  • Peer Reviewed
• [Journal Article] Generation of Functional Oocytes by Xeno-Ectopic Transplantation of Primordial Germ Cells (2014)
  • Author(s): Hayama T, Yamaguchi T, Kato-Itoh M, Hamanaka S, Kawarai M, Sanbo M, Tamura C, Lee YS, Yanagida A, Murayama H, Mizuno N, Umino A, Sato H, Yamazaki S, Masaki H, Kobayashi T, Hirabayashi M, Nakauchi H
  • Journal Title: Biol Reprod Volume: 91(4) 89 Issue: 4 Pages: 1-9
  • DOI: 10.1095/biolreprod.114.121640
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] A retrospective analysis of germline competence in rat embryonic stem cell Lines (2013)
  • Author(s): Hirabayashi M
  • Journal Title: Transgenic Res. Volume: 22 Issue: 2 Pages: 411-416
  • DOI: 10.1007/s11248-012-9638-7
  • Peer Reviewed
• [Presentation] ドナー核注入後に時間をおいて除核する方法で作製したラットクローン胚盤胞 (2015)
  • Author(s): 原 弘真, 滝澤 明子, 後藤 哲平, 三宝 誠, 小林 俊寛, 中内 啓光, 保地 眞一, 平林 真澄.
  • Organizer: 第108回日本繁殖生物学会
  • Place of Presentation: 宮崎大学木花キャンパス （宮崎県宮崎市）
  • Year and Date: 2015-09-19
• [Presentation] 臓器再生に向けたCRISPR/Cas9法による腎臓欠損ラットの作出 (2015)
  • Author(s): 後藤 哲平, 原 弘真, 保地 眞一, 平林 真澄.
  • Organizer: 第108回日本繁殖生物学会
  • Place of Presentation: 宮崎大学木花キャンパス （宮崎県宮崎市）
  • Year and Date: 2015-09-17
• [Presentation] Production of cloned rat blastocysts with CAG/Venus-transgenic donor cells and Rosa26-H2B/tdTomato knock-in recipient oocytes. (2015)
  • Author(s): Hara H, Takizawa A, Goto A, Sanbo M, Kobayashi T, Nakauchi H, Hochi S, Hirabayashi M.
  • Organizer: The 48th Annual Meeting of the Society for the Study of Reproduction
  • Place of Presentation: Puerto Rico Convention Center (プエルトリコ,U.S.A.)
  • Year and Date: 2015-06-20
  • Int'l Joint Research
• [Presentation] CRISPR/Cas9法により作出したFoxN1遺伝子ノックアウトラットの解析 (2015)
  • Author(s): 後藤 哲平, 原 弘真, 保地 眞一, 平林 真澄.
  • Organizer: 第62回日本実験動物学会
  • Place of Presentation: 京都テルサ (京都府京都市)
  • Year and Date: 2015-05-29
• [Presentation] The role of kisspeptin for defeminization/masculinization of sexual (2014)
  • Author(s): Nakamura S, Uenoyama Y, Ikegami K, Tamura C, Sambo M, Hirabayashi M, Tsukamura H, Maeda K.
  • Organizer: The 3rd Meeting of World Congress on
  • Place of Presentation: Edinburgh International Conference Centre（スコットランド エジンバラ）
  • Year and Date: 2014-09-03
• [Presentation] ラット性行動の発現制御におけるキスペプチンの役割 (2014)
  • Author(s): 中村 翔, 上野山 賀久, 池上 花奈, 田村 千尋, 三宝 誠, 平林真 澄, 束村 博子, 前多 敬一郎.
  • Organizer: 第107回日本繁殖生物学会
  • Place of Presentation: 帯広畜産大学 (北海道帯広市)
  • Year and Date: 2014-08-13
• [Presentation] 染色体工学技術とケノム編集技術によるヒト化薬物代謝モテル動物の作製 (2014)
  • Author(s): 香月 康宏, 小林 カオル, 平林 真澄, 佐久間 哲史, 久世 治郎, 阿部 智志, 滝口 正人, 平松 敬, 本間 和久, 梶谷 尚世, 嵩原 昇子, 香月 加奈子, 千葉 寛, 山本 卓, 押村 光雄
  • Organizer: 第61回日本実験動物学会
  • Place of Presentation: 札幌コンベンションセンター(北海道札幌市)
  • Year and Date: 2014-05-16
• [Presentation] 単為発生胚に由来するラットES細胞株の樹立
  • Author(s): 平林 真澄, 田村 千尋, 後藤 哲平, 三宝 誠, 保地 眞一
  • Organizer: 第60回日本実験動物学会
  • Place of Presentation: つくば国際会議場（茨城県つくば市）
• [Presentation] ラットの内部細胞塊および胚性幹細胞におけるインプリント遺伝子のDNAメチル化解析
  • Author(s): 後藤 哲平, 田村 千尋, 三宝 誠, 保地 眞一, 平林 真澄
  • Organizer: 第60回日本実験動物学会
  • Place of Presentation: つくば国際会議場（茨城県つくば市）
• [Presentation] フォルスコリンの2i培地への添加はラットES細胞株の樹立に有効か？
  • Author(s): 平林 真澄, 田村 千尋, 後藤 哲平, 三宝 誠, 保地 眞一
  • Organizer: 第106回日本繁殖生物学会
  • Place of Presentation: 東京農工大学農学部府中キャンパス（東京都府中市）
• [Presentation] Successful derivation of rat embryonic stem cell lines from parthenogenetically developing blastocysts.
  • Author(s): Hirabayashi M, Tamura C, Sanbo M, Goto T, Kato-Itoh M, Sato H, Kobayashi T, Nakauchi H, Hochi S.
  • Organizer: The 64th AALAS national meeting
  • Place of Presentation: Baltimore Convention Center（アメリカ合衆国メリーランド州ボルティモア）
• [Presentation] DNA methylation status of imprinted genes in rat ICM cells, rat ES cells and mouse ES cells.
  • Author(s): Goto T, Tamura C, Sanbo M, Kato-Itoh M, Kobayashi T, Nakauchi H, Hochi S, Hirabayashi M.
  • Organizer: The 64th AALAS national meeting
  • Place of Presentation: Baltimore Convention Center（アメリカ合衆国メリーランド州ボルティモア）
• [Presentation] Effect of leukemia inhibitory factor and forskolin on establishment of rat embryonic stem cell lines.
  • Author(s): Hara H, Tagiri M, Hirabayashi M, Hochi S.
  • Organizer: The 40th IETS Annual Conference
  • Place of Presentation: John Ascuaga's Nugget Hotel（アメリカ合衆国ネバダ州リノ）
• [Remarks] 遺伝子改変動物作製室ホームページ
  • URL: http://www.nips.ac.jp/mamtg/
• [Remarks] 遺伝子改変動物作製室ホームページ
  • URL: http://www.nips.ac.jp/mamtg/