Creation of Knock-out rabbit models for the study of atherosclerosis

• Project/Area Number: 25670190
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Experimental pathology
• Research Institution: University of Yamanashi
• Principal Investigator: FAN Jianglin 山梨大学, 総合研究部, 教授 (60272192)
• Project Period (FY): 2013-04-01 – 2016-03-31
• Project Status: Completed (Fiscal Year 2015)
• Budget Amount: ¥3,640,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥840,000); Fiscal Year 2015: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000); Fiscal Year 2014: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2013: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
• Keywords: 遺伝子改変ウサギ / 動脈硬化 / ノックアウト

Outline of Final Research Achievements

In this project, we attempted to create knock-out and knock-in rabbit models using TALEN and CRISPR-Cas9 methods. We first generated apoE KO rabbits using CRISPR-Cas9 and obtained two founders. After several breeding, we were able to get both heterozygous and homozygous apoE KO rabbits. Compared with wild-type rabbits, chow-fed apoE KO rabbits exhibited high levels of plasma total cholesterol and triglycerides with no changes in HDL-C levels. When these rabbits were fed with a diet containing a 0.3% cholesterol for 2 weeks, apoE KO showed more prominent hyperlipidemia than wild-type rabbits with increased plasma remnant lipoproteins and apoB48 contents. These rabbits also showed more aortic atherosclerosis than wild-type rabbits. These apoE KO rabbits should become a useful model for the study of human type III hyperlipidemia. We also created apoAII knock-in rabbits and are in the process of breeding and characterization.

Research Products

• [Int'l Joint Research] ミシガン大学/心臓血管研究所(米国)
• [Int'l Joint Research] 西安交通大学/動脈硬化研究所(中国)
• [Journal Article] AMPK Suppresses Connexin43 Expression in the Bladder and Ameliorates Voiding Dysfunction in Cyclophosphamide-induced Mouse Cystitis (2016)
  • Author(s): Zhang X, Yao J, Gao K, Chi Y, Mitsui T, Ihara T, Sawada N, Kamiyama M, Fan J, Takeda M
  • Journal Title: Sci Rep. Volume: 6 Issue: 1 Pages: 19708-19708
  • DOI: 10.1038/srep19708
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Production of human apolipoprotein(a) transgenic NIBS miniature pigs by somatic cell nuclear transfer (2016)
  • Author(s): Shimatsu Y, Horii W, Nunoya T, Iwata A, Fans J, Ozawa M.
  • Journal Title: Experimental Animals Volume: 65 Issue: 1 Pages: 37-43
  • DOI: 10.1538/expanim.15-0057
  • NAID: 130005125853
  • ISSN: 0007-5124, 1341-1357, 1881-7122
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] ApoE knockout rabbits: A novel model for the study of human hyperlipidemia. (2016)
  • Author(s): Niimi M, Yang D, Kitajima S, Ning B, Wang C, Li S, Liu E, Zhang J, Eugene Chen Y, Fan J.
  • Journal Title: Atherosclerosis Volume: 245 Pages: 187-193
  • DOI: 10.1016/j.atherosclerosis.2015.12.002
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Angiotensin II Destabilizes Coronary Plaques in Watanabe Heritable Hyperlipidemic Rabbits. (2016)
  • Author(s): Li S, Wang YN, Niimi M, Ning B, Chen Y, Kang D, Wang Z, Yu Q, Waqar AB, Liu E, Zhang J, Shiomi M, Chen YE, Fan J.
  • Journal Title: Arterioscler Thromb Vasc Biol Volume: 36 Issue: 5 Pages: 810-6
  • DOI: 10.1161/atvbaha.115.306871
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] LaGomiCs-Lagomorph Genomics Consortium: An International Collaborative Effort for Sequencing the Genomes of an Entire Mammalian Order (2016)
  • Author(s): Fontanesi L, Di Palma F, Flicek P, Smith AT, Thulin CG, Alves PC. LaGomiCs-Lagomorph Genomics Consortium
  • Journal Title: J Hered Volume: 107 Issue: 4 Pages: 295-308
  • DOI: 10.1093/jhered/esw010
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Plasma High-Mannose and Complex/Hybrid N-Glycans Are Associated with Hypercholesterolemia in Humans and Rabbits. (2016)
  • Author(s): Bai L, Li Q, Li L, Lin Y, Zhao S, Wang W, Wang R, Li Y, Yuan J, Wang C, Wang Z, Fan J, Liu E.
  • Journal Title: PloS one Volume: 11 Issue: 3 Pages: e0146982-e0146982
  • DOI: 10.1371/journal.pone.0146982
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Combined use of probucol and cilostazol with atorvastatin attenuates atherosclerosis in moderately hypercholesterolemic rabbits. (2015)
  • Author(s): Wang YL, Bai L, Lin Y, Chen YL, Guan H, Zhu NH, Li YF, Gao SC, Sun LJ, Zhao SH, Fan JL, Liu EQ.
  • Journal Title: Lipids in health and disease Volume: 14 Issue: 1 Pages: 82-82
  • DOI: 10.1186/s12944-015-0083-5
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] High-fructose and high-fat diet-induced insulin resistance enhances atherosclerosis in Watanabe heritable hyperlipidemic rabbits (2015)
  • Author(s): Ning, B, Wang X, Yu Y, Waqar AB, Yu Q, Koike T, Shiomi M, Liu E, Wang Y, Fan L.
  • Journal Title: Nutrition & Metabolism Volume: 12 Issue: 1
  • DOI: 10.1186/s12986-015-0024-3
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Rabbit models for the study of human atherosclerosis: from pathophysiological mechanisms to translational medicine (2015)
  • Author(s): Fan, J., Kitajima, S., Watanabe, T., Xuc, J., Zhang, J., Enqi Liu, E., Chen, Y.E.
  • Journal Title: Pharmacology & Therapeutics Volume: 146 Pages: 104-119
  • DOI: 10.1016/j.pharmthera.2014.09.009
  • Peer Reviewed / Open Access
• [Journal Article] Bisphenol a exposure induces metabolic disorders and enhances atherosclerosis in hyperlipidemic rabbits. (2015)
  • Author(s): Fang, C. Ning B, Waqar AB, Niimi M, Li S, Satoh K, Shiomi M, Ye T, Dong S, Fan J:
  • Journal Title: Journal of Applied Toxicology Volume: 10 Issue: 9 Pages: 1058-1070
  • DOI: 10.1002/jat.3103
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Carbonate ion-enriched hot spring water promotes skin wound healing in nude rats. (2015)
  • Author(s): Liang, J. Kang D, Wang Y, Yu Y, Fan J, Takashi E:
  • Journal Title: Plos One Volume: 10 Issue: 10 Pages: e110977-e110977
  • DOI: 10.1371/journal.pone.0110977
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Probucol suppresses macrophage infiltration and mmp expression in atherosclerotic plaques of whhl rabbits. (2014)
  • Author(s): Li, Shen. Liang J, Niimi M, Bilal Waqar A, Kang D, Koike T, Wang Y, Shiomi M, Fan J:
  • Journal Title: J Atheroscler Thromb Volume: 21 Pages: 648-658
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Urotensin ii promotes atherosclerosis in cholesterol-fed rabbits. (2014)
  • Author(s): Li, Y. Zhao S, Wang Y, Chen Y, Lin Y, Zhu N, Zheng H, Wu M, Cheng D, Bai L, Fan J, Liu E:
  • Journal Title: Plos One Volume: 9 Issue: 4 Pages: e95089-e95089
  • DOI: 10.1371/journal.pone.0095089
  • Peer Reviewed / Open Access
• [Journal Article] Effects of Antisense Oligonucleotides against C-Reactive Protein on the Development of Atherosclerosis in WHHL Rabbits (2014)
  • Author(s): Qi Yu, Zhengcao Liu, Ahmed Bilal Waqar, Bo Ning, Xianghong Yang, Masashi Shiomi, Mark J. Graham, Rosanne M. Crooke, Enqi Liu, Sijun Dong, and Jianglin Fan
  • Journal Title: Mediators of Inflammation Volume: 2014 Pages: 1-8
  • DOI: 10.1155/2014/979132
  • Peer Reviewed / Open Access
• [Journal Article] Demonstration of an add-on effect of probucol and cilostazol on the statin-induced anti-atherogenic effects. (2014)
  • Author(s): Wang, Y. Bai L, Lin Y, Guan H, Zhu N, Chen Y, Li Y, Gao S, Zhao S, Fan J, Liu E:
  • Journal Title: Histol Histopathol Volume: 29 Pages: 1593-1600
  • Peer Reviewed / Open Access
• [Journal Article] Effective gene targeting in rabbits using RNA-guided Cas9 nucleases (2014)
  • Author(s): Yang D, Xu J, Zhu T, Fan J, Lai L, Zhang J, Chen YE.
  • Journal Title: J Mol Cell Biol Volume: none Issue: 1 Pages: 1-3
  • DOI: 10.1093/jmcb/mjt047
  • Peer Reviewed / Open Access
• [Journal Article] Animal Models of C-Reactive Protein, (2014)
  • Author(s): Michael Torzewski, Ahmed Bilal Waqar, and Jianglin Fan
  • Journal Title: Mediators of Inflammation Volume: 2014 Pages: 1-7
  • DOI: 10.1155/2014/683598
  • Peer Reviewed
• [Journal Article] Production of apolipoprotein C-III knockout rabbits using zinc finger nucleases (2013)
  • Author(s): Yang D, Zhang J, Xu J, Zhu T, Fan Y, Fan J, Chen YE.
  • Journal Title: J Vis Expt Volume: 81 Issue: 81
  • DOI: 10.3791/50957
  • Peer Reviewed
• [Presentation] Angiotensin II induces cornoary plaque rupture and myocardial infarction in hyperlipidemic rabbits (2015)
  • Author(s): Fan, L.
  • Organizer: The 4th Hsien Wu and Ray Wu symposium
  • Place of Presentation: （Peking, China）North Garden Hotel
  • Year and Date: 2015-08-10
  • Int'l Joint Research / Invited
• [Presentation] ApoE knockout rabbits: a novel model for the study of human hyperlipidemia (2015)
  • Author(s): Niimi, M, Ning B, Wang C, Li S, Zhang J, Chen, YE, Fan L.
  • Organizer: 第47回日本動脈硬化学会総会
  • Place of Presentation: 仙台国際センター（宮城県・仙台市）
  • Year and Date: 2015-07-16
• [Presentation] CETP deficiency in rabbits protects high fat high cholesterol diet induced atherosclerosis (2015)
  • Author(s): Zhang J, Xu J, Liang J, Yang D, Guo Y, Zhu T, Song J, Niimi M, Lai L, Fan L, Chen YE.
  • Organizer: ATVB 2015
  • Place of Presentation: （San Francisco, USA）Hilton San Francisco Union Square Hotel
  • Year and Date: 2015-05-03
  • Int'l Joint Research
• [Presentation] Normal calorific diet enriched fat and fructose leads to multiple metabolic disorders and enhances atherosclerosis in WHHL rabbits. (2014)
  • Author(s): Ning, B., Wang, X., Yu, Y., Waqar, AB., Shiomi, M., Fan, J.
  • Organizer: MSDA2014
  • Place of Presentation: Kyoto International Conference Center（京都府京都市）
  • Year and Date: 2014-09-13
• [Presentation] Recent progress in rabbit genome: LaGomiCs? (2014)
  • Author(s): Fan, J.
  • Organizer: 第二回中‐日ウサギ動物モデル研究会
  • Place of Presentation: 吉林大学和平校区（中国）
  • Year and Date: 2014-08-08
• [Presentation] Probucol inhibits the initiation of atherosclerosis in cholesterol-fed rabbits. (2014)
  • Author(s): Niimi, M.
  • Organizer: 第二回中‐日ウサギ動物モデル研究会
  • Place of Presentation: 吉林大学和平校区（中国）
  • Year and Date: 2014-08-08
• [Presentation] Revised protocol to analyze rabbit coronary atherosclerosis. (2014)
  • Author(s): Li, S., Waqar AB., Fan, J.
  • Organizer: 第3回ウサギバイオサイエンス研究会
  • Place of Presentation: 山梨大学甲府キャンパス（山梨県甲府市）
  • Year and Date: 2014-08-02
• [Presentation] Hypertension enhances atherosclerosis and destabilizes plaques. (2014)
  • Author(s): Waqar, AB., Ning, B., Liu, Z., Shiomi, M., Fan, J.
  • Organizer: 第46回日本動脈硬化学会総会・学術集会
  • Place of Presentation: 京王プラザホテル（東京都新宿区）
  • Year and Date: 2014-07-11
• [Presentation] Conditional knockout of macrophage MED1 increases atherosclerosis in low-density lipoprotein receptor-deficient mice. (2014)
  • Author(s): Bai, L., Li, Q., Liu, R., Lin, Y., Wang, Y., Chen, Y., Li, Y., Zhao, S., Wang, W., Jia, Y., Fan, J., Reddy, JK., Liu, E.
  • Organizer: 第46回日本動脈硬化学会総会・学術集会
  • Place of Presentation: 京王プラザホテ（東京都新宿区）
  • Year and Date: 2014-07-11
• [Presentation] Human apolipoprotein AII protects against atherosclerosis: an old guy exerts a novel function?
  • Author(s): 範 江林
  • Organizer: 第8回Aso International Meeting
  • Place of Presentation: ホテルグリーンピア南阿蘇（熊本県阿蘇郡）
  • Invited
• [Presentation] Rabbit Models for the study of human atherosclerosis.
  • Author(s): 範 江林
  • Organizer: 第45回日本動脈硬化学会総会・学術集会
  • Place of Presentation: 京王プラザホテル（東京都新宿区）
• [Book] The Genetics and Genomics of the Rabbit (2015)
  • Author(s): Fan, J.
  • Total Pages: 500
  • Publisher: CABI
• [Remarks] 山梨大学大学院総合研究部医学域基礎医学系分子病理学講座
  • URL: http://www.med.yamanashi.ac.jp/clinical_basic/pathol01/
• [Remarks] 山梨大学大学院医学工学総合研究部分子病理学講座
  • URL: http://www.med.yamanashi.ac.jp/clinical_basic/pathol01/
• [Remarks] 山梨大学大学院医学工学総合研究部分子病理学講座
  • URL: http://www.med.yamanashi.ac.jp/clinical_basic/pathol01/gyouseki.html
• [Patent(Industrial Property Rights)] 急性心臓死ウサギモデルおよびその作成方法 (2013)
  • Inventor(s): 範 江林、塩見雅志、小池智也
  • Industrial Property Rights Holder: 山梨大学
  • Industrial Property Rights Type: 特許
  • Industrial Property Number: 2013-101525
  • Filing Date: 2013-05-13