Hpd基因修饰制备高酪氨酸血症Ⅲ型巴马小型猪模型  

Generation of a Bama minipig model of hereditary tyrosinemia type Ⅲ by modifying the Hpd gene

作　　者：顾鹏 陈傍柱 徐涛 刘闻[1] 邓俊成 徐名衬[1] 袁进[1] 陈恩生 顾为望[1,2,4] GU Peng;CHEN Bangzhu;XU Tao;LIU Wen;DENG Juncheng;XU Mingchen;YUAN Jin;CHEN Ensheng;GU Weiwang(Institute of Comparative Medicine & Laboratory Animal Center, Southern Medical University, Guangzhou 510515, China;Songshan Lake Pearl Laboratory Animal Science and Technology Ltd., Dongguan 523808;Integrated Traditional Chinese and Western Medicine Hospital, Southern Medical University, Guangzhou 510315;Wuyi University,Jiangmen 529020)

机构地区：[1]南方医科大学比较医学研究所暨实验动物中心,广州510515 [2]东莞松山湖明珠实验动物科技有限公司,广东东莞523808 [3]南方医科大学中西医结合医院,广州510315 [4]五邑大学,广东江门529020

出　　处：《中国比较医学杂志》2019年第5期11-16,共6页Chinese Journal of Comparative Medicine

基　　金：广东省科技计划项目(2013B060300013;2010A011200003;2016A020208004);国家自然科学基金项目(31271042);国家科技部国际合作项目(2011DFA33290);江门市创新团队项目(2017TD023290);广州市科技计划项目(201704020012);南方医科大学科研启动计划项目(PY2017N032)

摘　　要：目的利用CRISPR/Cas9技术敲除巴马小型猪4-羟基苯丙酮酸氧化酶(4-OH phenylpyruvate dioxygenase,4HPPD/HPD)制备高酪氨酸血症Ⅲ型巴马小型猪模型。方法分别从质粒pGL3-U6-gRNA-PGK-puromycin和pST1374-NLS-flag-linker-Cas9中通过PCR扩增sgRNA-Hpd和Cas9体外转录用模板,接着体外转录出Cas9 mRNA和sgRNA-Hpd,最后将Cas9 mRNA和sgRNA-Hpd共注射入巴马小型猪受精卵的胞质内,以制备Hpd基因敲除巴马小型猪。结果胞质内共注射后,共移植20枚受精卵至2只代孕母猪。获得子代Hpd基因敲除巴马小型猪共4只。结论本研究成功制备了高酪氨酸血症Ⅲ型巴马小型猪模型,将为研究酪氨酸代谢通路提供更好的模型。Objective To prepare a Bama minipig model of hereditary tyrosinemia type Ⅲ, the hydroxyphenylpyruvate dioxygenase (Hpd) gene was chosen to be edited by the CRISPR/Cas9 technology. Methods Templates (used as in vitro transcripts for Cas9 mRNA and sgRNA) were amplified from pST1374-NLS-flag-linker-Cas9 and pGL3-U6-gRNA-PGK-puromycin by PCR, respectively, and subsequently transcribed in vitro into Cas9 mRNA and sgRNA-Hpd. Finally, cas9 mRNA and sgRNA-Hpd were co-injected into the cytoplasm of single cell embryos to generate an Hpd-modified Bama minipig. Results Twenty embryos of Bama minipigs co-injected with cas9 mRNA and sgRNA-Hpd were transplanted into two pseudopregnant mothers. We obtained four offsprings with a modified Hpd gene. Conclusions In this study, we have successfully generated Hpd knockout Bama minipigs with hereditary tyrosinemia type Ⅲ using the CRISPR/ Cas9 technique, which will be a valuable model for research of the tyrosine metabolic pathway.

关 键 词：4-羟基苯丙酮酸氧化酶 CRISPR/ Cas9 高酪氨酸血症Ⅲ型 巴马小型猪 

分 类 号：R-33[医药卫生]