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2025年8月8日 星期五
农业生物技术学报  2019, Vol. 27 Issue (10): 1858-1868    DOI: 10.3969/j.issn.1674-7968.2019.10.016
  研究资源与技术改进 本期目录 | 过刊浏览 | 高级检索 |
利用TALEN技术构建猪内源性基因Tiki1打靶猪模型
吴彩霞1,2, 刘朝明1,2, 颜泉梅2,*, 欧阳振2, 赵宇2, 张全军2, 樊娜娜2, 赖良学2,*
1 吉林大学 动物医学学院,长春 130062;
2 中国科学院 广州生物医药与健康研究院, 广州 510530
Construction of Porcine Endogenous Gene Tiki1 Targeted Pig (Sus scrofa) Model Using TALEN Technology
WU Cai-Xia1,2, LIU Zhao-Ming1,2, YAN Quan-Mei2,*, OUYANG Zhen2, ZHAO Yu2, ZHANG Quan-Jun2, FAN Na-Na2, LAI Liang-Xue2,*
1 College of Veterinary Medicine, Jilin University, Changchun 130062;
2 Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530
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摘要 Tiki1基因(TraB domain containing protein 2A)在蛙(Xenopus laevis)头部诱导发生过程中起着决定性的作用,但Tiki1在哺乳动物乃至人(Homo sapiens)的早期发育中的功能尚不清楚,与人类在生理学、病理学上都极为相近的猪(Sus scrofa)成为研究Tiki1功能的理想哺乳动物模型。本研究利用转录激活因子样效应物核酸酶(transcription activator-like effector nuclease, TALEN)基因编辑技术针对猪内源性基因Tiki1外显子4设计了1对TALEN质粒,并在猪孤雌激活胚胎水平上进行了TALEN质粒的体外活性检测,比较了设计的TALEN质粒以不同浓度注射时打靶效率及对胚胎发育的影响,并进行了细胞的转染和筛选,结合体细胞核移植技术构建了Tiki1基因打靶猪模型。本研究共计构建了1 922枚基因编辑克隆胚胎并植入8头代孕母猪子宫内,其中3头怀孕到期并顺利分娩,共计获得12头活猪和1头死胎,经酶切鉴定和测序鉴定结果表明其中有4头属于预期的Tiki1基因打靶阳性猪模型。将Tiki1基因打靶阳性猪模型和WT配种,经过足月后顺产分娩获得5头F1代成活仔猪,进一步测序鉴定基因敲除情况,结果表明构建的Tiki1基因打靶阳性猪模型是可以生殖系遗传的,不是嵌合体。本研究首次采用TALEN技术对猪基因组内源性基因Tiki1进行定点修饰,成功且高效率地构建了猪内源性基因Tiki1敲除的大动物模型,为实现大动物高效的基因编辑提供了有力的工具,也为将来建立各种生物医药模型和农业基因改良猪提供了技术基础。
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作者相关文章
吴彩霞
刘朝明
颜泉梅
欧阳振
赵宇
张全军
樊娜娜
赖良学
关键词 转录激活因子样效应物核酸酶(TALEN)基因编辑技术Tiki1基因打靶猪模型    
Abstract:Functional research experiments on frogs (Xenopus laevis) have confirmed that TraB domain containing 2A (Tiki1) plays a decisive role in the process of head induction, but the function of Tiki1 in the early development of mammals and even humans (Homo sapiens) is not yet clear. Pigs (Sus scrofa) that are very similar to humans in physiology and pathology have become ideal mammalian models for studying Tiki1 functions. In this study, a pair of transcription activator-like effector nuclease (TALEN) plasmids tageting exon 4 of the pig endogenous gene Tiki1 were designed by TALEN technology. TALEN plasmids were validated at the level of porcine parthenogenetic embryos. The targeting efficiency and the affect on embryo development of TALEN plasmids injected at different concentrations were compared. Then plasmids transfection and screening of cells were carried out. In this study, a total of 8 sows were transplanted. 3 sows were pregnant and delivered smoothly, and a total of 12 live pigs and 1 stillborn fetus were obtained. The results showed that 4 of them belonged to the Tiki1 gene targeting positive pig model that expected by enzyme digestion and sequencing. 3 live cloned piglets were obtained from the first sow, and one of the 3 pigs (33.3%) was identified by enzyme digestion and sequencing as the expected Tiki1 gene targeting positive pig model. 6 live pigs and 1 stillbirth were obtained from the second sow. 2 of the 7 piglets (28.6%) were identified by the enzyme digestion and sequencing as the expected Tiki1 gene targeting positive pig model. 3 live cloned piglets were obtained from the third sow. One of the 3 piglets (33.3%) was identified by enzyme digestion and sequencing as the expected Tiki1 gene targeting positive pig model. The Tiki1 gene targeting positive pig model and WT mating were used to obtain 5 live F1 generation piglets. The sequencing analysis showed that the constructed Tiki1 gene targeting positive pig model was inherited by the germline, not chimera. In this experiment, TALEN technology was used to modify the endogenous gene Tiki1 of pig genome for the first time. It provides a powerful tool for efficient gene editing in large animals and a technical basis for the establishment of various biomedical models and agricultural genetically modified pigs in the future.
Key wordsTranscription activator-like effector nuclease (TALEN) gene editing technology    Tiki1    Gene targeting    Swine model
收稿日期: 2019-03-22     
ZTFLH:  S828.9+9  
基金资助:国家重点研发计划(No. 2017YFA0105100)和国家973计划(No. 2011CB944200)
通讯作者: lai_liangxue@gibh.ac.cn;yan_quanmei@gibh.ac.cn   
引用本文:   
吴彩霞, 刘朝明, 颜泉梅, 欧阳振, 赵宇, 张全军, 樊娜娜, 赖良学. 利用TALEN技术构建猪内源性基因Tiki1打靶猪模型[J]. 农业生物技术学报, 2019, 27(10): 1858-1868.
WU Cai-Xia, LIU Zhao-Ming, YAN Quan-Mei, OUYANG Zhen, ZHAO Yu, ZHANG Quan-Jun, FAN Na-Na, LAI Liang-Xue. Construction of Porcine Endogenous Gene Tiki1 Targeted Pig (Sus scrofa) Model Using TALEN Technology. 农业生物技术学报, 2019, 27(10): 1858-1868.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2019.10.016     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2019/V27/I10/1858
 
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