神经退行性疾病转基因猴模型的构建及相关技术体系建立  

Construction of a transgenic monkey model of neurodegenerative diseases and establishment of its related technical system

作　　者：吴彩霞 刘朝明 张全军 颜泉梅 赵宇 张秀娟[3] 刘晓明[3] 欧阳振 樊娜娜 赖良学 WU Caixia;LIU Zhaoming;ZHANG Quanjun;YAN Quanmei;ZHAO Yu;ZHANG Xiujuan;LIU Xiaoming;OUYANG Zhen;FAN Nana;LAI Liangxue(Guangzhou Institutes of Biomedicine and Health,Chinese Academy of Sciences,Guangzhou 510530,China;College of Veterinary Medicine of Jilin-University,Changchun 130062,China;Guangdong Institute of Entomology,Guangzhou 510530,China)

机构地区：[1]中国科学院广州生物医药与健康研究院,广东广州510530 [2]吉林大学动物医学学院,吉林长春130062 [3]广东省昆虫研究所,广州510530

出　　处：《畜牧与兽医》2020年第5期123-127,共5页Animal Husbandry & Veterinary Medicine

基　　金：国家重点研发计划(2017YFA0105100);国家973计划(2011CB944200);广东省科技计划项目(2019A030317010)。

摘　　要：非人灵长类动物因与人有最近亲缘关系而成为研究人类生命领域最高级模式动物。本研究采用慢病毒载体技术结合胚胎工程技术,拟建立一套高效简便转基因猴制作技术体系,建立小脑共济失调转基因猴模型,为该病研究提供最为理想动物模型,同时为建立其他疾病转基因猴模型提供技术路线。利用卵泡浆内单精子显微注射技术(Introeytoplasmiesperm injection,ICSI)技术总共构建88个体外受精胚胎,经过体外培养共计32枚发育到原核期并进行病毒注射后移入7只受体猴输卵管内。B超妊娠检测,怀孕3只,其中:1只未能发育到期中间流产,1只发育到期生1死胎,1只顺产生1只活试管猴,经过人工喂养奶粉后健康存活,但经检测转基因为阴性。本研究虽然未能通过病毒注射获得阳性转基因猴,但对食蟹猴(Macaca fascicularis)超数排卵及B超监测卵泡发育技术、卵母细胞回收及体外成熟培养技术、电刺激采精及精子获能技术、食蟹猴体外受精技术和胚胎体外培养技术以及胚胎移植等技术体系进行多次探索和优化,并成功建立相关技术平台,将为后续利用试管猴技术结合高效的TALEN和CRISPR/Cas9等基因编辑工具获得各种疾病模型奠定基础。Non-human primates are the most advanced model animals in the research field of human life because they are closely related to human beings.In this study,the lentivirus vector technology was combined with the embryo engineering technology to establish a transgenic monkey model of cerebellar ataxia,providing the most ideal animal model for the study of this disease,and a technical route for the establishment of transgenic monkey models of other diseases.A total of 88 in vitro fertilized embryos were constructed using the ICSI(Introeytoplasm sperm injection)technology.After in vitro culture,32 embryos developed into the prokaryotic stage,were injected with the virus,and were transferred into the oviducts of 7 recipient monkeys.B-ultrasound pregnancy test showed three monkeys were pregnant,one of which miscarried before developing into maturity,another developed to maturity but gave birth to a stillborn fetus,and the third one gave birth to a live test-tube monkey which survived by artificial feeding of milk powder,but the genetically modified test showed the baby monkey to be negative.This study failed to obtain positive transgenic monkeys by viral injection,but the techniques of superovulation and B ultrasound monitoring follicular development,oocyte recovery and in vitro maturation,electric stimulation sperm collection and sperm capacitation,in vitro fertilization,embryo culture and embryo transfer of cynomolgus monkeys were explored and optimized for many times,and the related technology platforms were successfully established;all of which laid the foundation for the subsequent use of test tube monkey technology combined with efficient genetic editing tools such as the TALEN and CRISPR/Cas9 systems to obtain various disease models.

关 键 词：卵胞浆内单精子显微注射 体外受精 猴 疾病模型 

分 类 号：S829.9[农业科学—畜牧学]