摘要
目的 为了获得白化的C57BL/6N小鼠,扩大其在皮肤移植和胚胎干细胞方面研究中的应用。方法 通过体外设计合成Cas9 mRNA和系列sgRNA(single guide RNAs),注射到小鼠的受精卵内,破坏合成C57BL/6N小鼠黑色素生成必须的酪氨酸酶(tyrosinase,TYR)基因序列的第1和第2外显子,产生基因突变,获得F0代白化的C57BL/6N小鼠,然后经过重复回交和自交,形成白化C57BL/6N小鼠近交系。结果 经过注射两对sgRNA,成功的获得了F0代的白化小鼠,在Tyr基因的第1和第2外显子中均发生了缺失突变,小鼠可以将突变基因传递给后代,并在其后代中产生了纯合的白色C57BL/6N小鼠,最后对白化小鼠突变类型进行了分析。结论 通过CRISPR-Cas9技术破坏了小鼠Tyr基因,成功地建立了白化C57BL/6N小鼠近交系,为将来的嵌合体制作、组织移植提供了新的研究工具。
Objective To obtain albino C57BL/6N mice and expand their application in research of skin transplantation and embryonic stem cells.Methods Cas9 mRNA and a series of single guide RNAs(sgRNAs)were synthesized in vitro and injected into the fertilized eggs of C57BL/6N mice.The gene encoding tyrosinase(TYR),an enzyme necessary for melanin production in C57BL/6N mice,was destroyed in exon 1 and exon 2,and gene mutations were generated to obtain albino F0 generation.Albino C57BL/6N mice were then subjected to repeated backcrossing and inbreeding to produce C57BL/6N albino mouse inbreds.Results Two pairs of sgRNA were injected into mice,and the F0 generation of albino mice was successfully obtained.Deletions in both exon 1 and exon 2 of the Tyr gene were confirmed.The albino mice transmited the mutant gene to offsprings,and homozygous white C57BL/6N mice as offspring were confirmed.The mutation types of albino mice were analyzed.Conclusions The mouse Tyr gene is disrupted by CRISPRCas9 technology,and the C57BL/6N albino mouse inbred line is successfully established.This mouse line provides a new research tool for future chimera preparation and tissue transplantation.
作者
常秋荣
刘丽丽
王会阳
付丽
邢凤英
李垚
陈学进
李善刚
CHANG Qiurong;LIU Lili;WANG Huiyang;FU Li;XING Fengying;LI Yao;CHEN Xuejin;LI Shangang(Department of Laboratory Animal Science,School of Medicine,Shanghai Jiaotong University,Shanghai 200025,China;Institute of Primate Translational Medicine,Kunming University of Science and Technology,Kunming 650500)
出处
《中国实验动物学报》
CAS
CSCD
北大核心
2019年第4期493-500,共8页
Acta Laboratorium Animalis Scientia Sinica
基金
国家重点研发计划(2016YFC1304805)
上海市科研计划项目(15140901700)~~
