利用CRISPR／Cas9系统建立Xist基因敲除猪模型  

Establishment of porcine Xist knockout model using CRISPR/Cas9 system

作　　者：李国玲[1] 钟翠丽[1] 倪生[1] 刘德武[1] 蔡更元[1] 李紫聪[1] 杨化强[1] 吴珍芳[1] 

机构地区：[1]华南农业大学动物科学学院,国家生猪种业工程技术研究中心,广州510642

出　　处：《遗传》2016年第12期1081-1089,共9页Hereditas（Beijing)

基　　金：国家转基因重大专项(编号:2016ZX08006002);广东省科学技术部(编号:2016B0202330064,2015TX01N081)资助~~

摘　　要：体细胞核移植技术在家畜良种繁育、基因修饰动物生产、濒危动物的拯救和人类疾病的治疗等领域具有重要的应用价值,但目前克隆动物生产效率较低,平均不超过5%。低下的克隆效率极大地限制了该技术的快速发展。在影响克隆猪生产效率的诸多因素中,X染色体的异常失活是导致克隆效率低下的重要原因,而与X染色体失活密切相关的一个重要基因是Xist基因,这表明Xist基因可能直接或间接地影响猪的克隆效率。本文以CRISPR/Cas系统为基础,在Xist基因上设计5个CRISPR/Cas系统打靶位点,并筛选出有效的Target 3、Target4 sg RNA,在细胞水平切割效率为1%和3%,在胚胎水平为75%和85.7%。同时将有效的sg RNA体外转录并显微注射至胚胎体内,发现Target 3和Target 4组合效果最好,敲除效率为100%。通过胞浆注射和胚胎移植方法生产出6头克隆猪,有2头活仔实现完全敲除。本文成功建立Xist基因敲除猪模型,为后续通过敲除猪Xist基因提高克隆效率的研究奠定了基础。Somatic cell nuclear transfer technique has great applications in livestock breeding, production of ge- netically modified animals, rescue of endangered species and treatment of human diseases. However, the currently low efficiency in animals cloning, an average of less than 5%, greatly hindered the rapid development of this tech- nique. Among many factors which affect the efficiency of cloning pigs, X chromosome inactivation is an important one. Moreover, Xist gene is closely related to X chromosome inactivation, suggesting that Jt may directly or indirectly affects cloning efficiency. In this study, multiple sgRNAs were designed based on the CRISPR/Cas system, and twosites （Target 3 and Target 4） whose mutation efficiency were 1% and 3% at the cellular level were selected. We suc- cessfully knocked out Xist with 100% efficiency by microinjecting sgRNAs for Target 3 and Target 4 in embryo. Fi- nally, 6 cloning piglets were born including two Xist-fully-knockout piglets. The follow-up studies on increasing cloning efficiency can be carried out based on the Xist-knockout model.

关 键 词：XIST CRISPR/Cas 克隆效率 猪 

分 类 号：Q78[生物学—分子生物学]