利用CRISPR/Cas9技术成功建立基因敲除克隆猪

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      中国科学院广州生物医药与健康研究院研究员赖良学博士的研究团队利用最新的CRISPR/Cas9基因敲除技术成功地培育出两种基因敲除克隆小型猪，即酪氨酸酶基因敲除猪和PARK2和PINK1双基因敲除猪，建立了人类白化病和帕金森综合征两种猪模型。该研究成果于10月2日在线发表在《细胞分子生命科学》（Cellular and Molecular Life Sciences）上。  

　　课题组杨化强博士、博士研究生周小青和信吉阁，针对猪的酪氨酸酶、PARK2和PINK1基因的外显子设计了Cas9 打靶质粒，将其分别转染版纳小型猪的胎儿成纤维细胞和巴马小型猪的胎儿成纤维细胞后，获得了纯合的酪氨酸酶基因敲除以及PARK2和PINK1双基因敲除的细胞系。细胞系用于体细胞核移植后获得15头酪氨酸酶基因敲除克隆猪，20头PARK2和PINK1双基因敲除克隆猪。酪氨酸酶基因敲除后，本来是黑色的版纳小型猪，变成了白猪，表现出典型的白化病特征；而缺失PARK2和PINK1双基因的敲除克隆猪可以作为帕金森症大动物模型，用于研究其发生机制和评估相关治疗药物的有效性和安全性。

　　赖良学研究团队长期从事猪等大动物的基因修饰研究，曾率先利用锌指核酸酶、TALEN等基因打靶技术成功制作多种基因修饰猪。本次研究又将新兴的CRISPR/Cas9技术与体细胞克隆相结合，不仅使猪基因打靶效率更高、更为精确，而且在一个世代内首次实现了大动物双基因的等位敲除。该技术路线的建立将大大加速多基因修饰猪制备，对农业而言，可促进猪的生产性能的改良；与此同时，在生物医药领域，也可加速大动物疾病模型的建立和异种器官移植研究进展。

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