Li Ze, Yang Hai-Yuan, Wang Ying, Zhang Man-Ling, Liu Xiao-Rui, Xiong Qiang, Zhang Li-Ning, Jin Yong, Mou Li-Sha, Liu Yan, Li Rong-Feng, Rao Yi, Dai Yi-Fan
Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing, Jiangsu 211166, China.
Shenzhen Xenotransplantation Medical Engineering Research and Development Center, Institute of Translational Medicine, Shenzhen Second People's Hospital, First Affiliated Hospital of Shenzhen University, Shenzhen, Guangdong 518035, China.
J Biomed Res. 2017 Sep 26;31(5):445-452. doi: 10.7555/JBR.31.20170026.
Unbalanced brain serotonin (5-HT) levels have implications in various behavioral abnormalities and neuropsychiatric disorders. The biosynthesis of neuronal 5-HT is regulated by the rate-limiting enzyme, tryptophan hydroxylase-2 (TPH2). In the present study, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system was used to target theTph2 gene in Bama mini pig fetal fibroblasts. It was found that CRISPR/Cas9 targeting efficiency could be as high as 61.5%, and the biallelic mutation efficiency reached at 38.5%. The biallelic modified colonies were used as donors for somatic cell nuclear transfer (SCNT) and 10Tph2 targeted piglets were successfully generated. These Tph2 KO piglets were viable and appeared normal at the birth. However, their central 5-HT levels were dramatically reduced, and their survival and growth rates were impaired before weaning. TheseTph2 KO pigs are valuable large-animal models for studies of 5-HT deficiency induced behavior abnomality.
大脑血清素（5-羟色胺，5-HT）水平失衡与多种行为异常和神经精神疾病有关。神经元5-HT的生物合成受限速酶色氨酸羟化酶-2（TPH2）调控。在本研究中，利用成簇规律间隔短回文重复序列（CRISPR）/CRISPR相关蛋白（Cas）系统靶向巴马小型猪胎儿成纤维细胞中的Tph2基因。结果发现，CRISPR/Cas9靶向效率高达61.5%，双等位基因突变效率达38.5%。将双等位基因修饰的细胞集落用作体细胞核移植（SCNT）的供体，成功获得了10头Tph2基因靶向仔猪。这些Tph2基因敲除仔猪出生时存活且外观正常。然而，它们中枢神经系统的5-HT水平显著降低，断奶前的存活和生长速率受损。这些Tph2基因敲除猪是研究5-HT缺乏诱导行为异常的有价值的大型动物模型。
