Title: Generation of tryptophan hydroxylase 2 gene knockout pigs by CRISPR/Cas9-mediated gene targeting
Authors: Ze Li1, , Hai-Yuan Yang1, , Ying Wang1, Man-Ling Zhang1, Xiao-Rui Liu1, Qiang Xiong 1, Li-Ning Zhang1, Yong Jin1, Li-Sha Mou3, Yan Liu2, Rong-Feng Li1, Yi Rao2, Yi-Fan Dai1
Institutions: 1Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing, Jiangsu 211166, China; 2Peking-Tsinghua Center for Life Sciences, PKU-IDG/McGovern Institute for Brain Research, the School of Life Sciences, Peking University, Beijing 100871, China; 3Shenzhen 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.
Abstract: 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 the Tph2 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 10 Tph2 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. These Tph2 KO pigs are valuable large-animal models for studies of 5-HT deficiency induced behavior abnomality.
Keywords: CRISPR/Cas9, tryptophan hydroxylase-2 gene, serotonin, Bama mini pigs
Full Text: JBR-2017-0026.pdfJBR-2017-0026-supplementary.pdf
J Biomed Res published on 18 May, 2017, doi:10.7555/JBR.31.20170026