Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing, 211166, P.R. 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.
Sci Rep. 2017 Jul 10;7(1):5009. doi: 10.1038/s41598-017-05400-2.
Complement protein C3 is the pivotal component of the complement system. Previous studies have demonstrated that C3 has implications in various human diseases and exerts profound functions under certain conditions. However, the delineation of pathological and physiological roles of C3 has been hampered by the insufficiency of suitable animal models. In the present study, we applied the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system to target the C3 gene in porcine fetal fibroblasts. Our results indicated that CRISPR/Cas9 targeting efficiency was as high as 84.7%, and the biallelic mutation efficiency reached at 45.7%. The biallelic modified colonies were used as donor for somatic cell nuclear transfer (SCNT) technology to generate C3 targeted piglets. A total of 19 C3 knockout (KO) piglets were produced and their plasma C3 protein was undetectable by western blot analysis and ELISA. The hemolytic complement activity and complement-dependent cytotoxicity assay further confirmed that C3 was disrupted in these piglets. These C3 KO pigs could be utilized as a valuable large animal model for the elucidation of the roles of C3.
补体蛋白 C3 是补体系统的关键组成部分。先前的研究表明，C3 与多种人类疾病有关，并在某些条件下发挥着深远的作用。然而，由于缺乏合适的动物模型，C3 的病理和生理作用的描绘一直受到阻碍。在本研究中，我们应用成簇规律间隔短回文重复（CRISPR）/CRISPR 相关（Cas）系统靶向猪胎儿成纤维细胞中的 C3 基因。我们的结果表明，CRISPR/Cas9 的靶向效率高达 84.7%，并且双等位基因突变效率达到 45.7%。将双等位基因修饰的克隆作为供体用于体细胞核移植（SCNT）技术，以产生 C3 靶向的小猪。共产生了 19 头 C3 敲除（KO）小猪，其血浆 C3 蛋白通过 Western blot 分析和 ELISA 检测不到。溶血补体活性和补体依赖性细胞毒性测定进一步证实了这些小猪中 C3 的缺失。这些 C3 KO 猪可用作阐明 C3 作用的有价值的大动物模型。
