Laboratory of Animal Cloning, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China;
Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany;
J Immunol. 2014 Aug 1;193(3):1496-503. doi: 10.4049/jimmunol.1400915. Epub 2014 Jun 27.
Pigs share many physiological, biochemical, and anatomical similarities with humans and have emerged as valuable large animal models for biomedical research. Considering the advantages in immune system resemblance, suitable size, and longevity for clinical practical and monitoring purpose, SCID pigs bearing dysfunctional RAG could serve as important experimental tools for regenerative medicine, allograft and xenograft transplantation, and reconstitution experiments related to the immune system. In this study, we report the generation and phenotypic characterization of RAG1 and RAG2 knockout pigs using transcription activator-like effector nucleases. Porcine fetal fibroblasts were genetically engineered using transcription activator-like effector nucleases and then used to provide donor nuclei for somatic cell nuclear transfer. We obtained 27 live cloned piglets; among these piglets, 9 were targeted with biallelic mutations in RAG1, 3 were targeted with biallelic mutations in RAG2, and 10 were targeted with a monoallelic mutation in RAG2. Piglets with biallelic mutations in either RAG1 or RAG2 exhibited hypoplasia of immune organs, failed to perform V(D)J rearrangement, and lost mature B and T cells. These immunodeficient RAG1/2 knockout pigs are promising tools for biomedical and translational research.
猪在生理、生化和解剖学上与人类有许多相似之处，已成为生物医学研究中有价值的大型动物模型。考虑到免疫系统相似性、合适的体型和用于临床实际和监测目的的寿命方面的优势，携带功能失调 RAG 的 SCID 猪可以作为再生医学、同种异体和异种移植以及与免疫系统相关的重建实验的重要实验工具。在本研究中，我们使用转录激活因子样效应核酶报告了 RAG1 和 RAG2 敲除猪的产生和表型特征。使用转录激活因子样效应核酶对猪胎儿成纤维细胞进行基因工程改造，然后将其用于提供体细胞核移植的供体核。我们获得了 27 头活克隆仔猪；在这些仔猪中，9 头仔猪的 RAG1 发生了双等位基因突变，3 头仔猪的 RAG2 发生了双等位基因突变，10 头仔猪的 RAG2 发生了单等位基因突变。RAG1 或 RAG2 发生双等位基因突变的仔猪表现出免疫器官发育不良，无法进行 V(D)J 重排，并且失去成熟的 B 和 T 细胞。这些免疫缺陷型 RAG1/2 敲除猪是生物医学和转化研究的有前途的工具。
