Lei Shaohua, Ryu Junghyun, Wen Ke, Twitchell Erica, Bui Tammy, Ramesh Ashwin, Weiss Mariah, Li Guohua, Samuel Helen, Clark-Deener Sherrie, Jiang Xi, Lee Kiho, Yuan Lijuan
Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA 24061, USA.
Department of Animal and Poultry Sciences, College of Agriculture and Life Sciences, Virginia Tech, Blacksburg, VA 24061, USA.
Sci Rep. 2016 Apr 27;6:25222. doi: 10.1038/srep25222.
Application of genetically engineered (GE) large animals carrying multi-allelic modifications has been hampered by low efficiency in production and extended gestation period compared to rodents. Here, we rapidly generated RAG2/IL2RG double knockout pigs using direct injection of CRISPR/Cas9 system into developing embryos. RAG2/IL2RG deficient pigs were immunodeficient, characterized by depletion of lymphocytes and either absence of or structurally abnormal immune organs. Pigs were maintained in gnotobiotic facility and evaluated for human norovirus (HuNoV) infection. HuNoV shedding lasted for 16 days in wild type pigs, compared to 27 days (until the end of trials) in RAG2/IL2RG deficient pigs. Additionally, higher HuNoV titers were detected in intestinal tissues and contents and in blood, indicating increased and prolonged HuNoV infection in RAG2/IL2RG deficient pigs and the importance of lymphocytes in HuNoV clearance. These results suggest that GE immunodeficient gnotobiotic pigs serve as a novel model for biomedical research and will facilitate HuNoV studies.
与啮齿动物相比，携带多等位基因修饰的基因工程（GE）大型动物的应用受到生产效率低下和妊娠期延长的阻碍。在这里，我们通过将CRISPR/Cas9系统直接注射到发育中的胚胎中，快速培育出RAG2/IL2RG双敲除猪。RAG2/IL2RG缺陷猪存在免疫缺陷，其特征是淋巴细胞耗竭以及免疫器官缺失或结构异常。猪饲养在无菌设施中，并评估其感染人诺如病毒（HuNoV）的情况。野生型猪的HuNoV排泄持续16天，而RAG2/IL2RG缺陷猪则持续27天（直至试验结束）。此外，在肠道组织、内容物和血液中检测到更高的HuNoV滴度，表明RAG2/IL2RG缺陷猪中HuNoV感染增加且持续时间延长，以及淋巴细胞在HuNoV清除中的重要性。这些结果表明，基因工程免疫缺陷无菌猪可作为生物医学研究的新型模型，并将促进HuNoV研究。
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