Biotechnology Research Institute, Mgenplus Co., Ltd., Seoul, Korea.
Department of Animal and Poultry Sciences, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, USA.
Transgenic Res. 2018 Jun;27(3):289-300. doi: 10.1007/s11248-018-0074-1. Epub 2018 Apr 24.
Diabetes mellitus is a chronic disease with accompanying severe complications. Various animal models, mostly rodents due to availability of genetically modified lines, have been used to investigate the pathophysiology of diabetes. Using pigs for diabetic research can be beneficial because of their similarity in size, pathogenesis pathway, physiology, and metabolism with human. However, the use of pigs for diabetes research has been hampered due to only few pig models presenting diabetes symptoms. In this study, we have successfully generated insulin-deficient pigs by generating the indels of the porcine INS gene in somatic cells using CRISPR/Cas9 system followed by somatic cell nuclear transfer. First, somatic cells carrying a modified INS gene were generated using CRISPR/Cas9 system and their genotypes were confirmed by T7E1 assay; targeting efficiency was 40.4% (21/52). After embryo transfer, three live and five stillborn piglets were born. As expected, INS knockout piglets presented high blood glucose levels and glucose was detected in the urine. The level of insulin and c-peptide in the blood serum of INS knockout piglets were constant after feeding and the expression of insulin in the pancreas was absent in those piglets. This study demonstrates effectiveness of CRISPR/Cas9 system in generating novel pig models. We expect that these insulin-deficient pigs can be used in diabetes research to test the efficacy and safety of new drugs and the recipient of islet transplantation to investigate optimal transplantation strategies.
糖尿病是一种伴有严重并发症的慢性疾病。各种动物模型，由于可获得遗传修饰系，主要是啮齿动物，已被用于研究糖尿病的病理生理学。使用猪进行糖尿病研究是有益的，因为它们在大小、发病机制途径、生理学和新陈代谢方面与人类相似。然而，由于只有少数猪模型出现糖尿病症状，因此使用猪进行糖尿病研究受到了阻碍。在这项研究中，我们通过使用 CRISPR/Cas9 系统在体细胞中生成猪 INS 基因的插入/缺失，然后进行体细胞核移植，成功地生成了胰岛素缺乏的猪。首先，使用 CRISPR/Cas9 系统生成携带修饰的 INS 基因的体细胞，并通过 T7E1 测定法确认其基因型；靶向效率为 40.4%（21/52）。胚胎移植后，有三只活产和五只死产小猪出生。正如预期的那样，INS 敲除小猪表现出高血糖水平，尿液中检测到葡萄糖。INS 敲除小猪的血清胰岛素和 C 肽水平在喂食后保持不变，并且这些小猪的胰腺中没有胰岛素表达。这项研究证明了 CRISPR/Cas9 系统在生成新型猪模型方面的有效性。我们预计这些胰岛素缺乏的猪可用于糖尿病研究，以测试新药的疗效和安全性，以及胰岛移植的受体，以研究最佳的移植策略。
