Department of Animal and Avian Sciences, University of Maryland, College Park, MD, 20742, USA.
Animal Bioscience and Biotechnology Laboratory, USDA, ARS, Beltsville, MD, 20705, USA.
Sci Rep. 2018 Feb 26;8(1):3582. doi: 10.1038/s41598-018-22050-0.
The domestic pig is an attractive model for biomedical research because of similarities in anatomy and physiology to humans. However, key gaps remain in our understanding of the role of developmental genes in pig, limiting its full potential. In this publication, the role of NEUROGENIN 3 (NGN3), a transcription factor involved in endocrine pancreas development has been investigated by CRISPR/Cas9 gene ablation. Precomplexed Cas9 ribonucleoproteins targeting NGN3 were injected into in vivo derived porcine embryos, and transferred into surrogate females. On day 60 of pregnancy, nine fetuses were collected for genotypic and phenotypic analysis. One of the piglets was identified as an in-frame biallelic knockout (Δ2/Δ2), which showed a loss of putative NGN3-downstream target genes: NEUROD1 and PAX4, as well as insulin, glucagon, somatostatin and pancreatic polypeptide-Y. Fibroblasts from this fetus were used in somatic cell nuclear transfer to generate clonal animals to qualify the effect of mutation on embryonic lethality. Three live piglets were born, received colostrum and suckled normally, but experienced extreme weight loss over a 24 to 36-hour period requiring humane euthanasia. Expression of pancreatic endocrine hormones: insulin, glucagon, and somatostatin were lost. The data support a critical role of NGN3 in porcine endocrine pancreas development.
家猪作为生物医学研究的理想模型，具有与人类相似的解剖学和生理学特征。然而，在理解发育基因在猪中的作用方面，仍然存在一些关键的差距，限制了其充分发挥潜力。在本研究中，通过 CRISPR/Cas9 基因敲除技术，研究了参与内分泌胰腺发育的转录因子 NEUROGENIN 3（NGN3）的作用。针对 NGN3 的预复合物 Cas9 核糖核蛋白被注射到体内衍生的猪胚胎中，并转移到代孕雌性体内。在妊娠第 60 天，收集了 9 只胎儿进行基因型和表型分析。其中一只仔猪被鉴定为框内双等位基因敲除（Δ2/Δ2），其表现出假定的 NGN3 下游靶基因：NEUROD1 和 PAX4 以及胰岛素、胰高血糖素、生长抑素和胰多肽-Y 的缺失。从这个胎儿的成纤维细胞中进行体细胞核移植，以产生克隆动物，以确定突变对胚胎致死性的影响。有三只活仔猪出生，接受了初乳并正常哺乳，但在 24 至 36 小时内经历了极度体重减轻，需要人道安乐死。胰腺内分泌激素的表达：胰岛素、胰高血糖素和生长抑素均丢失。数据支持 NGN3 在猪内分泌胰腺发育中的关键作用。
