Targeted mutation of NGN3 gene disrupts pancreatic endocrine cell development in pigs

Authors: TELUGU, BHANU - University Of Maryland; SHEETS, TIMOTHY - University Of Maryland; PARK, KI-EUN - University Of Maryland; PARK, CHI-HUN - University Of Maryland; Swift, Steven; POWELL, ANNE - US Department Of Agriculture (USDA); DONOVAN, DAVID - Retired ARS Employee

Submitted to: Journal of Animal Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/15/2018
Publication Date: 2/26/2018
Citation: Telugu, B.P., Sheets, T.P., Park, K., Park, C., Swift, S.M., Powell, A., Donovan, D.M. 2018. Targeted mutation of NGN3 gene disrupts pancreatic endocrine cell development in pigs. Journal of Animal Science. 8(1):3582. https://doi.org/10.1038/s41598-018-22050-0.
DOI: https://doi.org/10.1038/s41598-018-22050-0

Interpretive Summary: The domestic pig is an attractive model for biomedical research because of the 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 as a biomedical model. In this publication, the role of Neurogenin 3 (NGN3), a factor involved in regulating endocrine pancreas development has been investigated using the techniques of gene editing. Specialized proteins targeting NGN3 were injected into pig embryos, and transferred into adult female pigs. On day 60 of pregnancy, nine fetuses were collected for analysis. One of the piglets was identified as being successfully gene edited so that it possessed an interruption in the NGN3 gene which resulted in a loss in expression of genes regulated by NGN3: NeuroD1 and Pax4, as well as insulin, glucagon, somatostatin and pancreatic polypeptide-Y. Cells from this fetus were used to generate clonal animals to examine the effect of mutation on embryonic mortality. 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 hormones by the pancreas were lost, including insulin, glucagon, and somatostatin which are all essential to normal glucose metabolism by the body; thus, demonstrating a critical role for NGN3 in porcine endocrine pancreas development.

Technical Abstract: The domestic pig is an attractive model for biomedical research because of the 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 (delta2/delta2), 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.