Abstract
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.
Highlights
Our fundamental understanding of pancreatic development and the role of key genes comes from studies in mice
NGN3 is reactivated in human cultured exocrine tissue prepared from clinical islet preparations[24]
Difficulties still remain when dealing with large animals, as highlighted by the lack of a reported sequence for NGN3 in the current pig genome build[41]
Summary
Our fundamental understanding of pancreatic development and the role of key genes comes from studies in mice. Several experimental models of in vivo differentiation have been performed to exploit regenerative signaling, as illustrated by the partial duct ligation model, partial pancreatectomy, and chemical-mediated tissue injury models[18,19,20,21,22] In these models, subpopulations of low level Ngn3/Sox9+ epithelial cells have been shown to give rise to an elevated level of Ngn[3] endocrine precursors[23,24]. Once a modification of interest is achieved, subsequent somatic cell nuclear transfer (SCNT) can be used to generate a cohort of animals harboring the desired targeted genome modifications Using this experimental pipeline, we set to investigate the role of NGN3 in a pig model. Our hypothesis is that loss of NGN3 will result in the ablation of endocrine but not exocrine pancreas
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