Generation of viable PDX1 gene‐edited founder pigs as providers of nonmosaics

Abstract

Pancreatic duodenal homeobox 1 (PDX1) is a crucial gene for pancreas development during the fetal period. PDX1-modified pigs have the potential to be used as a model of diabetes mellitus. However, the severe health problems caused by the PDX1 mutation limit phenotypic studies of PDX1-modified pigs as diabetes models. In this study, we generated PDX1-modified pigs by the CRISPR/Cas9 system introduced into zygotes via electroporation and investigated the mosaicism, phenotypes, and inheritance of the resulting pigs. After the embryo transfer of PDX1-modified zygotes, nine mutant piglets were delivered. Two piglets were apancreatic biallelic mutants. For the other seven piglets, the ratio of mutant alleles to total alleles was 17.5-79.7%. Two mutant piglets with high mutation rates (67.7% and 79.7%) exhibited hypoplasia of the pancreas, whereas the other five piglets were healthy.One of the male mutant piglets was further analyzed. The ejaculated semen from the pig contained PDX1-mutant spermatozoa and the pig showed normal reproductive ability. In conclusion, the frequency of the PDX1 mutation is presumed to relate to pancreas formation, and PDX1 mutant founder pigs generated from zygotes introduced to the CRISPR/Cas9 system can serve as providers of nonmosaics to contribute to medical research on diabetes mellitus. K E Y W O R D SCRISPR/Cas9, diabetes mellitus, electroporation, IVF, PDX1-mutant pigs SUPPORTING INFORMATIONAdditional supporting information may be found online in the Supporting Information section.

Efficient generation of GGTA1-deficient pigs by electroporation of the CRISPR/Cas9 system into in vitro-fertilized zygotes

“…We observed that the piglet harbored mutations, as observed in the ear biopsy analysis, in its major organs at various frequencies. Our previous study demonstrated mosaicism of major organs with frequencies similar to those observed in the results of the ear biopsy analysis [35]. The frequency of genotype varies with organ in genetically modified F0 pigs, indicating that a careful observation of WT sequence/in-frame mutation in the genomic DNA is an essential step to apply these organs for transplantation.…”

Current status of the application of gene editing in pigs

“…In mice, electroporation is widely used to introduce gene editors [ 51 ]. Gene editing via electroporation has also been applied to porcine zygotes [ 52 ], with successful gene modification (knockout) [ 52 , 53 , 54 , 55 ]. Electroporation-mediated gene editing requires no specialized equipment and benefits from a simple process and high zygote viability.…”

“…(2019) [ 107 ] PDX1 EP CRISPR Tanihara et al . (2020) [ 54 ] Duchenne muscular dystrophy DMD CMI CRISPR Yu et al . (2016) [ 130 ] Familial hypercholesterolemia LDLR SCNT TALEN Carlson et al .…”

Genetically engineered pigs manifesting pancreatic agenesis with severe diabetes

“…In contrast, pigs with low mutation rates were healthy. 37 In our preliminary trial, we realized that 20-30 copies of the Pdx1-Hes1 transgene in midgestational Pathophysiology/complications fetuses resulted in anomalies in organs, including anal atresia. As shown by our qPCR results, endogenous expression of Pdx1 on day one after birth resulted in a 30% reduction in expression compared with that in the WT group.…”