动物造模 z-bio 2022-10-11 589

　　Objective: The shortage of donors is the main problem faced by organ transplantation, and the preparation of humanized organs is one of the effective ways to solve this problem. Lack of fumarate acetoacetate hydrolase can cause hereditary tyrosinemia typeⅠ, HTⅠ), resulting in hepatocyte apoptosis and liver damage. Gene editing of pigs (Sus scrofa) to establish FAH gene knockout Bama miniature pigs, combined with human stem cells will facilitate the preparation of humanized stem cell livers . In this study, based on the α-1,3-galactosyltransferase gene (α-1,3galactosyltransferase, GGTA1) knockout in Bama minipigs, the FAH gene knockout (FAH+/-, FAH) was prepared by CRISPR/Cas9 technology. FAH-/-) miniature pigs, and their health and reproductive status were analyzed.

　　Methods: A single-stranded guide RNA (sgRNA) was designed for porcine FAH gene to construct pX330 expression vector, which was transfected into ear fibroblasts of Bama minipigs (BMEF) and the knockout efficiency was verified. Positive cells were selected as nuclear donors, and FAH gene knockout Bama minipigs were prepared by somatic cell nuclear transfer technology. Genomic DNA of piglets was extracted, and the mutation type was obtained after PCR sequencing; blood of 5-month-old FAH+/- Bama minipigs was collected, Blood biochemical and blood routine indexes were detected and liver histological stained sections were observed; when FAH+/-Bama miniature boars were sexually mature, they were bred with wild-type (WT) sows, and the reproductive ability was evaluated by litter size. situation.

　　RESULTS: FAH+/-Bama minipigs were obtained, Western blot showed that the expression of FAH in the liver and kidney of FAH+/-Bama minipigs decreased; compared with wild type, FAH+/-pig blood There was no significant difference in physical and chemical parameters; histological section staining found that FAH+/- pig liver histological changes showed vacuolar-like changes; wild sows mated with FAH+/- boars had normal litter size; FAH+/- pigs had normal health status and reproductive capacity.

　　Conclusion: FAH single-gene knockout miniature pigs were prepared in this study, and the health status and reproductive ability were normal. The single-gene knockout pigs provided a good foundation for the preparation of biallelic knockout pigs and humanized livers in the future.