Delayed body development with reduced triglycerides levels in leptin transgenic pigs.

Leptin is a well-known adipokine that plays critical role in adiposity. To further investigate the role of leptin in adiposity, we utilized leptin overexpressing transgenic pigs and evaluated the effect of leptin on growth and development, fat deposition, and lipid metabolism at tissue and cell level. Leptin transgenic pigs were produced and divided into two groups: elevated leptin expression (leptin ( +)) and normal leptin expression group (control). Results indicated that leptin ( +) pigs had elevated leptin protein and mRNA expression levels and exhibited sluggish growth and development followed by decreased subcutaneous fat thickness, low serum triglycerides, saturated, unsaturated fatty acids and high cholesterol esters (p < 0.05). There were differences in the lipid metabolism related genes at different fat depots, including upregulation of PPARγ, AGPAT6, PLIN2, HSL and ATGL in subcutaneous, PPARγ in perirenal, and FAT/CD36 and PLIN2 in mesenteric adipose tissues and downregulation of AGPAT6 and ATGL in perirenal and AGPAT6 in mesenteric adipose tissues (p < 0.05). Additionally, in-vitro cultured leptin ( +) preadipocytes exhibited upregulation of PPARγ, FAT/CD36, ACACA, AGPAT, PLIN2, ATGL and HSL as compared to control (p < 0.05). These findings suggested that homeostasis imbalance in lipolysis and lipogenesis at adipose tissue and adipocytes levels led to low subcutaneous fat depots in leptin overexpression pigs. These pigs can act as model for obesity and related metabolic disorder.

Efficient Generation of P53 Biallelic Mutations in Diannan Miniature Pigs Using RNA-Guided Base Editing.

The base editing 3 (BE3) system, a single-base gene editing technology developed using CRISPR/Cas9n, has a broad range of applications for human disease model construction and gene therapy, as it is highly efficient, accurate, and non-destructive. P53 mutations are present in more than 50% of human malignancies. Due to the similarities between humans and pigs at the molecular level, pig models carrying P53 mutations can be used to research the mechanism of tumorigenesis and improve tumor diagnosis and treatment. According to pathogenic mutations of the human P53 gene at W146* and Q100*, sgRNAs were designed to target exon 4 and exon 5 of the porcine P53 gene. The target editing efficiencies of the two sgRNAs were 61.9% and 50.0%, respectively. The editing efficiency of the BE3 system was highest (about 60%) when C (or G) was at the 5th base. Puromycin screening revealed that 75.0% (21/28) and 68.7% (22/32) of cell colonies contained a P53 mutation at sgRNA-Exon5 and sgRNA-Exon4, respectively. The reconstructed embryos from sgRNA-Exon5-5# were transferred into six recipient gilts, all of which aborted. The reconstructed embryos from sgRNA-Exon4-7# were transferred into 6 recipient gilts, 3 of which became pregnant, resulting in 14 live and 3 dead piglets. Sequencing analyses of the target site confirmed 1 P53 monoallelic mutation and 16 biallelic mutations. The qPCR analysis showed that the P53 mRNA expression level was significantly decreased in different tissues of the P53 mutant piglets (p < 0.05). Additionally, confocal microscopy and western blot analysis revealed an absence of P53 expression in the P53 mutant fibroblasts, livers, and lung tissues. In conclusion, a porcine cancer model with a P53 point mutation can be obtained via the BE3 system and somatic cell nuclear transfer (SCNT).

Improving porcine SCNT efficiency by selecting donor cells size.

Considerable advancements have recently been achieved in porcine somatic cell nuclear transfer (SCNT), but the efficiency remains low. Donor cell size might play an important role in SCNT, but its effects in pigs remain unclear. This study aimed to evaluate the efficiency of porcine SCNT by selecting donor cells of suitable size. Porcine fetal fibroblasts (PFFs) were divided into three groups, group S (small, d ≤ 13 µm), group M (medium, 13 µm 18 µm), and their biological characteristics were analyzed. Next, SCNT was performed using PFFs of different sizes to evaluate the developmental potential of reconstructed embryos. The data showed that PFFs in groups S, M and L accounted for 17.5%, 47.7% and 34.8% of cells, respectively. Morphologically, cells in group S exhibited clear and regular cell membranes and nuclei, whereas cells in groups M and L displayed varying degrees of cell membrane protuberance, karyo-pyknosis, autophagy and mitochondrial abnormalities. In addition, the growth status and proliferation capabilities of cells in group S were significantly better than those of group M and group L. The percentage of cells at G0/G1 in group S and M were significantly greater than group L. The senescence rate of group S was lower than group M and group L. The apoptosis rate of group S was significantly lower than that of group L but comparable to that of group M . The cleavage rate of group S was also significantly greater than that of group M but comparable to that of group L . The blastocyst rate of group S was significantly greater than that of group M and group L. The blastocyst cell numbers of group S were also significantly greater than those of group M and group L. These findings suggested that small PFFs with a diameter of less than 13 µm are more suitable donor cells for SCNT in pigs.Abbreviations: DMEM: Dulbecco's modified Eagle's medium; FBS: Fetal bovine serum; PBS: Phosphate buffer saline; PFFs: Porcine fetal fibroblast cells; SCNT: Somatic cell nuclear transfer.