Generation of genome-edited dogs by somatic cell nuclear transfer.

BACKGROUND: Canine cloning technology based on somatic cell nuclear transfer (SCNT) combined with genome-editing tools such as CRISPR-Cas9 can be used to correct pathogenic mutations in purebred dogs or to generate animal models of disease. RESULTS: We constructed a CRISPR-Cas9 vector targeting canine DJ-1. Genome-edited canine fibroblasts were established using vector transfection and antibiotic selection. We performed canine SCNT using genome-edited fibroblasts and successfully generated two genome-edited dogs. Both genome-edited dogs had insertion-deletion mutations at the target locus, and DJ-1 expression was either downregulated or completely repressed. CONCLUSION: SCNT successfully produced genome-edited dogs by using the CRISPR-Cas9 system for the first time.

Effect of Passage Number of Conditioned Medium Collected from Equine Amniotic Fluid Mesenchymal Stem Cells: Porcine Oocyte Maturation and Embryo Development.

Oocyte in vitro maturation (IVM) is the most important first step in in vitro embryo production. One prerequisite for the success of IVM in oocytes is to provide a rich culture microenvironment that meets the nutritional needs of developing oocytes. We applied different equine amniotic fluid mesenchymal stem cell conditioned medium (eAFMSC-CM) from passages 7, 18, and 27 to porcine oocytes during IVM to determine its effects on oocyte development and subsequent embryo development, specifically. The eAFMSC-CM from passage 7 (eAFMSC-CMp7) has a considerable impact on 9 genes: BAX, BCL2, SOD2, NRF2, TNFAIP6, PTGS2, HAS2, Cx37, and Cx43, which are associated with cumulus cell mediated oocyte maturation. GSH levels and distribution of mitochondrial and cortical granules were significantly increased in oocytes incubated with eAFMSC-CMp7. In addition, catalase and superoxide dismutase activities were high after IVM 44 h with eAFMSC-CMp7. After in vitro fertilization, blastocyst quality was significantly increased in the eAFMSC-CMp7 group compared to control. Lastly, the antioxidant effect of eAFMSC-CMp7 substantially regulated the expression of apoptosis, pluripotency related genes and decreased autophagy activity in blastocysts. Taken together, this study demonstrated that the eAFMSC-CMp7 enhanced the cytoplasmic maturation of oocytes and subsequent embryonic development by generating high antioxidant activity.

Accomplishment of canine cloning through in vitro matured oocytes: a pioneering milestone.

The in vitro maturation (IVM) rate of canine oocytes remains low compared to other mammals due to their unique reproductive characteristics. This study aimed to explore the effect of hormone supplementation during the IVM of canine immature oocytes on nuclear maturation and subsequently assess its potential application in canine somatic cell nuclear transfer (SCNT). Immature oocytes were collected and cultured in an IVM medium supplemented with hormones (follicle-stimulating hormone [FSH] and progesterone [P4]) or without hormones (control) for 24 hours. The maturation rates of oocytes in the hormone-treated group (94.92 ± 3.15%) were significantly higher than those in the control group (61.01 ± 4.23%). Both in vitro and in vivo matured oocytes underwent NT to evaluate their utility, and the fusion rates were higher in the in vitro matured group than those in the vivo matured group, not significant between in vivo and in vitro matured group (73.28% and 82.35%, respectively). As a result, 14 fused embryos from the in vitro matured group were transferred into two surrogates, with one surrogate achieving a successful pregnancy and delivering four puppies. Whereas in the in vivo matured group, 85 fused embryos were transferred to 8 surrogate mothers, leading to three surrogates becoming pregnant and delivering one, four, and two puppies. The pregnancy rates were not significant between both groups (50% and 37.50%), but the number of offspring exhibited a significant difference (28.57% and 8.23%). In conclusion, we achieved a remarkable milestone by successfully producing cloned puppies using in vitro matured oocytes, underscoring the feasibility of canine cloning from in vitro recovered oocytes. It is important to note that this study focused only on immature oocytes after ovulation and only during the estrus stage. Further research targeting other stages of the estrous cycle could potentially enhance canine cloning efficiency.