• Journal of Animal Reproduction and Biotechnology
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• v.35 no.4
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• pp.323-328
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• 2020

Direct injection of genome editing tools such as CRISPR/Cas9 system into developing embryos has been widely used to generate genetically engineered pigs. The approach allows us to produce pigs carrying targeted modifications at high efficiency without having to apply somatic cell nuclear transfer. However, the targeted modifications during embryogenesis often result in mosaicism, which causes issues in phenotyping founder animals and establishing a group of pigs carrying intended modifications. This study was aimed to establish a genomic PCR and sequencing system of a single blastomere in the four-cell embryos to detect potential mosaicism. We performed genomic PCR in four individual blastomeres from four-cell embryos. We successfully amplified target genomic region from single blastomeres of 4-cell stage embryo by PCR. Sanger sequencing of the PCR amplicons obtained from the blastomeres suggested that PCR-based genotyping of single blastomere was a feasible method to determine mutation type generated by genome editing technology such as CRISPR/Cas9 in early stage embryos. In conclusion, we successfully genotyped single blastomeres in a single 4-cell stage embryo to detect potential mosaicism in porcine embryos. Our approach offers a simple platform that can be used to screen the prevalence of mosaicism from designed CRISPR/Cas9 systems.

• Korean Journal of Veterinary Research
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• v.57 no.2
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• pp.131-133
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• 2017

A 11-year-old, female Russian Blue cat was presented with anorexia, vomiting, and diarrhea lasting for 3 days. Abdominal ultrasonography revealed a hypoechoic, non-circumferential, and eccentrically formed intestinal loop with altered wall layering and thickening of the tunica muscularis. After surgical resection, histopathologic examination confirmed an infiltrative, round-cell neoplasm composed of sheets and cords of neoplastic mast cells within a fibrotic, edematous stroma. The cat was alive and healthy 6 months after surgery. To the best of our knowledge, this is the first reported case of an intestinal mast cell tumor in a Russian Blue cat in South Korea.

• Korean Journal of Veterinary Service
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• v.36 no.2
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• pp.127-132
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• 2013

An 11-year-old poodle bitch was presented for investigation of multicentric mammary masses. Abdominal sonography and radiography demonstrated abnormal enlargement of uterus and ovaries. Blood analysis revealed high progesterone concentration. The ovariohysterectomy and mastectomy were performed. Histopathologically, the mammary masses revealed complex carcinoma-tubulopapillary carcinoma with papillary pattern and tubule pattern. In the uterus, cystic endometrial hyperplasia was observed. Scattered inflammatory cells were observed in the endometrial stroma and mucinous material was protruded from endometrial surface. Also, in the ovaries, bilateral ovary granulosa cell tumor was detected. The bitch made a complete recovery following the ovariohysterectomy and mastectomy. This case was a very rare multiple tumor occurrence with bilateral ovary granulosa cell tumor and mammary complex carcinoma. High progesterone concentration was characterized clinically in the bitch.

• Korean Journal of Animal Reproduction
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• v.27 no.4
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• pp.339-347
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• 2003

The purpose of this study was to investigate the development of bovine nuclear transfer (NT) embryos cultured in serum-free conditions. Bovine NT embryos cultured in various culture conditions were compared blastocyst development, total cell number and apoptosis using TUNEL assay. In experiment 1, blastocyst rates of NT embryos were significantly higher (P<0.01) in FBS (22.0％) and BSA (26.6％) groups than in PVA (6.3％) group. Total cell number was significantly higher in FBS (78.4$\pm$19.4) and BSA (90.9$\pm$29.1) groups than in PVA group (46.0$\pm$0.0). Apoptotic cell number was significantly fewer in FBS (3.1$\pm$1.4) and BSA (1.7$\pm$1.4) groups than in PVA group (7.0$\pm$20.0) However, all of results were not different between the FBS and BSA group. In experiment 2, blastocyst rates of NT embryos were significantly higher (P<0.05) in fatty acid free-BSA (FAF-BSA) group (26.8％) than in fraction V-BSA group (11.2％). Total cell number were somewhat higher in FAF-BSA group (89.8$\pm$30.7) than in fraction V-BSA group (88.1$\pm$19.3). Apoptotic cell number were somewhat fewer in FAF-BSA (1.7$\pm$1.5) group than in fraction V-BSA group (4.2$\pm$2.9). These findings suggest that serum free condition were effective for the in vitro development of bovine NT embryos. Therefore, we concluded that fatty acid free-BSA has beneficial effect in development bovine NT embryos and can be use as a serum substitute.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2003.10a
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• pp.121-121
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• 2003

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.2
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• pp.166-171
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• 2007

The objective of this study was to assess the association of polymorphisms in ${\kappa}$-cn, ${\beta}$-lg, ${\beta}$-lg 5′ flanking region, CSN1S2, and IGFBP-3 genes with milk production traits and mastitis-related traits in Chinese Holstein. Traits analyzed were 305 day standard milk yield, protein percentage, fat percentage, the ratio of fat percentage and protein percentage, pre-somatic cell count, somatic cell count, and somatic cell score, respectively. CSN1S2 locus was uninformative because only one genotype BB was found in Chinese Holstein. Allele frequencies of A and B in IGFBP-3 gene were 0.5738 and 0.4262 in Chinese Holstein population, which was different from reported Qinchuan cattle population. The genotypes of animals at IGFBP-3 locus significantly affected 305 day standard milk yield, protein percentage, and somatic cell score. The ${\beta}$-lg genotypes had a significant effect on protein percentage and the ratio of fat percentage and protein percentage. Polymorphism in ${\beta}$-lg 5′ flanking region was associated with 305 day standard milk yield, protein percentage, fat percentage, pre-somatic cell count, and somatic cell count. No significant associations of the polymorphism in ${\kappa}$-cn gene were observed for any trait.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.66-66
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• 2001

Since the first birth of pig derived from embryonic cells by nuclear transfer, many researches to produce cloned pig have been carried out. Recently, two reports about the birth of somatic cell cloned pigs using in vivo oocytes and also Betthauser et al. (2000) reported the birth of somatic cell cloned pigs using in vitro oocytes. So here we investigated the effect of activation method and culture medium on in vitro development of porcine nuclear transfer embryo using fetal fibroblast. Oocytes derived from slaughter house obtained ovaries were matured for 42 to 44 h in TCM 199. Matured oocytes were denuded using 0.1% hyaluronidase and then Oocytes with the first polar body were used for enucleation by aspirating the first polar body and adjacent cytoplasm in TCM 199 supplemented with 7.5 $\mu\textrm{g}$ cytochalasin B. Petal fibroblast cells were prepared from 35 days old fetus. To be used as donor cells, fetal fibroblast cells were serum starved for 3 to 5 days and then isolated into single co:1 by trypsinization. Nuclear transfer embryos were fused using 2 times 1.25㎸ for 30$mutextrm{s}$. Fused NT embryos were activated with calcium ionophore (CI) and 6-dimethyl-aminopurine (6-DMAP). Activated oocytes were cultured in NCSU 23 or BECM 3 for 6 days. There was no significant difference between chemical activation and no chemical activation for blastocyst development rate(11.6 vs. 14.8%). However, cell number was significantly higher when NT embryos were activated with CI and 6-DMAP (31.2 vs. 22.6). When NT embryos were cultured in NCSU 23 or BECM 3, blastocyst development rate was 16.4 and 13.2%, respectively, and cell number was 31.5 and 24.1, respectively. These results suggest that chemical activation after fusion and culture in NCSU 23 could increase cell number of porcine NT embryos.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.5
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• pp.626-631
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• 2005

Nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) is involved in cell apoptosis, which contributes to luteal regression and luteolysis in some species. In large domestic animals, no direct evidence for the relationship between NO and cell apoptosis in the process of corpus luteum regression is reported. The present study was conducted to investigate the localization of iNOS on porcine corpora lutea (CL) during the oestrus cycle and its relation to cell DNA fragmentation and CL regression. According to morphology, four luteal phases throughout the estrous cycle were defined as CL1, CL2, CL3 and CL4. By isoform-specific antibody against iNOS, the immunochemial staining was determined. Luteal cell DNA fragmentation was determined by flow cytometry. The results showed that no positive staining for iNOS was in CL1 and that iNOS was produced but limited to the periphery of CL2, while in the CL3, the spreading immunochemical staining was found inside the CL. No iNOS positive staining was detected in CL4. Meanwhile, DNA fragmentation increased dramatically when CL developed from CL2 to CL3 (p<0.05). In CL4, higher proportion of luteal cells still had fragmented DNA than that of luteal cells from CL1 or CL2 (p<0.05). These results indicate that iNOS expression is closely related to luteal cell apoptosis and then to luteal regression.

• Applied Microscopy
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• v.22 no.1
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• pp.15-32
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• 1992

This study was an attempt to investigate and compare the fine structure of small cells in the space of Disse of various animal livers. All animal livers contained small cells with or without lipid droplets and the one with lipid droplet seemed to be more developed and show an abundance of activity in function. The fine structure of the small cells observed in the nonmammals was similar to that of Ito cell in the mammal. The electron density of the small cells was similar to that of other cell types in the same animal liver. The cisternal dilation of rough endoplasmic reticulum and Golgi apparatus was more predominant in the mammalian Ito cells. In the nonmammalian, aquatic vertebrates, however, lysosomes and filaments are much more abundant in the Ito cell and its abundant cytoplasmic processes rich in filaments were usually extended between the parenchymal cells. The disparity in size of organelles and numbers of lipid droplets in the small cells showed a tendency similar to those of other cell types in the same animal. From these results, it is considered that the small cells in the space of Disse is a Ito cell and the Ito cell without lipid droplets differentiates into the one containing lipid droplets according to the characteristics of the different animals respectively, and that the Ito cells in the mammals are more active in metabolic function, while those in the nonmammalian aquatic vertebrates are abundant in support of parenchyme.

• Korean Journal of Animal Reproduction
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• v.23 no.4
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• pp.393-398
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• 1999

Somatic cell nuclear transfer is an efficient technique for the multiplication of elite livestock, engineering of transgenic animals, cell therapy and xenotransplantation, and analyzing the interactions between nucleus and cytoplasm, for various agricultural, biomedical and research purposes. Since the first somatic cell clone lamb was born, tremendous progress has been made toward developing technology for animal cloning. Viable farm animals and mice have now been produced by nuclear transfer using various fetal and adult somatic cells as nuclei donors. Transgenic clones were also produced from nuclear transfer of transfected somatic cells. In the future, somatic cell nuclear transfer will provide more numerous opportunities, both in basic and appled research as well as immediate uses in the generations of superior clone and transgenic animals. However, further technology refinement and improved understanding of the process are essential for commercial and basic research applications.