• Korean Journal of Plant Tissue Culture
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• v.22 no.3
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• pp.157-164
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• 1995

Intact mature zygotic embryos or their excised cotyledons of ginseng, were cultured on media containing various growth regulators such as auxin (2,4D, IAA) and cytokinin(BAP kinetin). In the culture of intact zygotic embryos, auxin inhibited germination but cytokinin did not Somatic embryogenesis occurred only from those of ungerminated embryos. In the culture of cotyledon segment, medium without growth regulators was the most appropriate to somatic embryogenesis. Somatic embryos were produced sporadically over the surfaces of zygotic embryos on medium containing auxin, while on medium without growth regulators, or media containing cytokinin, somatic embryos formed only on the proximal region of cotyledon. on medium containing 2,4-D, somatic embryos originated from multiple cells which comprised epidermal and subepidermal layers of cotyledon, which resulted in poly-somatic embryogenesis. When these somatic embryos were cultured on the same medium, the primary somatic embryos procured secondary embryos, which arose from epidermal or subepidermal single cells.

• Journal of Embryo Transfer
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• v.17 no.2
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• pp.109-115
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• 2002

The present study was conducted for the production of transgenic cloned cows those secrete human lactoferricin into milk by somatic cell nuclear transfer (NT). To estimate detrimental effects of gene transfection on transgenic cloned embryo production, development rates of NT embryos were compared between transfected and non-transfected cumulus and ear fibroblast cells. An expression plasmid for human lactofericin (pbeta-LFC) was constructed by inserting a bovine beta-casein promoter, a green fluorescent protein (GFP) marker gene, and human lactoferricin target gene into a pcDNA3 plasmid. Two bovine somatic cell lines (cumulus cell and ear fibroblast) were established and transfected with the expression plasmid using a liposomal transfection reagent, Fugene6 as a carrier. Cumulus cell and ear fibroblast were transfected at the passage of 2 to 4, trypsinized and GFP-expressing cells were randomly selected and used for somatic cell NT. Developmental competences (rates of fusion, cleavage, and blastocyst formation) in bovine transgenic somatic cell NT embryos reconstructed with non-transfectecd cells were significantly higher than those from transfected cells in cumulus cell and ear fibroblast (P＜0.05). This study indicated that transfection of done. cell has detrimental effect on embryo development in bovine transgenic NT.

• The Korean Journal of Zoology
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• v.39 no.1
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• pp.75-88
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• 1996

OTF9-63 (OTF9) and P19S1O1A1 (P19) embryonal carcinoma (EC) cells were examined for their ability to produce the readivation of inactive X chromosomes from somatic cells. They were hybridized with various somatic cells and resulting HATr EC-somatic cell clones were analysed for their morphology, chromosomal replication pafterns and expression proffies of X-linked and distantiy located genes, Hprt and Pgk-1. The results demonstrated that 0RF9 cells could reactivate the inactive X chromosome whereas P19 cells could not. In adition, EC-somatic cell hybrids tended to reduce the number of sex chromosomes in long-term culture, resulting m 1:2 ratio of sex chromosomes to autosomes The use of EC cell hybrids provides an experimental system for studying the mechanism(s) of the X-reactivatio that is initiated and maintained from meiotic prophase of oogenesis to early embryogenesis.

• Genomics & Informatics
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• v.12 no.2
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• pp.50-57
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• 2014

We present a new next-generation sequencing-based method to identify somatic mutations of lung cancer. It is a comprehensive mutation profiling protocol to detect somatic mutations in 30 genes found frequently in lung adenocarcinoma. The total length of the target regions is 107 kb, and a capture assay was designed to cover 99% of it. This method exhibited about 97% mean coverage at $30{\times}$ sequencing depth and 42% average specificity when sequencing of more than 3.25 Gb was carried out for the normal sample. We discovered 513 variations from targeted exome sequencing of lung cancer cells, which is 3.9-fold higher than in the normal sample. The variations in cancer cells included previously reported somatic mutations in the COSMIC database, such as variations in TP53, KRAS, and STK11 of sample H-23 and in EGFR of sample H-1650, especially with more than $1,000{\times}$ coverage. Among the somatic mutations, up to 91% of single nucleotide polymorphisms from the two cancer samples were validated by DNA microarray-based genotyping. Our results demonstrated the feasibility of high-throughput mutation profiling with lung adenocarcinoma samples, and the profiling method can be used as a robust and effective protocol for somatic variant screening.

• Asian-Australasian Journal of Animal Sciences
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• v.15 no.10
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• pp.1517-1522
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• 2002

The effect of post milking teat dipping on somatic cell count (SCC) of milk was determined in 20 Crossbred cows and 20 Murrah buffaloes selected from institute's herd. The animals were divided into two groups of 10 each. Animals of Group I (control) were teat washed with water before the milking while Group II animals were applied teat dipping solution after the completion of milking. The cows were milked 3 times a day while buffaloes were milked twice a day. The milk samples were collected from control and treated animals on day 0, 5, 10, 15, respectively. The milk samples were analyzed for milk constituents like fat, protein, lactose, chloride, IgG, NEFA, pH and EC and total and differential somatic cell counts. The changes in milk composition and somatic cell counts were significantly different (p<0.01) between the animals and between the breeds. However SCC, chloride content (p<0.05) and epithelial cells (p<0.01) varied during different days of study. The alterations in SCC, epithelial cells, TLC, lymphocyte, neutrophil, IgG, and protein content were significantly different (p<0.01) between control and treated groups. The pH, EC, protein, SCC, epithelial cells, lymphocyte and neutrophil cells of milk declined significantly (p<0.05) after the application of teat dipping, the respective values were 6.5 vs 6.40, 2.28 vs 2.37 mhos, 3.33 vs 4.04%, 1.00 vs $0.87{\times}10^5cells/ml$, 0.39 vs 0$0.34{\times}10^5cells/ml$, 0.36 vs $0.31{\times}1,000cells/ml$ and 0.17 vs $0.14{\times}1,000cells/ml$ in cows. However in buffaloes, epithelial cells, lymphocytes, neutrophils, EC and SCC declined (p<0.05) after application of teat dipping, the values being 0.37 vs $0.29{\times}10^5cells/ml$, 0.37 vs $0.25{\times}1,000cells/ml$, 0.14 vs $0.11{\times}1,000cells/ml$, 2.56 vs 2.37 mhos and 0.94 vs $0.73{\times}10^5cells/ml$, respectively. The study indicated that post milking teat dipping could be used as an effective method for the lowering of SCC in milk of crossbred cows and buffaloes.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.7-8
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• 2003

Since it was first reported in 1997, somatic cell cloning has been demonstrated in several other mammalian species. On the mouse, it can be cloned from embryonic stem (ES) cells, fetus-derived cells, and adult-derived cells, both male and female. While cloning efficiencies range from 0 to 20%, rates of just 1-2% are typical (i.e. one or two live offspring per one hundred initial embryos). Recently, abnormalities in mice cloned from somatic cells have been reported, such as abnormal gene expression in embryo (Boiani et al., 2001, Bortvin et al., 2003), abnormal placenta (Wakayama and Yanagimachi 1999), obesity (Tamashiro et ai, 2000, 2002) or early death (Ogonuki et al., 2002). Such abnormalities notwithstanding, success in generating cloned offspring has opened new avenues of investigation and provides a valuable tool that basic research scientists have employed to study complex processes such as genomic reprogramming, imprinting and embryonic development. On the other hand, mouse ES cell lines can also be generated from adult somatic cells via nuclear transfer. These 'ntES cells' are capable of differentiation into an extensive variety of cell types in vitro, as well assperm and oocytes in vivo. Interestingly, the establish rate of ntES cell line from cloned blastocyst is much higher than the success rate of cloned mouse. It is also possible to make cloned mice from ntES cell nuclei as donor, but this serial nuclear transfer method could not improved the cloning efficiency. Might be ntES cell has both character between ES cell and somatic cell. A number of potential agricultural and clinical applications are also are being explored, including the reproductive cloning of farm animals and therapeutic cloning for human cell, tissue, and organ replacement. This talk seeks to describe both the relationship between nucleus donor cell type and cloning success rate, and methods for establishing ntES cell lines. (중략)

• Journal of Veterinary Science
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• v.23 no.2
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• pp.40.1-40.13
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• 2022

Background: Somatic cell nuclear transfer (SCNT) is used widely in cloning, stem cell research, and regenerative medicine. The type of donor cells is a key factor affecting the SCNT efficiency. Objectives: This study examined whether urine-derived somatic cells could be used as donors for SCNT in pigs. Methods: The viability of cells isolated from urine was assessed using trypan blue and propidium iodide staining. The H3K9me3/H3K27me3 level of the cells was analyzed by immunofluorescence. The in vitro developmental ability of SCNT embryos was evaluated by the blastocyst rate and the expression levels of the core pluripotency factor. Blastocyst cell apoptosis was examined using a terminal deoxynucleotidyl transferase dUTP nick end-labeling assay. The in vivo developmental ability of SCNT embryos was evaluated after embryo transfer. Results: Most sow urine-derived cells were viable and could be cultured and propagated easily. On the other hand, most of the somatic cells isolated from the boar urine exhibited poor cellular activity. The in vitro development efficiency between the embryos produced by SCNT using porcine embryonic fibroblasts (PEFs) and urine-derived cells were similar. Moreover, The H3K9me3 in SCNT embryos produced from sow urine-derived cells and PEFs at the four-cell stage showed similar intensity. The levels of Oct4, Nanog, and Sox2 expression in blastocysts were similar in the two groups. Furthermore, there is a similar apoptotic level of cloned embryos produced by the two types of cells. Finally, the full-term development ability of the cloned embryos was evaluated, and the cloned fetuses from the urine-derived cells showed absorption. Conclusions: Sow urine-derived cells could be used to produce SCNT embryos.

• Journal of Plant Biotechnology
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• v.3 no.1
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• pp.33-38
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• 2001

Protoplasts isolated from inbred lines of Brassica oleracea L. var capitata (cabbage) and Brassica campestris L. ssp. pekinensis (Chinese cabbage) were fused by PEG-mediated method, and somatic hybrid cells were differentiated into plants. for the identification of somatic hybrid plants, ploidy level, plant morphology, and cytological analysis were performed. All of the regenerated plants derived from fused protoplasts were shown to be 2X-4X, or higher ploidy level, presumably due to somatic hybridization or chromosome doubling. The morphology of leaves, petioles, and flowers showed an intermediate phenotype between Chinese cabbage and cabbage. Chromosome numbers in these somatic hybrids ranged mostly from 33 to 38. According to Genomic in situ hybridization (GISH) pattern, signals from both fusion parents of B.campestris or B.oleracea were detected in different colors when chromosomes of putative somatic hybrids were observed.

• Reproductive and Developmental Biology
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• v.38 no.2
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• pp.71-77
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• 2014

The specific genetic modification in porcine somatic cells by gene targeting has been very difficult because of low efficiency of homologous recombination. To improve gene targeting, we designed three kinds of knock-out vectors with ${\alpha}1,3$-galactosyltransferase gene (${\alpha}1,3$-GT gene), DT-A/pGT5'/neo/pGT3', DT-A/NLS/pGT5'/neo/pGT3' and pGT5'/neo/ pGT3'/NLS. The knock-out vectors consisted of a 4.8-kb fragment as the 5' recombination arm (pGT5') and a 1.9-kb fragment as the 3' recombination arm (pGT3'). We used the neomycin resistance gene (neo) as a positive selectable marker and the diphtheria toxin A (DT-A) gene as a negative selectable marker. These vectors have a neo gene insertion in exon 9 for inactivation of ${\alpha}1,3$-GT locus. DT-A/pGT5'/neo/pGT3' vector contain only positive-negative selection marker with conventional targeting vector. DT-A/NLS/pGT5'/neo/pGT3' vector contain positive-negative selection marker and NLS sequences in upstream of 5' recombination arm which enhances nuclear transport of foreign DNA into bovine somatic cells. pGT5'/neo/pGT3'/NLS vector contain only positive selection marker and NLS sequence in downstream of 3' recombination arm, not contain negative selectable marker. For transfection, linearzed vectors were introduced into porcine ear fibroblasts by electroporation. After 48 hours, the transfected cells were selected with $300{\mu}g/ml$ G418 during 12 day. The G418-resistant colonies were picked, of which 5 colonies were positive for ${\alpha}1,3$-GT gene disruption in 3' PCR and southern blot screening. Three knock-out somatic cells were obtained from DT-A/NLS/ pGT5'/neo/pGT3' knock-out vector. Thus, these data indicate that gene targeting vector using nuclear localization signal and negative selection marker improve targeting efficiency in porcine somatic cells.

• Journal of Plant Biotechnology
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• v.40 no.3
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• pp.141-146
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• 2013

This study was conducted to examine suspended embryogenic cells growth with days of culture, effects of various kinds/concentrations of osmoticum for induction of somatic embryos (SEs), following somatic embryos germination or plantlet regeneration. The proliferation pattern of embryogenic cells in suspension culture is characterized by settled cells volume (SCV) increased with the duration of culture with marked the maxium of SCV (10.1 ml) in 18 days of culture, however the SCV of cells gradually decreased after that. In comparison of kinds/concentrations of osmoticum on somatic embryo induction, the highest induction number (352.3/g FW) of the SE was showed in 0.2 M sucrose, in addition, we also observed some effects with treatments of 0.2 M maltose (203.7) and 0.3 M maltose (193.7), respectively. However, no somatic embryos produced in treatments of 7.5% PEG plus 0.15 M sucrose or maltose. In comparison of germination efficiency of SEs which occurred from the treatments of various kinds/ concentrations of osmoticum, the highest induction frequency of cotyledon (25.2%) was obtained from SEs that produced 0.3 M maltose, however, the best occurrence rates of hypocotyl (39%), radicle (30.3%) and plantlet regeneration (3.5%) were observed from the 0.2 M sucrose treatment, respectively.