• Journal of Embryo Transfer
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• v.17 no.2
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• pp.101-108
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• 2002

Human Prourokinase (proUK) offers potential as a novel agent with improved fibrin specificity and, as such, may offer advantages as an attractive alternative to urokinase that is associated with clinical benefits in patients with acute peripheral arterial occlusion. For production of transgenic cow as human proUK bioreacotor, we conducted this study to establish efficient production system for bovine transgenic embryos by somatic cell nuclear transfer (NT) using human prourokinase gene transfected donor cell. An expression plasmid for human prourokinase was constructed by inserting a bovine beta-casein promoter, a green fluorescent protein (GFP) marker gene, and human prourokinase target gene into a pcDNA3 plasmid. Cumulus cells were used as donor cell and transfected with the expression plasmid using the Fugene 6 as a carrier. To increase the efficiency for the production of transgenic NT, development rates were compared between non-transfected and transfected cell in experiment 1, and in experiment 2, development rates were compared according to level of GFP expression in donor cells. In experiment 1, development rates of non-transgenic NT embryos were significantly higher than transgenic NT embryos (43.3 vs. 28.4％). In experiment 2, there were no significant differences in fusion rates (85.4 vs. 78.9％) and cleavage rates (78.7 vs. 84.4％) between low and high expressed cells. However, development rates to blastocyst were higher in low expressed cells (17.0 vs. 33.3％), and GFP expression rates in blastocyst were higher in high expressed cells (75.0 vs. 43.3％), significantly.

• Journal of Plant Biotechnology
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• v.29 no.4
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• pp.241-246
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• 2002

Somatic embryos or adventitious buds were formed from the segments of zygotic embryo of Lycium chinense Mill. on Murashige and Skoog (MS) medium supplemented with auxins (2,4-D, NAA, IAA) and / or cytokinins (zeatin, kinetin, BAP). Embryogenic callus formed on MS medium containing 0.5 mg/L 2,4-D and then differentiated into somatic embryos without transfer to hormone free medium. On the other hand, adventitious buds were formed on medium with 0.01 mg/L 2,4-D and 0.5 mg/L zeatin. Among various parts of zygotic embryos, the morphogenic potential was higher in the cotyledonary region, and the most organogenic potential was found in cotyledon followed by radicle, hypocotyl, and whole embryo. Histologically, bipolar structure of the heart-shaped embryos were confirmed and in adventitious buds only shoot apical meristems were found to exist.

• Journal of Embryo Transfer
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• v.22 no.4
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• pp.235-243
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• 2007

Our goal was to examine the effects of early denudation on the enucleation efficiency and developmental competence of embryos following somatic cell nuclear transfer (SCNT) and parthenogenetic activation (PA). Oocytes were denuded following 30 h of in vitro maturation (IVM) and then cultured with (D+) or without (D-) their detached cumulus cells for additional $10{\sim}14$ h. Control oocytes were denuded after $40{\sim}44$ h of IVM. The size of the perivitelline space was larger at 40 h of IVM ($11.7{\sim}11.8{\mu}m$) than at 30 h ($8.9{\mu}m;$ p<0.01). The distances between the metaphase II (M II) plates and the polar bodies (PBs) were shorter in D+ ($19.4{\mu}m$) and D- oocytes ($18.9{\mu}m$) than in control oocytes ($25.5{\mu}m;$ p<0.01). Enucleation rates following blind aspiration at 40 h of IVM were higher (p<0.01) in D+ (92%) and D- oocytes (93%) compared to controls (82%). Early denudation did not affect oocyte maturation or the in vitro development of SCNT and PA embryos. When SCNT embryos from D+ oocytes were transferred to four gilts, pregnancy was established in two pigs, and one of them farrowed three live piglets. In conclusion, early denudation of oocytes at 30 h of IVM could improve the enucleation efficiency by maintaining the M II plate and the PB within close proximity and support the in vivo development of SCNT embryos to term.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.64-64
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• 2001

To facilitate the widespread application of somatic cell cloning, improvements in blastocyst production efficiency and subsequent fetal viability are required. Area where technical improvements are needed include donor cell treatments, starvation and passage numbers. This study was carried out to investigate the effect of serum-starvation and passage on the development of ear skin fibroblast cells cloned embryos. A skin biopsy was obtained from the ear of a 2-year-old Korean Hanwoo female. The cells were cultured in 10% FBS＋DMEM up to 2-3 months(up to 10 passages) and then used. In Experiment 1, the Korean bovine Ear Skin Fibroblast cells (KbESF) were either serum starved (culture in 0.05% FBS＋DMEM) or serum fed (10% FBS＋DMEM) for 4-7 days Prior to NT In Experiment 2, the KbESF cells used for nuclear transfer in these experiments were from passages 2 to 10. The development of 208 nuclear transfer (NT) embryos reconstructed from either serum starved or serum fed ear skin fibroblast was assessed. NT embryos reconstructed from serum starved and serum fed cells showed the same developmental rate (cleavage 80.16 vs. 85.37%; blastocyst 20.63 vs. 19,51%). The development of 590 nuclear transfer (NT) embryos reconstructed from passage 2 to 10 was assessed. We observed the same developmental rates for embryos derived from later Passages as compared with those embryos from early passages(blastocyst from 16.69 to 27.91%, average 20.17%). There was no significant difference between serum-fed and serum-starved donor cells. We observed no difference in developmental rates for embryos derived from 2 to 10 passages. These data show that prolonged culture and serum starvation does not affects the cloning competence of adult somatic cells.

• Korean Journal of Animal Reproduction
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• v.25 no.1
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• pp.51-61
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• 2001

This study was conducted to determine developmental ability of reconstructed embryos by nuclear transfer using somatic cell of Korean bull with high genetic value. Fibroblast cells obtained from ear biopsy of the bull were cultured in Dulbecco's Modified Eagle's medium (DMEM) at 37$^{\circ}C$ in air containing 5% $CO_2$. The cummulus-oocyte complexes were collected from slaughterhouse and were matured in vitro for 20 h in TCM 199 culture medium and the oocytes were then enucleated in modified phosphate buffered saline with cytochalasin B. Matured bovine oocytes were enucleated by aspirating the first polar body and metaphase chromatin using a beveled pipette in modified phosphate buffered saline. The ear fibroblast cells were fused into enucleated oocyte by electrical stimulation. The reconstructed oocytes were activated with ionomycine and 6-dimethylaminopurine, and then cultured in CR1aa medium for 7.5 days. Out of 524 bovine eggs reconstructed by nuclear transfer 65.6%(277/422) embryos were cleaved, and 30.7% (85/277) cleaved embryos were developed to the morula to blastocysts. There was no difference of developmental ability in vitro of reconstructed embryos regardless of donor cell passages. In order to determine fate of foreign mitochondria of donor nucleus, the Mito Tracker stained cells were fused into enucleated oocytes. The donor mitochondria were detected early stage of embryos, but disappeared rapidly. The developmental ability of reconstructed embryos was not impaired by Mito Tracker treatments. The results indicate that viable reconstructed embryos can be producted by nuclear transfer using somatic cell of Korean bulls.bulls.

• Journal of Embryo Transfer
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• v.33 no.3
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• pp.119-127
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• 2018

The purpose of this study is to examined the electrofusion and activation conditions for the production of porcine somatic cell nuclear transfer (SCNT) embryos. In this study, immature oocytes were cultured in TCM-199 with and without hormones for 22 hours. Skin fibroblasts cells of porcine were transferred into the perivitelline space of enucleated in vitro matured oocytes. Cell fusion was performed with two different pulses that each one pulse (DC) of 1.1 kV/cm or 1.5 kV/cm for $30{\mu}sec$. After fusion subsequent activation were divided into three groups; non-treatment (control) and treatment with 2 mM 6-DMAP or $7.5{\mu}g/ml$ cytochalasin B for 4 hours. Transferred embryos were cultured in PZM-3 (Porcine Zygote Medium-3) in $5%\;CO_2$ and 95% air at $39^{\circ}C$ for 7 day. Apoptosis-related genes (Caspase-3, BCL-2, mTOR, and MMP-2) were analyzed by immunofluorescence staining. There was no significant difference between two different electrofusion stimuli in the cleavage rate; $64.9{\pm}4.8%$ in 1.1 kV/cm and $62.7{\pm}4.0%$ in 1.5 kV/cm. However, blastocyst formation rate (%) was significantly different among three different activation groups (no treatment, 2 mM 6-DMAP or $7.5{\mu}g/ml$ cytochalasin B) combined with electrofusion of 1.1 kV/cm. The blastocyst formation rate was $12.6{\pm}2.5$, $20.0{\pm}5.0$, and $34.9{\pm}4.3%$ in control, 2 mM 6-DMAP, and $7.5{\mu}g/ml$ cytochalasin B, respectively. Immunofluorescence data showed that expression levels of caspase-3 in SCNT embryos undeveloped to blastocyst stage were higher than those in the blastocyst stage embryos. Expression levels of Bcl-2 in blastocyst stage embryos were higher than those in the arrested SCNT embryos. These results showed that the combination of an electric pulse (1.1 kV/cm for $30{\mu}sec$) and $7.5{\mu}g/ml$ cytochalasin B treatment was effective for production of the porcine SCNT embryos.

• Korean Journal of Plant Tissue Culture
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• v.28 no.3
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• pp.129-134
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• 2001

Winter bud explants from 15 individual angelica tree (Aralia elata) were cultured in vitro to find out optimal conditions for somatic embryo induction as well as plant regeneration. Calli are induced and grown on MS medium supplemented with 1.0 mg/L 2,4-D for 4 weeks and subcultured on a half-strength MS medium without phytohormones to induce somatic embryos. Inter-simple sequence repeat (I-SSR) markers were analyzed with total DNAs extracted from the trees. Genotype effects on somatic embryo induction were examined by cluster analysis. Callus induction rate varied from 58.5 to 100% among the genotypes. Somatic embryo induction rate also greatly varied from 0 to 100% among the genotypes. There was a significant difference in somatic embryo induction rate even among the individual trees that showed close genetic relationships each other. This suggested that somatic embryo induction rate in Aralia elata be influenced by a few major specific genes rather than whole genomic similarity among individual trees. Four individuals of Ulneong-7, Cheju-1, Shingu and China, which are recalcitrant to somatic embryo induction, turned out to have a close genetic relationship, suggesting that both physiological and genetic factors affect somatic embryo induction. The results suggest that genotype selection be the most important factor to achieve an efficient propagation, although cultural optimization through medium and explant manipulation may also play crucial roles in somatic embryogensis as well as plant regeneration of these species.

• Journal of Plant Biotechnology
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• v.31 no.1
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• pp.67-72
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• 2004

An efficient protocol has been developed for rapid mass propagation of sweetpotato from shoot-tips derived embryogenic callus. Optimal embryogenic callus was induced from shoot apical meristem explants on Murashige and Skoog (MS) medium supplemented with 1mg/L 2,4-D. The addition of casein hydrolysate in the media increased the embryogenesis efficiency of sweetpotato. Somatic embryos were easily induced from the embryogenic callus on MS basal medium containing 300-500mg/L casein hydrolysate without phytohormon. Treatment of casein hydrolysate (100∼300mg/L) with 1mg/L 2,4-D also improved the secondary embryonic efficiency from somatic embryos below 2mm in length. Plant regeneration was achieved via somatic embryogenesis and direct organogenesis. Regenerated planlets with well developed shoots and roots on MS basal medium were successfully transferred to soil.

• Korean Journal of Plant Tissue Culture
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• v.22 no.5
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• pp.299-306
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• 1995

The objective of this study is to establish an efficient celll culture system for somatic embryogenesis in Ostericum koreanum and Angelica purpuraefolia. The highest frequency of embryogenic callus on immature seeds of O. konanum and A, purpuraefolia. was obtained when seeds were cultured on MS medium containing 0.1 mg/L 2,4-D and 0.1 mg/L BA. However somatic embryos were formed directly from the edge of cotyledon and hypocotyl of plant which regenerated on medium supplemented with 0.1-3.0 mg/L NAA. Immature seed explane cultured at 25$\pm$2$^{\circ}C$ after 10 days treatment at 5$^{\circ}C$ produced embryogenic callus and somatic embryos, and these differentiated into whole plane. Addition of glutamine and coconut milk to media did not enhance the frequency of somatic embryogenesis in immature seed cultures of A. purpuraefolia. However in immature seed culture of O. koreanum, the frequency of somatic embryogenesis were increased on media supplemented with glutamine and 10% coconut milk. Especially addition of glutamine to the medium substituted effect of NH$_4$N0$_3$ in constast to coconut milk. The highest frequency of conversion somatic embryos into plantlet was 89.1% on MS basal medium Embryogenic calli were grown vigorously when maintained on medium with 0.01 mg/L 2,4-D and 0.01 mg/L BA.

• Plant Biotechnology Reports
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• v.2 no.2
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• pp.153-161
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• 2008

The factors influencing somatic embryo maturation, high frequency somatic embryo germination, and plantlet formation were studied in Terminalia chebula Retz. Maturation of somatic embryo were influenced by a number of factors such as in vitro culture passage, concentrations of sucrose, levels of abscisic acid (ABA), basal media and media additive combinations. Maximum frequency of somatic embryo maturation ($57.22{\pm}2.02$), was obtained on MS medium supplemented with 50 g/l sucrose. Different factors such as strengths of MS nutrients, plant growth regulators, media additives and their combinations controlling somatic embryo germination and plantlet formation were studied. High frequency of germination and plantlet formation ($58.80{\pm}1.47$) were achieved by subsequent subculture of mature somatic embryos on MS medium containing 30 g/l sucrose and 0.5 mg/l benzyl-adenine (BA). However, although duration of in vitro passage of the callus tissue was critical, contribution of the combinations of plant growth regulators and media additives showed nugatory effect on somatic embryo maturation and germination as evident from variable responses.