• Asian-Australasian Journal of Animal Sciences
• /
• v.21 no.5
• /
• pp.648-656
• /
• 2008

The objective of this study was to establish conditions for transfection of a foreign gene into somatic cells using cationic lipid reagents and to evaluate the effects of transfection on in vitro development of somatic cell nuclear transfer (SCNT) embryos. Green fluorescent protein (GFP) gene was used as a foreign gene and a non-transfected somatic cell was utilized as a control karyoplast. Monolayers of porcine cells were established and subsequently transfected with a GFP-expressing gene (pEGFP-N1) using three types of transfection reagents (LipofectAMINE PLUS, FuGENE 6 or ExGen500). Donor cells used for SCNT included transfected fetal or adult fibroblasts and oviduct epithelial cells, either serum-fed or serum-starved. Oocytes matured in vitro for 42 h were reconstructed with either transfected or non-transfected porcine somatic cells by electric fusion and activation using a single DC pulse of 1.8 kV/cm for $30{\mu}s$ in $Ca^{2+}$ and $Mg^{2+}-containing$ 0.26 M mannitol solution. Reconstructed oocytes were subsequently cultured in NCSU-23 medium for 168 h and the developmental competence and cell number in blastocyst were compared. There were no significant differences (P>0.05) in fusion, cleavage rates or development to the blastocyst stage between non-transfected, transfected, serum-fed and serum-starved cells. However, the rates of GFP-expressing blastocysts were higher in the FuGENE 6 group (71.4%) among transfection reagents and in the fetal fibroblasts group (70.4%) for donor cells. These results indicate that fetal fibroblasts transfected with FuGENE 6 can be used as donor cells for porcine SCNT and that GFP gene can be safely used as a marker of foreign genes in porcine transgenesis.

• Korean Journal of Medicinal Crop Science
• /
• v.15 no.5
• /
• pp.346-351
• /
• 2007

An efficient somatic embryogenesis and plant regeneration protocol was developed for Schisandra chinensis Baill, using embryogenic cell suspensions and optimized media conditions. Friable embryogenic callus was induced from cotyledonary leaf and hypocotyl explants of 7 days old seedlings on MS agar medium supplemented with 1.0 to $4.0\;mg\;l^{-1}$ of 2,4-dichlorophenoxyacetic acid (2,4-D). Fast growing and well dispersed embryogenic cell suspensions were developed within two months when embryogenic calli were transferred to MS liquid medium containing $1.0\;mg\;l^{-1}\;2,4-D$. One third strength of MS medium was the best for both overall growth and development of somatic embryos in liquid culture. Over 3400 viable somatic embryos were produced from each 150 ml flask with an initial cell density of 30 mg in 30 ml medium. Germinated somatic embryos developed in liquid medium converted into plantlets after transferred to half-strength MS semi-solid medium. Approximately 90% of the converted plantlets were successfully transplanted to soil and grew into fertile plants.

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

• Journal of Plant Biology
• /
• v.37 no.3
• /
• pp.365-370
• /
• 1994

Excised cotyledon segments of ginseng zygotic embryos at various developmental stages were cultured on MS basal medium from which somatic embryos were directly induced. The frequency of somatic embryo formation on the segments declined with the advancing zygotic embryo maturity. All of the cells in the cotyledons of immature zygotic embryos were smaller and more densely cytoplasmic than those in mature embryos. Histological examinations revealed that the poly-somatic embryos formed on immature embryos were of multi-cell originand derived from the epidermal and subepidermal cell layers. However, in the cotyledon of germinating zygotic embryos, only theepidermal cells were densely cytoplasmic and singularly competent to develop into somatic embryos resulting into single embryos at a frequency of 100%.

• BMB Reports
• /
• v.49 no.4
• /
• pp.197-198
• /
• 2016

Although three different research groups have reported successful derivations of human somatic cell nuclear transfer-derived embryonic stem cell (SCNT-ESC) lines using fetal, neonatal and adult fibroblasts, the extremely poor development of cloned embryos has hindered its potential applications in regenerative medicine. Recently, however, our group discovered that the severe methylation of lysine 9 in Histone H3 in a human somatic cell genome was a major SCNT reprogramming barrier, and the overexpression of KDM4A, a H3K9me3 demethylase, significantly improved the blastocyst formation of SCNT embryos. In particular, by applying this new approach, we were able to produce multiple SCNT-ES cell lines using oocytes obtained from donors whose eggs previously failed to develop to the blastocyst stage. Moreover, the success rate was closer to 25%, which is comparable to that of IVF embryos, so that our new human SCNT method seems to be a practical approach to establishing a pluripotent stem cell bank for the general public as well as for individual patients.

• Korean Journal of Plant Tissue Culture
• /
• v.26 no.3
• /
• pp.143-148
• /
• 1999

This study was conducted to elucidate the effects of ascorbic acid and dehydroascorbic acid on somatic embryogenesis from the cultured cells of carrot. Ascorbic acid in culture medium merely stimulated the proliferation of non-embryogenic cells but dehydroascorbic acid in medium induced embryogenic cells from non-embryogenic cells accompanying the inhibition of cell proliferation. Ascorbic acid in medium inhibited somatic embryogenesis from embryogenic cells while dehydroascorbic acid in medium enhanced somatic embryogenesis from the cells as well as non-embryogenic cells. This enhancement was limited to globular embryos and the maturation to cotyledonary embryos was inhibited by dehydroascorbic acid treatment. From the above results it is suggested that carrot callus cultures on medium containing dehydroascorbic acid could quickly induce embryogenic cells. In addition after brief culture of embryogenic cells on development medium containing dehydroascorbic there by acid the subculture of the cells to MS basal medium resulted in the high frequency production of somatic embryos.

• Plant Biotechnology Reports
• /
• v.2 no.1
• /
• pp.87-92
• /
• 2008

An improved protocol for high frequency plant regeneration via somatic embryogenesis from zygotic embryo-derived cell suspension cultures of watershield (Brasenia schreberi) was developed. Zygotic embryos formed pale-yellow globular structures and white friable callus at a frequency of 80% when cultured on halfstrength MS medium supplemented with $0.3mg\;l^{-1}$ 2,4-D. However, the frequency of formation of pale-yellow globular structures and white friable callus decreased slightly with increasing concentrations of 2,4-D up to $3mg\;l^{-1}$, where the frequency reached ~50% of the control. Cell suspension cultures from zygotic embryoderived white friable callus were established using half-strength MS medium supplemented with $0.3mg\;l^{-1}$ 2,4-D. Upon plating of cell aggregates on half-strength MS basal medium, approximately 8.3% gave rise to somatic embryos and developed into plantlets. However, the frequency of plantlet development from cell aggregates was sharply increased (by up to 55%) when activated charcoal and zeatin were applied. Regenerated plantlets were successfully transplanted to potting soil and grown to normal plants in a growth chamber. The distinctive feature of this study is the establishment of a high frequency plant regeneration system via somatic embryogenesis from zygotic embryo-derived cell suspension cultures of water-shield, which has not been previously reported. The protocol for plant regeneration of watershield through somatic embryogenesis could be useful for the mass propagation and transformation of selected elite lines.

• Proceedings of the Korean Society of Developmental Biology Conference
• /
• 2003.10a
• /
• pp.7-8
• /
• 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 Animal Reproduction and Biotechnology
• /
• v.35 no.1
• /
• pp.21-27
• /
• 2020

Somatic cell nuclear transfer derived embryonic stem cells (NT-ESCs) have significant advantages in various fields such as genetics, embryology, stem cell science, and regenerative medicine. However, the poor establishment of NT-ESCs hinders various research. Here, we applied fasudil, a Rho-associated kinase (ROCK) inhibitor, to develop somatic cell nuclear transfer (SCNT) embryos and establish NT-ESCs. In the study, MII oocytes were isolated from female B6D2F1 mice and performed SCNT with mouse embryonic fibroblasts (MEFs). The reconstructed NT-oocytes were activated artificially, and cultured to blastocysts in KSOM supplemented with 10 μM fasudil. Further, the blastocysts were seeded on inactivated MEFs in embryonic stem cell medium supplemented with 10 μM fasudil. A total of 26% of embryos formed into blastocysts in the fasudil treated group, while this ratio was 44% in the fasudil free control group. On the other hand, 30% of blastocysts were established NT-ESCs after exposure of fasudil, which was significantly higher than the control group (10%). The results suggest that fasudil reduced blastocyst development after SCNT due to inhibition of 2 cell cleavage while improved the establishment of NT-ESCs through the anti-apoptotic pathway.

• Journal of Animal Science and Technology
• /
• v.48 no.1
• /
• pp.39-48
• /
• 2006

This study utilized test day of somatic cell score data of dairy cattle from 2000 to 2004. The number of data used were 124,635 of first parity, 134,308 of second parity, 77,862 of third parity, 41,787 of forth parity and 37,412 of fifth parity. The data was analyzed by least square mean method using GLM to estimate the effects of calving year, age, lactation stage, parity and season on somatic cell score. Variance component estimation using test day model was determined by using expectation maximization algorithm- restricted maximum likelihood (EM-REML) analysis method. In each parity, somatic cell score was low for younger group and was relatively high in older groups. Likewise, for lactation stage, the score was low in early-lactation and high in late-lactation in first parity and second parity. Nevertheless, for the third, fourth and fifth parity, however, high somatic cell score was observed in mid-lactation. Generally, the score was high in the peak. Although in fourth and fifth parity, the score was low in late-lactation. Environmental effect of season, somatic cell score was generally low from September to November for all parities. The score was high between June and August when the milk production is usually low. The heritability in each parity were 0.05, 0.09, 0.10, 0.05 and 0.05 for parity 1, 2, 3, 4, 5, respectively. Genetic variance value was estimated to be high in second, third and fifth parity in early-lactation and to be low in first and forth parity.