• Korean Journal of Animal Reproduction
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• v.19 no.1
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• pp.55-63
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• 1995

The putative totipotency germ cells has a relative abundance of alkaline phosphatases. Thus, histological staining of AP activity offers a new route to isolate totipotent cells and also provides insights into culture systems of these cells. Furthermore, the AP staining technique is simple and fast, requires only the napthol AS/MS substrate in combination with trapping diazonium salts such as fast red or fast blue. However, our unexpected finding was that AP staining of mouse ES cells were detected in the undifferentiaed epiblast-derived cells as well as several types of differentiating cells. This findings are different from results of Talbot et al. (1993) reported usefulness of the AP staining and implies that histological staining of AP may not by useful to determine undifferentiaed state or totipotency of ES cells. Thus, we have investigated the patterns of AP expression by RT-PCR in order to identify a marker of undifferentiated ES/primordial germ (PG) cells. In RT-PCR analysis, embryonic (E)-AP was detected only in undifferentiated ES cells, but intestinal(I)-AP was not detected in all of the examined ES and PG cells. In addition, nonspecific (NS)-AP wasdetected in undifferentiated PG cell from day 7, 5 to 13 of gestation. Histological activity of AP in ES cells was completely suppressed by addition of L-phenylalanine (Phe), L-homoarginine (Har), and L-phenylalanylglycylglycine (PheGlyGly) as an inhibitor, but RT-PCR showed the same results as in the absence of an inhibitors. Our findings suggested that expression of E-AP and NS-AP may use as a marker to determine the undifferentiated status in ES and PG cells.

• Journal of Veterinary Clinics
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• v.13 no.2
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• pp.149-152
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• 1996

This study was carried out to evaluate the efficiency of production of cloned embryos by nuclear translatation (NT) when using 4-cell to compact morula stage embryos as nuclear donor. In micromanitulation and electrofusion of blastomeres from 4-cell to morula stage embryos, the successful injection rate was higher with late stage blastomeres, on the contrary the fusion rate was lower. The in vitro developmental rate of NT embryos was not significantly different between cell-stages of donor blastomeres. Although the overall rate of production of cloned embryos with 4-cell. 8-cell, early and late morula stage embryos was 14.0, 18.0, 15.3 and 14.1%, respectively, the mean number of blastocysts produced with a donor embryo was the most (4.51) with the compact morulae. Therefore, it can be suggested that the embryos at thelate stage is more beneficial for the mulciple production of cloned embryos, If the late stage blastomeres have maintained their totipotency to produce intact offspring.

• Korean Journal of Plant Tissue Culture
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• v.27 no.5
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• pp.411-413
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• 2000

The ability of microspores or young pollen grains (male gametophytes) to undergo developmetal switch to embryogenic (sporophytic) pathway exemplifies the concept of totipotency as applied to haploid posmeiotic cells. As a first step pollen is devoid of positional information provided in situ by the intact anther - by isolation and cultivation in vitro in artificial media. This is inevitably accompanied by some degree of stress response in microspore/pollen. It has been shown in both monocots and dicots that intentional stress treatment (mostly starvation or heat shock) greatly stimulates embryo induction rate. Using transgenic sHSP antisense Nicotiana tabacum we show that expression of small heat shock proteins is an integral part of successful embryo and later haploid plant production from pollen grains. Our recently published data show that sHSP chaperone function is optimal in the absence of ATP.

• Asian-Australasian Journal of Animal Sciences
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• v.13 no.2
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• pp.258-264
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• 2000

Production systems for cloned pigs are very important not only for an increase in production of superior animals but also for the production of knockout animals with organs that do not contain antigens for xenotransplantation or to analyze functions of isolated human genes. At present, however, effective systems have not been developed. We have tried to produce cloned pigs by transfering cultured cells into enucleated oocytes and obtained some cloned embryos. To develop a production system for cloned pigs, the basic technologies needed to support such an effort must be improved.

• Korean Journal of Animal Reproduction
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• v.18 no.4
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• pp.299-307
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• 1995

The goal of cell stem cell technology is to produce a viable and genetically normal animal. To achieve this goal various laboratories have followed 2 different pathways beginning with either the culture of 1) single or pooled ICMs grown with or without a feeder layer or 2) single or pooled 16-20 cell stage embryos grown with a feeder layer. Also, thus far embryonic cell cultures or lines have been established by several methods including loose suspension culture for short-term cultures and more commonly murine or bovine fibroblast feeder layers for long-term culture. Pluripotent lines have been derived from 16-cell through blastocyst inner cell mass stages. The efficiency of establishing cell lines and cell proliferation apper to be affected by the number of cells or embryos starting the line. Most attempts to produce offspring from long term STO cell feeder layer cultured ICM or morulae derived ES cells have resulted in pregnancy failure in the first trimester when ES cells were used in cuclear transfer or have failed to retain ES cells in the progeny produced by chimerization. The exception is 1 chimeric fetus from use of morula ES cells in the chimerization with early embryonic cells. There is much to be learned yet about ES cell culture requirements for maintenance of totipotency. If bovine ES cell lines loose imprinting pattern and totipotency with long-term culture and passage as suggested for mouse ES cells, we may be limited to the use of short-term cultures for multiplication of embryos and efficient production of transgenic animals. No bovine ES cell system has yet met all of the criteria indicated for a totipotent ES cell line.

• Korean Journal of Plant Tissue Culture
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• v.27 no.4
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• pp.245-258
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• 2000

Somatic embryogenesis has become a major tool in the study of plant embryology, as it is possible in culture to manipulate cells of many plant species to produce somatic embryos in a process that is remarkably similar to zygotic embryogenesis. Traditionally, the process has been studied by an examination of the ex vitro factors which influence embryo formation. Later structural, physiological and biochemical approaches have been applied. Host recently, molecular tools are being used. Together, these various approaches are giving valuable information on the process. This article gives an overview of somatic embryogenesis by reviewing information on the morphogenesis, physiology, biochemistry and molecular biology of the process. Topics covered include a brief description of the factors involved in the production of embryogenic cells. Carrot cell suspension is most commonly used, and the development of a high frequency and synchronous system is outlined. At the physiological and biochemical lev-els various topics, including the reactivation of the cell cycle, changes in endogenous growth regulators, amino acid, polyamine, DNA, RNA and protein metabolism, and embryogenic factors in conditioned medium are all discussed. Lastly, recent information on genes and molecular markers of the embryogenic process are outlined. Somatic embryogenesis, the best example of totipotency in plant cells, is not only an important tool in studies in basic biology, but is potentially of equal significance in the micropropagation of economically important plants.

• Development and Reproduction
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• v.7 no.2
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• pp.61-68
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• 2003

Zygote genome should entail a complex process of epigenetic reprogramming including a global DNA demethylation to reach a totipotency or pluripotency during early mammalian development. In this study, we have analyzed methylation patterns in cloned bovine embryos to monitor the epigenetic reprogramming process of donor genomic DNA. Aberrant DNA methylation patterns were observed in various genomic regions of cloned embryos except single-copy gene sequences. The overall genomic methylation status of cloned embryos was quite different from that of normal embryos produced in viかo or in vivo. Abnormal methylation profiles were also specifically represented in trophectoderm cells of cloned embryos, which probably result in widespread gene dysregulation in extraembryonic region or placental dysfunction familiar to cloned animals. Our findings suggest that developmental failures of cloned embryos are due to incomplete epigenetic reprogramming of donor genomic DNA. Understanding the epigenetic reprogramming processes of donor genome will clearly define the faulty development of cloned embryos.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.8
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• pp.1102-1107
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• 2003

The ES cell can provide a useful system for studying differentiation and development in vitro and a powerful tool for producing transgenic animalds. To investigate the culture condition of chicken embryonic stem (CES) cells which can retain their multipotentiality or totipotency, three kinds of feeder layer cells, SNL cells, primary mice embryonic fibroblasts (PMEF) cells and primary chicken embryonic fibroblasts (PCEF) cells, were used as the feeder cells in media of DMEM supplemented with leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and stem cell factor (SCF) for co-culture with blastoderm cells from stage X embryos of chicken. The alkaline phosphatase (AKP) test, differentiation experiment in vitro and chimeric chicken production were carried out. The results showed that culture on feeder layer of PMEF yielded high quality CES cell colonies. The typical CES cells clone shape revealed as follows: nested aggregation (clone) with clear edge and round surface as well as close arrangement within the clone. Strong alkaline phosphatase (AKP) reactive cells were observed in the fourth passage cells. On the other hand, the fourth passage CES cells could differentiate into various cells in the absence of feeder layer cells and LIF in vitro. The third and fourth passage cells were injected into the subgerminal cavity of recipient embryos at stage X. Of 269 Hailan embryos injected with CES cells of Shouguang Chickens, 8.2% (22/269) survived to hatching, 5 feather chimeras had been produced. This suggests that an effective culture system established in this study can promote the growth of CES cells and maintain them in the state of undifferentiated and development, which lays a solid foundation for the application of CES cells and may provide an alternative tool for genetic modification of chickens.

• Journal of Plant Biology
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• v.38 no.1
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• pp.55-62
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• 1995

Embryogenic cell (EC) suspension cultures derived from mature seed-embryo of rice (Oryza sativa L cv. Kye Hwa) were used for the expression patterns of isozyme and enzyme activity. EC suspension cultures were composed of cells that were densely cytoplasmic, potentially embryogenic. However, nonembryogenic cell (NEC) cultures were composed of large, elongated and vacuolated cells. These cells were analyzed for the isozyme pattern and enzyme activity of EC and NEC. Isozyme patterns of peroxidase, esterase, acid phosphatase and malate dehydrogenase exhibited striking difference in the total number of bands, specificity and intensity of band. Also, these isozymes showed very high activity in the EC. Specific band, band activity and higher enzyme activity of isozyme in EC was absent or low in NEC, which may indicate an association of these specific isozymes with morphological characterization and totipotency of embryogenic cells. These results indicate that specific pattern and activity of enzyme in EC could probably be used as a biochemical marker of EC in rice.n rice.

• Korean Journal of Plant Tissue Culture
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• v.24 no.3
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• pp.167-173
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• 1997

The embryogenic callus (EC), from which somatic embryos could be induced, was compared with nonembryogenic callus(NE) to study the origin and features of totipotent cell in water dropwort (Oenanthe stolonifera DC). To induce and maintain of EC and the NE, meristematic stem and immature floret were inoculated in MS media supplemented with 1 mg/L 2,4-D, and with 2.5 mg/L NAA and 5mg/L BA, respectively, The EC was not induced from the NE even after subculturing in MS medium supplemented with 1 mg/L 2,4-D. Plantlets were not regenerated from the NE in hormone-free medium. In histochemical comparison of the EC with the NE by light microscopy, the EC had smaller cells in size, dense cytoplasm, and more starch granules of cells compared to the NE cells. The cell from the EC, as observed by transmission electron microscopy, had smaller vaculoes, well developed ribosomes, mitochondria, and endoplasmic reticulum, whereas the cells from the NE had larger vacuoles and underdeveloped organelles. In protein pattern from NE, EC and Somatic embryo (SE), as analyzed by SDS polyacrylamide gel electrophoresis, different proteins specific for tissue were observed: 17 and 28 KD for NE, 50, 52, 57, 66, 68 KD for EC and 20 KD for SE. DNA polymorphism was also observed between EC and NE as analyzed by RAPD (randomly amplified polymorphic DNA) method. The origin of totipotent stem cell and the relationship between irreversible genomic change arose in differentiation and the loss of totipotency in plant were discussed.