• BMB Reports
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• v.43 no.6
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• pp.438-444
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• 2010

Dnd (dead end) gene encodes an RNA binding protein and is specifically expressed in primordial germ cells (PGCs) as a vertebrate-specific component of the germ plasma throughout embryogenesis. By utilizing a technique of specific nucleic acids associated with proteins (SNAAP), 13 potential target mRNAs of zebrafish Dnd (ZDnd) protein were identified from 8-cell embryo, and 8 target mRNAs have been confirmed using an RT-PCR analysis. Of the target mRNAs, the present study is focused on the regulation of geminin, which is an inhibitor of DNA replication. Using electrophoretic mobility shift assay (EMSA), we demonstrated that ZDND protein bound the 67-nucleotide region from 864 to 931 in the 3'UTR of geminin mRNA, a sequence containing 60.29% of uridine. Results from a dual-luciferase assay in HEK293 cells showed that ZDND increases the translation of geminin. Taken together, the identification of target mRNA for ZDnd will be helpful to further explore the biological function of Dnd in zebrafish germ-line development as well as in cancer cells.

• Proceedings of the Korea Society of Poultry Science Conference
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• 2001.11a
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• pp.71-73
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• 2001

In this study, we could improve transmission efficiency of germline chimeras by transfer of gonadal PGCs (gPGCs) cultured in vitro. Of hatched recipient chicks, 301 chickens (141 males and 160 females) were brought up to sexual maturity and these WLs (KOC) were mated with KOCs for testcross, resulting in 27 germline chimeras (15 males and 12 females) identified by black feather color of their progenies. The production efficiency of germline chimera production of experimental groups was observed (P=0.6831). The average transmission efficiency of proven germline chimeras was 0.6 ∼56.5% (15.0% on average). The transmission efficiency of experimental group which were transferred 10-days cultured gPGCs without Ficoll treatment was highest (49.7%) and that of experimental stock which transferred non-cultured gPGCs with Ficoll treatment was lowest (0.6%). The duration of in vitro culture before transferring was significantly important for the high efficiency of germline transmission. Transferring 10-days cultured gPGCs made the transmission efficiency higher rather than transferring non-cultured and 5-days cultured gPGCs, 50 times and 10 times, respectively (p<0.0001). However, Ficoll treatment for increasing the population ratio of gPGCs negatively affected the transmission efficiency and the effects of sexuality and the reciprocal interaction between treatments showed no significant differences. These findings demonstrated that the crucial factors for improving the germline transmission were the duration of in vitro culture prior to transfer. Thus, we developed the complete system for production of germline chimera using cultured gPGCs with highly improved efficiency and this system would be useful for genetic manipulation and obtaining the transgenic aves.

• Proceedings of the Korea Society of Poultry Science Conference
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• 2005.11a
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• pp.66-67
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• 2005

The domestic chicken (Gallus gallus) is an important model for research in developmental biology because its embryonic development occurs in ovo. To examine the mechanism of embryonic germ cell development, we constructed proteome map of gonadal primordial germ cells (gPGC) from chicken embryonic gonads. Embryonic gonads were collected from 500 embryos at 6 day of incubation, and the gPGC were cultured in vitro until colony formed. After 7-10 days in cultured gPGC colonies were separated from gonadal stroma cells (GSCs). Soluble extracts of cultured gPGCs were then fractionated by two-dimensional gel electrophoresis (pH 4-7). A number of protein spots, including those that displayed significant expression levels, were then identified by use of matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and LC-MS/MS. Of the 89 gPGC spots examined, 50 yielded mass spectra that matched avian proteins found in on-line databases. Proteome map of thistype will serve as an important reference for germ cell biology and transgenic research.

• Molecules and Cells
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• v.38 no.9
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• pp.743-749
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• 2015

Production of genome-edited animals using germline-competent cells and genetic modification tools has provided opportunities for investigation of biological mechanisms in various organisms. The recently reported programmed genome editing technology that can induce gene modification at a target locus in an efficient and precise manner facilitates establishment of animal models. In this regard, the demand for genome-edited avian species, which are some of the most suitable model animals due to their unique embryonic development, has also increased. Furthermore, germline chimera production through longterm culture of chicken primordial germ cells (PGCs) has facilitated research on production of genome-edited chickens. Thus, use of avian germline modification is promising for development of novel avian models for research of disease control and various biological mechanisms. Here, we discuss recent progress in genome modification technology in avian species and its applications and future strategies.

• Development and Reproduction
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• v.2 no.1
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• pp.69-74
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• 1998

The early gonadal development and sexual differentiation of Rhynchocypris oxycephalus are described from the stage of hatching to 150 days after hatching. During this peroid, the average length of the body grew from 0.64 cm to 5.96 cm. the primordial germ cells (PGCs), which could be recognized at the time of hatching, began to protrude into peritoneal cavity at a standard total length of 1.91 cm. At a standard total length of 2.29cm, initial ovarian differentiation wasidentified by the transformation of PGCs to meiotic oocytes. Finally, at the standard total length of 5.96 cm, the female gonads gradually developed towards migratory nucleus oocytes, characterisiing the maturation. Oocytes proliferated rapidly after sex differentiation while the testis entered a period of quiescence, as they continued to multiply but did not undergo growth until the standard total length of 4.00 cm. At a standard total length of 4.00 cm, spermatocytes arrested in thephase of interkinesis, Sertoli-like cells and sperm duct formation, with signs of meiotic activity, were observed. Therefore it may be concluded that R. oxycephalus belongs to the differentiated type of gonochoristic teleosts.

• Development and Reproduction
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• v.20 no.4
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• pp.289-296
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• 2016

This report describes the sex differentiation of the Korean rose bitterling, Rhodeus uyekii, from hatching to 170 days post-hatch (DPH) in relation to total length (TL), body weight (BW), and integral water temperature (IWT). The growth curve of TL from just hatching to 83 DPH was $5.144e^{0.045t}$ ($R^2=0.961$; t, time), and that of BW was $2.398e^{0.086t}$ ($R^2=0.725$). Primordial germ cells (PGCs) were observed at 17 DPH (7.9 mm TL, 3.74 mg BW, $374^{\circ}C$ IWT), and thereafter began to protrude into the peritoneal cavity. At 21 DPH ($9.2{\pm}0.14mm$ TL, $4.8{\pm}0.07mg$ BW, $462^{\circ}C$ IWT), some PGCs contained condensed chromatin and oocyte were observed in meiotic prophase. In contrast to the ovaries, which grew gradually after sexual differentiation, testes began multiplying at 25 DPH (10.1 mm TL, 5.42 mg BW, $550^{\circ}C$ IWT), when testicular differentiation was first identified, and multiplied continuously thereafter. At 33 DPH (11.2 mm TL, 10.5 mg BW, $726^{\circ}C$ IWT), the developing testes contained spermatogonia that exhibited mitotic activity. No spermatocyte or sperm cell was observed until 83 DPH (18.9 TL, 48.2 mg BW, $1,826^{\circ}C$ IWT). At 170 DPH (32.5 mm TL, 270.1 mg BW, $3,740^{\circ}C$ IWT), which was the end point of this study, the mature ovaries showed germinal vesicle breakdown, while the mature testes contained observable spermatocytes and sperm cells. These results allow us to identify the sex differentiation type of the Korean rose bitterling as differentiated gonochoristic.

• Korean Journal of Poultry Science
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• v.23 no.1
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• pp.9-17
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• 1996

This study was conducted to compare the survival rate of chick embryos among different eggshell window positions and to search for the most appropriate injection position. The eggshells were punctured at blunt-end, sharp-end and side-up with a sterilized fine forceps, respectively. The survival rate of sharp-end window was higher than the other window positions. Injection of Dulbecco’s modified eagle’s medium (DMEM) through blunt-end window (BE1) was impossible because inner cell membrane was obscure. The 2 ${\mu}$L DMEM was injected into 2.5 d-old embryo blood vessel through sharp end window. To prevent hemorrhages at the point of injection, the air bubbles were injected into the embryo blood vessel. The survival rate of chicks embryo in sharp end window was about 17.0％. Therefore, this sharp-end window system will be helpful for the production of germline chimera or transgenic chicken using primordial germ cells ( PGCs ).

• Korean Journal of Ichthyology
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• v.19 no.2
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• pp.107-111
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• 2007

Sex differentiation of the brown croaker Miichthys miiuy (Basilewsky) is described from hatching to the 120th day post-hatching (dph) (water temperature $24^{\circ}C$). Primordial germ cells (PGCs) were observed on the 20th dph (10.4 mm total length (TL), 0.14 g body weight (BW), and began to protrude into the peritoneal cavity from the 40th dph (19.4 mm TL, 0.39 g BW). On the 65th dph (31.3 mm TL, 0.93 g BW, $1,560D^{\circ}$ (degree-days)), initial ovarian differentiation was identified by the PGCs with condensed chromatin, and their transformation into meiotic oocytes. By the 120th dph (4.60 mm TL, 1.38 g BW, $2,880D^{\circ}$), the oocytes were in the perinucleolus stage and had increased from 20 to $40{\mu}m$ in diameter. While ovaries gradually grew after sex was differentiated, testes continued to multiply from the 65th dph. On the 80th dph (37.9 mm TL, 1.39 g BW, $1,920D^{\circ}$), the beginning of testis lobule formation was indicated by the occurrence of spermatogonial cysts enveloped by somatic cells in some of the testes. On the 120th dph, the testis lobules of some of the fish contained all germ cell stages through to the spermatocytes. Therefore, the sex differentiation type of the brown croaker is identified as gonochoristic.