• Asian-Australasian Journal of Animal Sciences
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• v.16 no.6
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• pp.800-805
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• 2003

The early embryos are obtained in different time after the former egg had been laid, and the aim of the present study was to observe the development law of chicken early embryo.The embryo development has been divided into the two periods according to morphology of blastodisc. Cleavage period, from 5.5 h (0 h uterine age) to 15.5 h (10-10.5 h uterine age) after the former egg had laid, formation blastodisc of 6-7 layers cell. Later blastocyst period, from 17.5 h (12-12.5 h uterine age) to area pellucida formation after the former egg had been laid. The first division took place at 5 h (0 h uterine age), morular at 11.5 h (6-6.5 h uterine age), and blastocyst at 15.5 h (10-10.5 h uterine age) after the former egg had been laid.

• Asian-Australasian Journal of Animal Sciences
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• v.8 no.6
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• pp.557-562
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• 1995

In this study, characteristics of chick primordial germ cells (PGCs), which is the founder cell of the germline, and gonadal development of the chick embryo between 12hrs and 6 day of incubation were investigated by transverse serial sections of chick embryos under the light microscopic observation. In embryo stage 20 (3 day of incubation), there are a lot of PGCs at the mesenchym, which were moving to the thickened epithelium (gonadal ridge). The PGCs arrive at both right and left gonad primordial in equal number prior to stage 24 (4 day of incubation), but in the following stages, the distribution of the PGCs became asymmetrical. More PGCs colonized the left than the right gonad, but the reason for the unequal distribution of PGCs is uncertain. The PGCs have mostly settled in the gonadal ridge (GR) at 6 day embryo. This study was conducted to investigate characteristics of the PGC migration and gonadal formation and observe the best condition for PGC isolation, culture and to attempt the possibility of the production for transgenic germline chimeras with manipulated PGCs.

• Journal of Microbiology and Biotechnology
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• v.24 no.7
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• pp.921-924
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• 2014

C31G is a potent antimicrobial agent and can disrupt the microbial membrane by the alkyl portion of the molecule. The objective of this study was to evaluate the virucidal effectiveness of C31G and mouthrinse containing C31G (Sense-Time) on seasonal influenza viruses. Evaluation of the virucidal activity against influenza viruses was performed with end-point titration in 10-day-old chicken embryos and Madin-Darby canine kidney cells. In vitro studies demonstrated that C31G and Sense-Time inhibited the growth of seasonal influenza viruses even in the presence of 5% organic material. Gargling with C31G or Sense-Time would enhance oropharyngeal hygiene, which would be helpful for reducing influenza transmission.

• Journal of Animal Science and Technology
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• v.55 no.5
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• pp.417-425
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• 2013

We sought to provide a method for freezing and preserving primordial germ cells, or an avian germ cell of a bird, as a material for developmental engineering or species preservation. The aim of this study was to compare the efficacy of slow freezing with a vitrification method for the cryopreservation of chicken primordial germ cells (PGCs). PGCs obtained from the germinal gonad of day 5.5-6 day (stage 28) cultured chick embryos, using the MACS method, were classified into two groups: slow freezing and vitrification. We examined the viability of PGCs after Cryopreservation. Four freezing methods were compared with each other, including the following: Method 1: The PGCs were frozen by a programmed freezer in a plastic straw, including 2.0 M ethylene glycol (EG) as cryoprotective additive (slow freezing) Method 2: The PGCs were vitrified in a plastic straw, including 8.0 M EG, plus 7% polyvinylpyrrolidone (PVP) (rapid freezing). Method 3: The slow freezing was induced with a cryotube including 2.0 M EG Method 4: The PGCs were frozen in a cryotube including 10% dimethyl suloxide (DMSO) (rapid freezing). After freezing and thawing, survival rates of the frozen-thawed PGCs from Method 1 to 4were 76.4%, 70.6%, 80.5% and 78.1% (p<0.05), respectively. The slow freezing ($-80^{\circ}C$ programmed freezer) method may provide better survival rates of frozen-thawed PGCs than the vitrification method for the cryopreservation of PGCs. Therefore, these systems may contribute to the cryopreservation of a rare avian species.

• Korean Journal of Veterinary Research
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• v.41 no.2
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• pp.133-138
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• 2001

The distributions and relative frequencies of the serotonin-immunoreactive cells were studied in dorsal, ventral, third and splenic lobes of developing chicken pancreas during embryonic periods (10 days of incubation to hatching) by immunohistochemical methods. The regions of pancreas were subdivided into three regions, exocrine, light and dark islets. Round and/or oval shaped serotonin-immunoreactive cells were detected in all four lobes. According to developmental stages, the types of lobes and the regions of pancreas, these immunoreactive cells were showed various distributions and relative frequencies. In exocrine portions, serotonin-immunoreactive cells were found in the splenic lobes at 13-14 days of incubation, in the third lobes from 10 days to 19 days of incubation, in the ventral lobes from 10 days of incubation to hatching and in the dorsal lobes from 11 days of incubation to hatching. In pancreatic islets, these cells were detected only in the dark islets of splenic lobes at 15 and 16 day of incubation with rare frequency. In conclusion, serotonin-immunoreactive cells decreased with developmental stages in all four lobes and their relative frequencies decreased with developmental stages.

• Proceedings of the Korea Society of Poultry Science Conference
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• 2001.11a
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• pp.74-76
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• 2001

Comparing to mammals, male bird has the homozygote ZZ and female has the heterozygote n. Therefore, the sex of fertilized eggs is defined by female chromosome constitution. Although this cytological observation had been established, the molecular and cellular mechanism of germ cell differentiation are essentially unknown in aves. Especially, the differentiation of germ cells in mixed-sex chimeras has not yet been clearly elucidated. Primordial germ cells, which are the progenitors of sperm or egg after sexual maturity, firstly arise in the epiblast and migrate to embryonic gonads through the blood vessel. During the embryo development, these PGCs differentiate in the pathway of mate or female, respectively and develop the sperm or egg cells after sexual maturity. In this paper, we confirmed that the female PGCs could migrate into the recipient male gonads after transferring and differentiate into germ cells in the embryonic stages. The primordial germ cells were isolated from the female embryonic gonads of 5.5-day-old incubation and re-injected into the male recipient embryos of 2-day-old incubation, which produced mixed-sex chimera in the germline. The finding in this study demonstrated the ability of migration and differentiation of gonadal primordial germ cells in mixed-sex chicken.

• Reproductive and Developmental Biology
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• v.32 no.3
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• pp.159-166
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• 2008

We report here the generation of transgenic chickens that produce human Thrombopoietin (hTPO) using replication-defective Moloney murine leukemia virus (MoMLV)-based vectors packaged with vesicular stomatitis virus G glycoprotein (VSV-G). For the retrovirus vectors, we used hCMV (human Cytomegalovirus) internal promoter to drive the hTPO gene. After confirming the expression of the hTPO gene in various target cells, the concentrated solution of recombinant retrovirus was injected beneath the blastoderm of non-incubated chicken embryos (stage X). The biological activity of the recombinant hTPO in target cell was significantly higher than its commercially available counterpart. Out of 132 injected eggs, 11 chicks hatched after 21 days of incubation and 4 hatched chicks were found to express vector-encoded hTPO gene. However, 3 out of the 4 transgenics died within one month of hatching. The major significance of this study is that it is one of the very few successful reports on the production of transgenic chickens as bioreactors aiming mass production of commercially valuable and biological active human cytokine proteins.

• Korean Journal of Poultry Science
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• v.36 no.2
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• pp.177-186
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• 2009

It is well-known that unregulated over-expression of foreign gene may have unwanted physiological or toxic effects in transgenic animals. To circumvent these problems, we constructed retrovirus vector designed to express the foreign gene under the control of the tetracycline-inducible promoter. However, gene expressions in the tetracycline-inducible expression system (Tet system) are not completely regulated but a little leaky due to the inherent defects in conventional Tet-based systems. A more tightly controllable regulatory system can be achieved when the advanced versions ($rtTA2^SM2$) of rtTA and a minimal promoter in responsive components (pTRE-tight) are used in combination therein. In this study, we tried to produce human thrombopoietin (hTPO) from various target cells and transgenic chickens using the retrovirus vector combined with Tet system. hTPO is the primary regulator of platelet production and has an important role in the survival and expansion of hematopoietic stem cells. In a preliminary experiment in vitro, higher hTPO expression and tighter expression control were observed in chicken embryonic fibroblast (CEF) cells. We also measured the biological activity of the hTPO using Mo7e cells whose proliferation is dependant on hTPO. The biological activity of the recombinant hTPO from CEF was higher than both its commercial counterpart and hTPO from other target cells. The recombinant retrovirus was injected beneath the blastoderm of non-incubated chicken embryos (stage X). Out of 138 injected eggs, 15 chicks hatched after 21 days of incubation. Among them, 8 hatched chicks were hTPO positive. When the Go transgenic chicken was fed doxycycline (0.5 mg per 1 gram of feed), a tetracycline derivative, hTPO concentration of the transgenic chicken blood was 200 ng/mL. Germline transmission of the transgene was confirmed in sperm of the Go transgenic roosters. These results are informative to establish transgenic chickens as bioreactors for the mass production of commercially valuable and biological active human cytokine proteins.

• Korean Journal of Poultry Science
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• v.41 no.4
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• pp.261-270
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• 2014

This study was conducted to establish the method for preserving chicken primordial germ cells (PGCs) that enables long-term storage in liquid nitrogen ($LN_2$) for developmental engineering or preservation of species. The purpose of this study is to clarify the effects of simple freeze-thaw treatment on viability of PGCs in chickens and to the optimal protocol for PGCs freezing. PGCs obtained from the germinal gonade of an early embryos of 5.5~6 day (stage 28) of Isa Brown, Korean Ogye (KO), White Leghorn and Commercial breeds, using the MACS method were suspended in a freezing medium containing a freezing and protecting agents (e.g. dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PG)). The gonadal cells, including PGCs, were then frozen in 1 of the following cryoprotectant treatments : 2.5%, 5%, 10%, 15%, and 0% cryoprotectant (DMSO, EG, PG) as a control. Effects of exposure to simple freezing, with different concentrations of the cryoprotectant solution, were examined. After simple freezing, the viability of PGCs after freeze-thawing was significantly higher for Commercial breeds ($88.7{\pm}2.4%$) than KO ($85.1{\pm}0.4%$), Isa Brown ($84.6{\pm}0.2%$) and White Leghorn ($85.9{\pm}0.1%$) (p<0.05) using 10% EG cryoprotectant. Therefore, these systems may contribute in the improvement of cryopreservation for a scarce species in birds preservation. This study established a method for preserving chicken PGCs that enables systematic storage and labeling of cryopreserved PGCs in liquid ($LN_2$) at a germplasm repository and ease of entry into a database.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.2
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• pp.158-164
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• 2005

A practical approach was proposed to produce transgenic chimeric chickens using blastodermal cells (BCs). The chicken BCs were mechanically dissociated and transferred into the recipient eggs that had been exposed to 500 rads irradiation of$^{60}Co$ and windowed on the equatorial plane. Chimeric chickens were generated using two models: the crosses (MXL) from Black Minors (ii,EE,b/b) ♂${\times}$Barred Leghorns (ii,ee,B/-) ♀ as donors and White Leghorns (WL, II) as acceptors (Model 1), or the Black Heifengs (BH, ii,EE,bb) as donors and Hua-xing white (HW, II) as recipients (Model 2). The treated eggs were incubated in their original shells in normal conditions until hatching. Green fluorescent protein (GFP) gene was transferred into the BCs derived from MXL and BH via lipofectamine and the pEGFP-C1, and transfection efficiency into the BCs was examined under a fluorescent microscope. Potential transgenic chimeras were selected based on the proposed methods in this study. Using the fresh BCs, the best rate of phenotypic chimeras was 6.7% and 26.0% in model-1 groups, and model-2 groups, respectively. We also described the optimized conditions for transfection. Although 30% of the BCs transfected in vitro emitted green light under an inverted fluorescent microscope, no embryos injected with the transfected BCs expressed foreign GFP gene at 3-4 days.