• Korean Journal of Animal Reproduction
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• v.27 no.3
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• pp.197-206
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• 2003

hFSH (human follicle stimulating hormone) is heterodimer consisting of $\alpha$ and $\beta$ subunits. Since assembly of the both subunits in the cell is often the rate-limiting step in production of functional hormone, single-chain hormones have been engineered by genetically linking two different cDNA fragments with a linker sequence. Using retrovirus vector system, the resulting recombinant hFSH gene was transferred in CHO cells and chicken embryos, and the expression of the gene was investigated. In CHO cells, protein synthesis from the single-chain FSH gene was 17 fold higher than that from the heterodimeric counterpart. In the study of transgenic chickens, ten of the eleven chicks hatched from 62 embryos manupulated with recombinant retrovirus stock was determined to carry transgenic genes. RT-PCR analyses confirmed transcription of the single-chain FSH gene, however, no recombinant FSH was detected from the blood samples.

• Korean Journal of Poultry Science
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• v.28 no.2
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• pp.125-133
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• 2001

The goal of this paper was to examine the qualify zygote-acquiring method for in-vitro culture and the in-vitro culture method of the acquired zygote from a technological perspective. We have reported the results on the introduction of foreign DNAs using the described culturing method. After performing in-vitro and surrogate eggshell culture on a zygote acquired from the abdomen of a hen, 25.8% hatchability was acquired. After microinjecting foreign DNAs into the acquired zygote and performing in-vitro and surrogate eggshell culture using the same method, 13.1∼11.7% hatchability was acquired. Having compared the developments of the control subjects and the experimental subjects, the viability of the experimental subjects on the 4∼5th day of culturing was much lower compared to that of the control subjects. This is a result that shows that the microinjection process of foreign DNAs might have a negative effect on the existence of the embryo; therefore, various technical attempts should be made to minimize such negative effects. Having microinjected foreign DNAs into the zygote of a hen to produce transgenic chickens, 3 transgenic founders were Produced and 70 G1 progeny were produced as a result of the progeny test that had been performed to the present.

• 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.

• Development and Reproduction
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• v.11 no.3
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• pp.219-226
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• 2007

This study was conducted to identify optimistic primordial germ cells'(PGCs) migration activity using heat activated busulfan treatment for the increasing germline chimerism. Donar PGCs viability tests of important conditions for useful germ line chimerism indicated approximately $70{\sim}80%$ viability was time dependent. Transplantation experiments of PGCs into recipient embryos after busulfun treatment, showed the treatment group having 23.5% viability. By comparison, the control group showed 4.8% viability. The 96 hour treatment group and the 118 hour treatment group of the cultured PGCs showed high migration activity. Generally, the transplantation method would consider morphological and physiological characteristics before transplantation. In the present study, the effect of busulfan on migration activity showed viability highest at 53.4% after 48-hour incubation time. However, a previous study showed the best condition for transplantation time to be prior to the 48-hour incubation period, when the chicken embryo does not yet have a developed blood vessel system. In conclusion, an important condition for the production of a transgenic chicken is that most donor PGCs migrate into the recipient embryo without any inhibitory factors. The present results suggest, perhaps by using this modified method of transplantation, it can produce a more efficient chimeric germ line, transgenic chicken.

• Korean Journal of Poultry Science
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• v.35 no.3
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• pp.241-245
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• 2008

Lentiviral vector system is efficient vehicles for the delivery of exogenous genes, and it is generally used in the generation of transgenic chickens. In this study, we used recombinant lentiviral vectors to generate transgenic chicks that express the human superoxide dismutase-3 gene driven by the chicken ovalbumin promoter. It is well known that superoxide dismutases(SODs) are believed to play a crucial role in protecting cells against oxygen toxicity. There are three forms of SOD proteins: cytosolic Cu-Zn SOD, mitochondrial Mn SOD, and extracellular SOD(SOD-3). The recombinant lentivirus containing the human SOD-3 gene was injected into the subgerminal cavity of freshly laid eggs. Subsequently, the embryos were incubated to hatch using phases II and III of the surrogate shell ex vivo culture system. From 341 injected embryos, the 78 chicks hatched after 21 days incubation. The hatched chicks were screened for the human SOD-3 gene by using PCR. Two of 47 male chickens that survived to sexual maturity contained the human SOD-3 gene in their semen. These results showed that our transgenic chicken generation system was completely established.

• Korean Journal of Poultry Science
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• v.27 no.1
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• pp.73-78
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• 2000

Embryonic stem (ES) cells are pluripotent cell lines, which derived from preimplantation embryo. These cells have been used as a vehicle of foreign DNA for production of transgenic mammals. this experiment was performed to examined the possible use of blastodermal cells derived from hen's egg for germline manipulation. Stage X blsdtodermal cells isolated from fertilized eggs were cultured in DMEM containing 15% fetal calf serum. Blastodermal cells wre co-cultured on the chicken embryonic fibroblast (CEF) or mouse embryonic fibroblast(MEF) cells. to examine the effects of growth factors on stem cell growth, bFGF and LIF were added. There was no significant difference in colony formation of putative ES cells between CEF and MEF as a feederlayer, but the addition of growth factors enhanced the proliferation and inhibited differentiation of blastodermal cells. To characterize the cell colonies as a putative ES cells, putative embryonic cell colonies were stained by periodic acid Schiffs (PAS) reagent. The putative ES cell colonies showed intensive positive reaction similar to the property of undifferentiated PGC upto 20days in vitro, but not in other cell types. this result demonstrates that PAS-positive cell colonies may be used for the study of establishment of chicken ES cell lines for the production of transgenic chicken.

• Proceedings of the Korea Society of Poultry Science Conference
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• 2001.11a
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• pp.40-46
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• 2001

The use of pluripotent stem cells has tremendous advantages for various purposes but these cell lines with proven germ-line transmission have been completely established only in the mouse. Embryonic germ (EG) cell lines are also pluripotent and undifferentiated stem cells established from primordial germ cells (PGCs). This study was conducted to establish and characterize the chicken EG cells derived from gonadal primordial germ cells. We isolated gonadal PGCs from 5.5-day-old (stage 28) White leghorn (WL) embryos and established chicken EG cells lines with EG culture medium supplemented with human stem cell factor (hSCF), murine leukemia inhibitory factor (mLIF), bovine basic fibroblast growth factor (bFGF), human interleukin-11 (hIL-11), and human insulin-like growth factor-I (hIGF-I). These cells grew continuously for 4 months (10 passages) on a feeder layer of mitotically active chicken embryonic fibroblasts. These cells were characterized by screening with the Periodic acid-Shiff＇s reaction, anti-SSEA-1 antibody, and a proliferation assay after several passages. As the results, the chicken EG cells maintained characteristics of undifferentiated stem cells as well as that of gonadal PGCs. When cultured in suspension, the chicken EG cells successfully formed an embryoid body and differentiated into a variety of cell types when re-seeded onto culture dish. The chicken EG cells were injected into blastodermal layer at stage X and dorsal aorta of recipient embryo at stage 14 (incubation of 53hrs) and produced chimeric chickens with various differentiated tissues derived from the EG cells. The germline chimeras were also successfully induced by using EG cells. Thus, Chicken EG cells will be useful for the production of transgenic chickena and for studies of germ cell differentiation and genomic imprinting.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.8
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• pp.1278-1284
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• 2007

Changes in the concentrations of biogenic amines (BAs) in fresh beef, pork, and chicken breast and leg were investigated during storage, and the relationship between the content of volatile basic nitrogen (VBN) and BAs was evaluated. As the storage period increased, the levels of putrescine (PUT), cadaverine (CAD) and tyramine (TYM) increased in all the meat samples, except for TYM in beef (p<0.001). The level of BAs in beef, pork and chicken changed but the extent of these changes was different among the kinds of BAs and meats. Measurement of the VBN content was confirmed as a good index for interpreting the specific BAs content in general, such as PUT, CAD, and TYM, as well as evaluating a meat's freshness during storage. However, the kinds of BAs which can be predicted from the VBN content varied in different meats (p<0.05).

• Korean Journal of Poultry Science
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• v.31 no.4
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• pp.293-298
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• 2004

Microinjection of DNA is a general method for generating transgenic animals, but the rate of transgenesis in chickens is very low. So it was carried out to investigate the efficiency of liposome-mediated gene transfer in stage one cell of chicken embryo with GFP expression vector. In order to determine efficiency and duration of the introduced foreign gene, it was microinjected DNA with liposome or naked DNA into the germinal disc of stage one cell or stage-X chicken embryo. Analysis of reporter gene expression in day-4 embryos showed that GFP expression was observed only in the liposome-mediate embryo groups and detectable up to day-8 embryos. The results suggest that stable integration of the introduced gene using liposome is a rare event. Nevertheless the liposome-mediated gene transfer may be a useful method to transfer a foreign gene into the stage one cell of chicken embryos.

• Molecules and Cells
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• v.19 no.2
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• pp.185-190
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• 2005

The chicken oviduct is a dynamic organ that produces secretory proteins such as ovalbumin and its cells undergo cell proliferation and differentiation. There has been no study of the cellular mechanism involved in cell death in the chicken oviduct. Therefore, this study has focused on the study of apoptosis in primary oviduct cells. Because ceramide is known to activate apoptosis in tumor cells and is produced in the oviduct, we used an exogenous ceramide analog to induce cell death. The viability of ceramide-treated chicken oviduct cells decreased in a dose-dependent manner and apoptotic cells were detected by staining with annexin V. The expression of apoptosis-related genes was assessed by RT-PCR and bcl-2 mRNA was found to decrease after exposure to ceramide while Bcl-x mRNA increased 12 h post-treatment. In addition, caspase-3 was expressed strongly in the early stages of apoptosis, while caspase-1 and -9 transcripts increased at later times. We conclude that ceramide induces apoptosis in oviduct-derived primary cells via a caspase- and bcl-2-dependent pathway.