• Journal of Life Science
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• v.21 no.12
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• pp.1726-1731
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• 2011

Human Stem Cell Factor (hSCF) is a cytokine that binds to the c-Kit receptor and plays an important role in hematopoiesis, spermatogenesis, and melanogenesis. To produce the human Stem Cell Factor (hSCF) recombinant protein, we constructed a germline transgenic silkworm using the piggyback vector. The expression of the hSCF gene was driven by the Drosophila heat shock protein 70 (dHsp70) promoter. 3XP3 promotor-driven EGFP was used as a marker which allowed us to rapidly distinguish the transgenic silkworm. A mixture of the donor and helper vector was micro-injected into 1,020 eggs of bivoltin silkworms, Keomokjam. We obtained approximately 22 G1 broods that were EGFP-positive. The expression of the hSCF gene in the transgenic silkworm was analyzed by SDS-PAGE and immunoblotting. Also, analysis of insertion sites into the silkworm genome using inverse PCR showed that exogenous DNA was inserted into the transgenic silkworm genome. These results show that successfully constructed transgenic silkworm expresses the hSCF recombinant protein.

• 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.249-259
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• 2014

Cryopreserving cells which are maintaining their viability are the very complex process. This study has been carried out in order to find the effects of cryopreservation steps and freezing media on the rates of viability of cryopreserved chicken primordial germ cells (PGCs). PGCs obtained from the germinal gonade of 5.5~6 day (stage 28) chick embryos of Korean Ogye (KO) and Commercial breeds (C), 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)). Gonads were harvested from stage 28 chick embryos and pooled in groups of 5, 10, 15, 20E embryos, contributing gonads to the cell suspension. 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 slow freezing and vitrification, with different concentrations of the cryoprotectant solution, were examined. After vitrification and slow freezing, survival rates of the frozen-thawed PGCs from the 10% EG plus FBS treatment were 85.63%, and 66.14% (p<0.05), respectively. The viability of PGCs after freeze-thawing was significantly higher for 10% EG plus FBS treatment than for 10% PG + FBS treatment (p<0.05) (85.63% vs 66.81%) by vitrification. 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 data base. In the future, the importance for this new technology is that poultry lines can be conserved while work is being conducted on improving the production of germline chimeras.

• Radiation Oncology Journal
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• v.21 no.2
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• pp.149-157
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• 2003

Purpose: The purpose of this study was to determine if there were prognostic differences between BRCA related and BRCA non-related Korean patients with early-onset breast carcinomas. Materials and Methods: Sixty women who had developed breast cancers before the age of 40, and who were treated at the Soonchunhyang University Hospital, were studied independently of their family histories. The age range was 18 to 40 with a median of 34.5 years. Lymphocyte specimens from peripheral blood were studied for the heterozygous mutations of BRCA1 and BRCA2 using direct sequencing methods. Immunohistochemistry was peformed on the paraffin-embedded tissue blocks that were available. Results: Eleven deleterious mutations (18.3%, 6 in BRCA1 and 5 in BRCA2) and 7 missense mutations of unknown significance (11.7%), were found among the 60 patients. More than half of the mutation were novel, and were not reported in the database. Most of the BRCA-associated patients had no history of breast cancer. No treatment related failures were observed in the BRCA carriers, with the exception of one patient that had experienced a new primary tumor of the contralateral breast. The seven year relapse free survival rate were 50 and 79% In the BRCA carrier and BRCA negative patients, respectively. Although the expression of estrogen and progesterone receptors were less common, and histological features more aggressive, in the BRCA associated tumors, the outcome of the patients with BRCA mutations was not poorer than that on the patients without deleterious mutations. Conclusion.: Despite the BRCA mutation carriers having adverse prognostic features, the recurrence rate was relatively lower than that in the BRCA non-carrying Korean patients wi4h early-onset breast carcinomas. In addition, although the prevalence of the BRCA mutation in Korean patients was higher than that in white patients, the penetrance of the cancer seemed to be relatively low in Korean women carrying BRCA mutations. A large population based study of the BRCA mutation, with a long-term follow-up of the study patients will be required to confirm these results.

• Korean Journal of Microbiology
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• v.43 no.3
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• pp.151-158
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• 2007

The possibility of inadvertent introduction of therapeutic gene expressing viral vectors has raised safety concerns about germ-line infection. Particularly, for indications such as prostate cancer and ovarian cancer, the proximity of the point of viral administration to organs of the reproductive system raises concerns regarding inadvertent germ-line transmission of genes carried by the virus vector. To evaluate the safety of in vivo adenovirus mediated gene transfer, we explored the biodistribution, persistance and potential germ-line transmission of p53-expressing adenovirus (Ad-CMV-p53). Both male and female Balb/c mice were injected with $1{\times}10^9$ PFU of Ad-CMV-p53. The PCR analysis showed that there were detectable vector sequences in liver, kidney, spleen, seminal vesicle, epididymis, prostate, ovary, and uterus. The RT-PCR analysis for detecting inserted gene, p53 showed that Ad-CMV-p53 viral RNA were present in spleen, prostate and ovary. Direct injected male and female mice of adenovirus vector into testis and ovary were mated and their of offspring were evaluated for germ-line transmission of the adenoviral vector. The PCR and RT-PCR analysis showed no evidence of germline transmission, although vector sequences were detected in DNA extracted from gonadal tissues. Real-time PCR result confirmed a significant decrease of adenovirus in gonad tissues 1 week after injection. We have also analysed the cell specific localization of viral DNA in gonad tissues by using in-situ PCR. Positive signals were detected in interstitial tissue but not in seminiferous tubule in sperm. In the case of ovary, adenovirus signal were localized to the stromal tissue, but no follicular signals were observed. Together, these data provide strong evidence that the risk of the Inadvertent germ-line transmission of vector sequences following intraperitoneal or direct injection into genito-urinary system of adenovirus is extremely low.

• Korean Journal of Poultry Science
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• v.40 no.3
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• pp.207-216
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• 2013

This study was conducted to establish the method for preserving PGCs that enables long-term storage in liquid nitrogen for developmental engineering or preservation of species. The purpose of this study is to clarify the effects of freeze-thaw treatment on viability of PGCs in chickens. PGCs were collected separately from a germinal gonad of an early embryo of 5.5~6 day (stage 28) of Isa brown, Korean Oge (KO), White Leghorn and Commercial breeds. PGCs separated from a germinal gonad of an early embryo of 5.5~6 day (stage 28) are suspended in a freezing medium containing a freezing and protecting agents (e.g. dimethyl sulfoxide (DMSO), ethylene glycol (EG) and glycerol). The PGCs were then purified using magnetic activated cell sorting (MACS) method. The viability of PGCs after thawing was $87.4{\pm}0.4%$ and $89.4{\pm}0.2%$ with the 10% EG treatments with no significant difference between the Isa brown and Commercial breeds. The viability of PGCs after freeze- thawing was significantly higher for Isa brown ($87.4{\pm}0.4%$) and Commercial breeds ($89.4{\pm}0.2%$) than Korean Oge (KO) ($77.6{\pm}1.1%$) and White Leghorn ($76.2{\pm}0.9%$)(p<0.05) using 10% EG cryoprotectant. This study established a method for pre- serving chicken PGCs that enables systematic storage and labeling of cryopreserved PGCs in liquid ($LN_2$) at agermplasm repository and ease of entry into a data base. In the future, the importance for this new technology is that poultry lines can be conserved while work is being conducted on improving the production of germline chimeras.

• Korean Journal of Poultry Science
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• v.40 no.3
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• pp.197-205
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• 2013

This study established a method for preserving chicken primordial germ cells (PGCs) that enables long-term storage in liquid nitrogen ($LN_2$) for preservation of the species. The purpose of this study was to compare the effects of Ethylene Glycol (EG) and Propylene Glycol (PG) on viability of cryopreserved PGCs with vitrification in Korean Native Chicken (Ogye), and to fine should be find or to the optimal protocol for PGCs freezing. One of the important components of cryopreservation process is cryopreservation medium that plays a vital role in preventing cellular injury during freeze-thawing. Cryoprotective agents have been known to improve cell viability after freeze-thawing. PGCs obtained from the germinal gonade of 5.5~6 day (stage 28) chick embryos, using the MACS method were suspended in a freezing medium containing a freezing and protecting agents. Gonads were harvested from stage 28 chick embryos and pooled in groups of 10E embryos, contributing gonads to the cell suspension. The gonadal cells, including PGCs, were then frozen in 1 of the following cryoprotectant treatments: 2.5% EG, 5% EG, 10% EG, 2.5% PG, 5% PG, 10% PG, and 0% cryoprotectant as a control. Effects of exposure to vitrification solution and vitrification, with different concentrations of the cryoprotectant solution, were examined. After freezing and thawing, survival rates of the frozen-thawed PGCs from the 0, 2.5, 5, 10 and 15% EG plus FBS treatment were 44.24%, 64.51%, 85.63%, 80.51% and 73.52% (p<0.05), respectively. The viability of PGCs after freeze-thawing was significantly higher for 10% EG plus FBS treatment than for 10% PG + FBS treatment (p<0.05)(85.63% vs 66.81%). 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 PGC that enables systematic storage and labeling of cryopreserved PGCs in liquid N at a germplasm repository and ease of entry into a database. In the future, the importance for this new technology is that poultry lines can be conserved while work is being conducted on improving the production of germline chimeras.