• Asian-Australasian Journal of Animal Sciences
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• 제15권9호
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• pp.1227-1231
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• 2002

A powerful tool for chicken transgenesis could be established by employing a germline chimera production through primordial germ cell transplantation. This study was conducted to examine whether foreign gene-transfected gonadal primordial germ cells (gPGCs) have a migration activity into the gonad after transfer to recipient embryos. In Experiment 1, gPGCs of Korean Ogol Chicken were retrieved from 5.5-day-old embryos and subsequently transferred to the dorsal aorta of 2.5-day-old White Leghorn embryos after being labeled with PKH26 fluorescent dye. To confirm migration activity after transplantation, recipient embryos were sacrificed and examined on 3 days after transfer. Sex determination was concomitantly undertaken to examine whether sex of recipient embryos could affect the migration activity of gPGCs. All of embryonic gonads examined showed positive signals with PKH26 fluorescence and W-chromosome specific band by polymerase chain reaction (PCR) was detected in male embryos when gPGCs with ZW chromosome were transferred to recipient embryos. In Experiment 2, retrieved gPGCs were transfected with LacZ gene-containing cytomegalovirus promoter ($pCMV{\beta}$) by electroporation and subsequently transferred to recipient embryos. LacZ gene expression was identified in the gonads of 6 or 10-day-old recipient embryos and hatched-chicks. A total of 20 embryos and 12 hatched-chicks were examined and 11 of them (10 embryos and one hatched chicken; 11/32=34.4%) expressed $\beta$-galactosidase, a marker substance of LacZ gene. The results of this study demonstrated that foreign gene-transfected gPGCs can migrate and settle down into the gonad after being transferred into the blood vessel of the recipient embryos. This established technique will contribute to developing a peer biotechnology for transgenic chicken.

• Asian-Australasian Journal of Animal Sciences
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• 제16권8호
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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.

• Asian-Australasian Journal of Animal Sciences
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• 제7권3호
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• pp.427-434
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• 1994

The primordial germ cells (PGCs) were transfected in vitro and expressed the exogenous RSVLTR/${\beta}G2$ plasmid, suggesting thaI PGC is a possible vector for direct gene transfer into the germ line. Transfection efficiency of cell suspensions containing PGCs was 1.5% by liposome mediated DNA transfection. By microinjection of the transfected PGCs into the host germinal crescent, PGCs migrated via blood vessel to the future gonad and these transfected PGCs resulted in the RSVLTR/${\beta}G2$ expression in the gonad. The results from the seeding of PGCs on the chorioallantoic membrane were insufficient to test the hypothesis that PGCs can penetrate or invade the chorioallantoic membrane for transport via the circulatory system.

• Reproductive and Developmental Biology
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• 제32권3호
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• pp.159-166
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• 2008

본 연구는 vesicular stomatitis virus G glycoprotein (VSV-G)으로 피막이 형성되는 replication-defective MoMLV-based vector를 이용한 hTPO 헝질전환 닭의 생산에 관한 연구이다. 실험에 사용한 retrovirus vector의 구조는 hTPO 유전자의 발현 조절을 위해 internal promoter인 hCMV promoter를 이용하였으며 외래 유전자의 발현을 증가시키기 위해 woodchurk hepatitis virus posttranascriptional regulatory element (WPRE) 서열을 도입하였다. 재조합한 vector는 GP2 293 포장세포에 도입하여 virus를 생산하였으며 이 virus를 이용하여 감염시킨 여러 표적세포에서 hTPO의 발현과 생물학적 활성을 확인하였다. 재조합 hTPO의 생물학적 활성은 시판되고 있는 재조합 hTPO에 비해 우월한 것으로 확인되었다. hTPO 형질전환 닭의 생산을 위하여 1,000배 이상 고농도로 농축된 virus를 stage X 단계의 계란의 배반엽 층에 미세주입하여 대리난각 방법으로 배양하였다. 미세주입한 132개의 계란 중 21일 후에 11개의 계란에서 병아리가 부화하였으며 그중 4마리가 형질전환 개체로 확인되었다. 그러나 생산된 4마리 중 3마리가 부화 후 1개월 이내에 원인불명으로 사망하였다. 본 연구의 의의는 상업적 이용 가능성이 있는 생물학적 활성을 가진 사람의 cytokine 단백질의 대량 생산을 위한 생체 반응기로서의 형질전환 닭 개발의 시례를 제공하는데 있다.

• 한국가금학회:학술대회논문집
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• 한국가금학회 2000년도 제17차 정기총회 및 학술발표
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• pp.88-90
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• 2000

Although primordial germ cells(PGCs) have been used in the production of germline chimera, efficiency has not been satisfactory. The Present study was conducted to improve efficiency of germline chimera production using the cultured gonadal PGCs(gPGCs). Germline chimeric chickens were produced by transfer of cultured gonadal primordial germ cells from Korean Ogol Chicken (KOC) to White Leghorn (5.5-day-old) and cultured in vitro for 10 days. Approximately 200 gPGCs (2-day-old) recipient embryos from which blood had been withdrawn via the dorsal aorta prior to the injection. Recipient embryos were incubated until hatching. Germline chimerism of the chickens reaching maturity was examined by mating them with Korean Ogol Chicken. Donor-derived offspring were identified as germline chimeric chickens based on their feather color. The frequency of germline transmission of donor PGCs ranged 1.9∼60.7%. There was no difference between both sexes. Therefore, it can be concluded that efficiency of germline chimerism can be improved via using cultured gPGCs.

• Asian-Australasian Journal of Animal Sciences
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• 제30권6호
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• pp.886-892
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• 2017

Objective: Transgenic technology is widely used for industrial applications and basic research. Systems that allow for genetic modification play a crucial role in biotechnology for a number of purposes, including the functional analysis of specific genes and the production of exogenous proteins. In this study, we examined and verified the cumate-inducible transgene expression system in chicken DF1 and quail QM7 cells, as well as loxP element-mediated transgene recombination using Cre recombinase in DF1 cells. Methods: After stable transfer of the transgene with piggyBac transposon and transposase, transgene expression was induced by an appropriate concentration of cumate. Additionally, we showed that the transgene can be replaced with additional transgenes by co-transfection with the Cre recombinase expression vector. Results: In the cumate-GFP DF1 and QM7 cells, green fluorescent protein (GFP) expression was repressed in the off state in the absence of cumate, and the GFP transgene expression was successfully induced in the presence of cumate. In the cumate-MyoD DF1 cells, MyoD transgene expression was induced by cumate, and the genes controlled by MyoD were upregulated according to the number of days in culture. Additionally, for the translocation experiments, a stable enhanced green fluorescent protein (eGFP)-expressing DF1 cell line transfected with the loxP66-eGFP-loxP71 vector was established, and DsRed-positive and eGFP-negative cells were observed after 14 days of co-transfection with the DsRed transgene and Cre recombinase indicating that the eGFP transgene was excised, and the DsRed transgene was replaced by Cre recombination. Conclusion: Transgene induction or replacement cassette systems in avian cells can be applied in functional genomics studies of specific genes and adapted further for efficient generation of transgenic poultry to modulate target gene expression.

• 한국가축번식학회지
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• 제21권1호
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• pp.1-7
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• 1997

형질 전환동물의 유선을 이용한 단백질의 생산이 보편화되고 있지만 원하는 단백질이 만들어지기 까지 많은 시간과 노력이 필요하며 기술적인 어려움이 항시 따른다. 그래서 보다 쉽게 재조합된 유전자의 발현 정도를 시험하는 in vitro에서의 시험방법의 개발은 중요한 의미가 있다고 하겠다. 따라서 본 연구에서는 인간의 유방암을 가진 환자의 유선에서 유래된 MCF7 cell line을 이용하여 유선 특징의 유전자 발현을 위한 세포 배양 모델을 개발하고자 하였다. 우선 소의 카제인의 cDNA를 MMTV-LTR의 통제하에 clone하였으며, 이것을 CaPO4의 침전법으로 MCF7 cell에 transfection 시킨 다음, HAT 배양액으로 선발하였으며, dexamethasone으로 유도시키고, 발현되는 정도를 면역 항체를 이용하여 분석하였다. 선발된 세포는 dexamethasone에 의하여 MMTV promoter가 유도되는 것을 확인할 수 있었다. 따라서 MCF7 cell과 같이 다양한 steroid receptor를 가지고 있는 세포는 유선 특정의 유전자 발현을 위한 세포 배양 모델에 유용하게 사용이 될 수 있을 것이다.

• 한국가금학회지
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• 제39권3호
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• pp.241-244
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• 2012

본 연구는 형질전환 닭에서 주입한 외래 유전자의 세대간 전이와 발현 양상을 조사하고자 하였다. 외래 유전자 전이 양상 조사는 제3세대(G2) GFP 형질전환 수탉을 최초 부계로 사용하여 최종적으로 제9세대(G8) 형질전환 닭을 연속적으로 생산하면서 GFP 유전자 전이 양상을 조사하였다. 형질전환 병아리 유전 분석은 자외선 램프 아래에서 부화한 병아리들의 날개, 부리와 다리에서 녹색형광단백질을 발현하는 병아리들만을 형질전환으로 선발하였다. 형질전환 닭에서 외래 유전자 전이율은 대략 38~58%이었다. 이는 유전자 전이가 멘델의 유전 법칙을 따르고 있음을 보여주고 있다. 연구 결과는 GFP 유전자가 유전자 침묵 없이 멘델의 유전 법칙에 따라 다음 세대로 계속 전이와 발현된다는 것을 보여 주고 있다.

• Asian-Australasian Journal of Animal Sciences
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• 제14권2호
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• pp.163-169
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• 2001

Despite the high potency of the retrovirus vector system in gene transfer, one of the main drawbacks of has been difficulty in preparing highly concentrated virus stock. Numerous efforts to boost the virus titer have ended in unsatisfactory results mainly due to fragile property of retrovirus envelope protein. In this study, to overcome this problem, we constructed our own retrovirus vector system producing vector viruses encapsulated with VSV-G (vesicular stomatitis virus G glycoprotein). Concentration process of the virus stock by ultracentrifuge did not sacrifice the virus infectivity, resulting in more than 108 to 109 CFU (colony forming unit) per ml on most of the target cell lines tested. Application of this high-titer retrovirus vector system was tested on chicken embryos. Injection of virus stock beneath the blastoderms of pre-incubated fertilized eggs resulted in chick embryos expressing E. coli LacZ gene with 100% efficiency. Therefore, our results suggest that it is possible to transfer the foreign gene into chicken embryo using our high-titer retrovirus vector.

• 한국가축번식학회지
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• 제25권2호
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• pp.171-180
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• 2001

형질전환 가금의 생산에 있어서 retrovirus vector를 이용하는 방법은 다양한 종류의 표적세포에 대하여 retrovirus 고유의 감염성에 의한 외래 유전자의 전이가 용이하고, 전이된 유전자가 진정염색질 영역 내로 선택적으로 도입될 수 있으며 유전적으로 안정성을 나타내므로 매우 효과적인 방법이다. 그러나 가금에서는 초기 배발달에 의한 급격한 세포의 수적 증가로 인해 고감염성의 virus의 획득이 요구되므로, 이를 위하여 virus stock의 농축에 있어 보다 안정적이고 pantropic인 vesicular stomatitis virus (VSV G) glycoprotein를 envelope로 가지는 pseudotyped retrovirus vector system을 이용하였으며, marker gene으로 eGFP gene이 발현되는 retrovirus를 생산하였다. 이 virus를 이용하여 여러 가지 표적세포와 primary culture한 CEF세포를 감염시켜 GFP의 발현을 확인하였으며, 농축한 virus stock은 stage X의 계란을 선택하여 windowed egg를 제작한 후 배하층에 주입하였다. 형질전환 닭은 정상 발생한 닭에 비하여 저조한 발생율을 보였으나 PCR을 이용하여 외래 유전자의 도입을 확인한 결과 100%인 것으로 나타났다. 또한 한 개체 내에서 유전자의 도입이 폐, 간, 정소, 소장 등의 여러 장기에서 확인되었다.