• 한국가금학회:학술대회논문집
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• 한국가금학회 2001년도 제18차 정기총회 및 학술발표 PROCEEDINGS
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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.

• Clinical and Experimental Reproductive Medicine
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• 제42권2호
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• pp.33-44
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• 2015

The generation of artificial gametes is a real challenge for the scientific community today. In vitro development of human eggs and sperm will pave the way for the understanding of the complex process of human gametogenesis and will provide with human gametes for the study of infertility and the onset of some inherited disorders. However, the great promise of artificial gametes resides in their future application on reproductive treatments for all these people wishing to have genetically related children and for which gamete donation is now their unique option of parenthood. This is the case of infertile patients devoid of suitable gametes, same sex couples, singles and those fertile couples in a high risk of transmitting serious diseases to their progeny. In the search of the best method to obtain artificial gametes, many researchers have successfully obtained human germ cell-like cells from stem cells at different stages of differentiation. In the near future, this field will evolve to new methods providing not only viable but also functional and safe artificial germ cells. These artificial sperm and eggs should be able to recapitulate all the genetic and epigenetic processes needed for the correct gametogenesis, fertilization and embryogenesis leading to the birth of a healthy and fertile newborn.

• Asian-Australasian Journal of Animal Sciences
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• 제13권5호
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• pp.587-594
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• 2000

At present embryonic stem (ES) cells with confirmed pluripotential properties are only available in the mouse. Recently, we were able to isolate, culture and genetically transform primordial germ cell (PGC)-derived cells from pig embryos and demonstrate their ability to contribute to chimera development in the pig. In order to determine whether the system we developed could be used to isolate embryonic germ (EG) cells from other mammalian species, we placed isolated PGCs from cattle, goats, rabbits and rats in culture. Briefly, PGCs were isolated from fetuses of cow (day 30-50), goat (day 25), rabbit (day 15-18) and rat (day 11-12), and plated on STO feeder cells in Dulbecco's modified Eagle's medium (DMEM): Ham's F10 medium (1:1) supplemented with 0.01 mM nonessential amino acids, 2 mM L-glutamine, 0.1 mM $\beta$ - mercaptoethnol, soluble recombinant human stem cell factor (SCF; 40ng/ml), human basic fibroblast growth factor (bFGF; 20ng/ml) and human leukemia inhibitory factor (LIF; 20ng/ml). For maintenance of the cells, colonies were passed to fresh feeders every 7-10 days. In all species tested, we were able to obtain and maintain colonies with ES-like morphology. Their developmental potential was tested by alkaline phosphatase (AP) staining and in vitro differentiation assay. For genetic transformation, cells were electroporated with a construct containing the green fluorescent protein (GFP) under the control of the cytomegalovirus (CMV) promoter. GFP-expressing colonies were detected in cattle, rabbits and rats. These results suggest that PGC-derived cells from cattle, goats, rabbits and rats can be isolated, cultured, and genetically transformed, and provide the basis for analyzing their developmental potential and their possible use for the precise genetic modification of these species.

• 한국가금학회지
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• 제26권2호
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• pp.119-129
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• 1999

In recent years, numerous researches have been carried out in author's laboratory to develop several kinds of methods for producing transgened chicken, leaving a lot of new findings. Some of them are very useful to search for new approaches necessary to improve the efficiency of hatchability and the survival rate of developing trasgened embryos. The results obtained hitherto might be summarized as follows: (1) foreign gene(Lac Z/ Miw Z) introduced into blastodermal cells of developing embryos was successfully transferred to embryos, leading to the production of primordial germ cells(PGCs) carrying foreign DNA. However, hatched hickens failed to show the incorporation of introduced gene into the gonads. (2) When foreign gene was introduced into germinal crescent region (GCR), the gene was also efficiently incorporated into germ cells, resulting in the production of transgened chickens(offspring) which produced fruther offspring having foreign gene in the gonads. In this case, 2nd and 3rd generations of chickens were obtained through the reproduction of transgened birds. (3) In another way, the gene was injected into blood vessels of developing embryos at stage 13∼15, creating PGCs having foreign gene, and produced some transgened chickens. In this work, the PGCs were transfered between embryos, resulting in the production of transgenic chickens. (4) in these experiments, PGCs were effectively employed for producing transgenic birds, developing some kinds of chimeric chickens from homo- or hetero-sexual transfer of the PGCs from embryos. This means that the gonads from donor PGCs developed in some degree to the stage of hatching. However, these gonads showed slightly abnormal tissues similar to ovotestis like organs through histological examination. (5) Avian Leukosis Virus(ALV) induced B cell line(DT40) successfully carried foreign genes into chicken embryos, suggesting the possibility of the cells as a vector in this field of study in the future. (6) Inter-embryonic transfer of the PGCs also gave us some.

• 한국발생생물학회지:발생과생식
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• 제2권1호
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• pp.69-74
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• 1998

부화 후부터 평균전장 0.64 cm를 나타내는 부화 후 150일까지의 버들치, Rhynchocypris oxycephalus를 대상으로 초기생식소 발달과 성분화를 조사하였다. 시언생식세포는 평균전장 0.64cm 자어에서 뚜렷이 나타나\ulcorner. 평균전장 1.91 cm자어에서 시원생식세포는 복강으로돌출되었으며, 평균전장 2.29 cm 자어에서 시원생식세포는 감수분열 난모세포로 전환되었고, 난소로의 분화가 최초 확인되엇따. 평균전장 5.96 cm 자어에서 암컷 생식소는 점진적으로 발달하였으며 성숙단계로 접어드는 핵이동 난모세포를 보였다. 성분화 후 난모세포는 빠르게 증식하는 반면 정소는 평균전장 4.00cm 자어까지는 성장이 중지된 채 증가만 하는 휴지상태이었다. 평규전장 4.00cm 자어에서의 정모세포는 중간기에서 발달이 정지되었으며, 감수분열이 활발하였고, Sertoli-like cell과 정소관이 형성되었다. 본 연구 결과 버들치의 성본화 양상은 분화형 장웅이체 (differentiated gonochorism)인 것으로 나타났다.