• Proceedings of the Korea Society of Poultry Science Conference
• /
• 2000.11a
• /
• pp.88-90
• /
• 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 Approximately 200 gPGCs (2-day-old) recipient embryos from which blood had been withdrawn via the dorsal aorta prior to the injection.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
• /
• v.7 no.4
• /
• pp.459-466
• /
• 1994

Primordial germ cells (PGCs) in aves are the progenitor cells for the gametes. These cells first appear in the epiblast (Eyal-Giladi et al.. 1981). Then translocate and concentrate to endoderm of germinal crescent area in the junction of the area opaca and area pellucida lateral to the primitive streak in stage 4 through 7. They separate from the endoderm, temporarily circulate via the blood vascular system, leave the blood vessels, and finally settle down in the gonadal anlagen at stage 20-24 where they rapidly proliferate to form germ cells. Recently, several attempts have been made to introduce foreign gene into the avian genome to form a transgenic chicken. The stem cells most readily available as vehicles for genetic manipulation of germline in avian species are the PGCs. PGCs have recently been manipulated genetically and used successfully as a vector for gene transfer.

• Proceedings of the Korea Society of Poultry Science Conference
• /
• 2006.11a
• /
• pp.43-58
• /
• 2006

As a bioreactor, bird has proved to be most efficient system for producing useful therapeutic proteins. More than half of the egg white protein content derives from the ovalbumin gene with four other proteins(lysozyme, ovomucoid, ovomucin and conalbumin) present at levels of 50 milligrams or greater. And the naturally sterile egg also contains egg white protein at high concentration allowing for a long shelf life of recombinant protein without loss in activity. In spite of these advantages, transgenic procedures for the bird have lagged far behind because of its complex process of fertilized egg and developmental differences. Recently, a system to transplant mouse testis cells from a fertile donor male to the seminiferous tubules of an infertile recipient male has been developed. Spermatogenesis is generated from transplanted cells, and recipients are capable of transmitting the donor haplotype to progeny. After transplantation, primitive donor spermatogonia migrate to the basement membrane of recipient seminiferous tubules and begin proliferating. Eventually, these cells establish stable colonies with a characteristic appearance, which expands and produces differentiating germ cells, including mature spermatozoa. Thus, the transplanted cells self-renew and produce progeny that differentiate into fully functional spermatozoa. In this study, to develop an alternative system of germline chimera production that operates via the testes rather than through developing embryos, the spermatogonial stem cell techniques were applied. This system consisted of isolation and in vitro-culture of chicken testicular cells, transfer of in vitro-maintained cells into heterologous testes, production of germline chimeras and confirmation of germline transmission for evaluating production of heterologous, functional spermatozoa.

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

• Asian-Australasian Journal of Animal Sciences
• /
• v.15 no.9
• /
• pp.1227-1231
• /
• 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.

• BMB Reports
• /
• v.46 no.8
• /
• pp.404-409
• /
• 2013

Extracellular superoxide dismutase (EC-SOD) is a metallo-protein and functions as an antioxidant enzyme. In this study, we used lentiviral vectors to generate transgenic chickens that express the human EC-SOD gene. The recombinant lentiviruses were 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. Of 158 injected embryos, 16 chicks (G0) hatched and were screened for the hEC-SOD by PCR. Only 1 chick was identified as a transgenic bird containing the transgene in its germline. This founder (G0) bird was mated with wild-type hens to produce transgenic progeny, and 2 transgenic chicks (G1) were produced. In the generated transgenic hens (G2), the hEC-SOD protein was expressed in the egg white and showed antioxidant activity. These results highlight the potential of the chicken for production of biologically active proteins in egg white.

• Proceedings of the Korea Society of Poultry Science Conference
• /
• 2001.11a
• /
• pp.71-73
• /
• 2001

In this study, we could improve transmission efficiency of germline chimeras by transfer of gonadal PGCs (gPGCs) cultured in vitro. Of hatched recipient chicks, 301 chickens (141 males and 160 females) were brought up to sexual maturity and these WLs (KOC) were mated with KOCs for testcross, resulting in 27 germline chimeras (15 males and 12 females) identified by black feather color of their progenies. The production efficiency of germline chimera production of experimental groups was observed (P=0.6831). The average transmission efficiency of proven germline chimeras was 0.6 ∼56.5% (15.0% on average). The transmission efficiency of experimental group which were transferred 10-days cultured gPGCs without Ficoll treatment was highest (49.7%) and that of experimental stock which transferred non-cultured gPGCs with Ficoll treatment was lowest (0.6%). The duration of in vitro culture before transferring was significantly important for the high efficiency of germline transmission. Transferring 10-days cultured gPGCs made the transmission efficiency higher rather than transferring non-cultured and 5-days cultured gPGCs, 50 times and 10 times, respectively (p<0.0001). However, Ficoll treatment for increasing the population ratio of gPGCs negatively affected the transmission efficiency and the effects of sexuality and the reciprocal interaction between treatments showed no significant differences. These findings demonstrated that the crucial factors for improving the germline transmission were the duration of in vitro culture prior to transfer. Thus, we developed the complete system for production of germline chimera using cultured gPGCs with highly improved efficiency and this system would be useful for genetic manipulation and obtaining the transgenic aves.

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

• Korean Journal of Poultry Science
• /
• v.27 no.1
• /
• pp.73-78
• /
• 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
• /
• 2001.11a
• /
• pp.40-46
• /
• 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.