• Asian-Australasian Journal of Animal Sciences
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• v.28 no.11
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• pp.1565-1572
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• 2015

Porcine embryonic stem cells (pESCs) have become an advantageous experimental tool for developing therapeutic applications and producing transgenic animals. However, despite numerous reports of putative pESC lines, deriving validated pESC lines from embryos produced in vitro remains difficult. Here, we report that embryo aggregation was useful for deriving pESCs from in vitro-produced embryos. Blastocysts derived from embryo aggregation formed a larger number of colonies and maintained cell culture stability. Our derived cell lines demonstrated expression of pluripotent markers (alkaline phosphatase, Oct4, Sox2, and Nanog), an ability to form embryoid bodies, and the capacity to differentiate into the three germ layers. A cytogenetic analysis of these cells revealed that all lines derived from aggregated blastocysts had normal female and male karyotypes. These results demonstrate that embryo aggregation could be a useful technique to improve the efficiency of deriving ESCs from in vitro-fertilized pig embryos, studying early development, and deriving pluripotent ESCs in vitro in other mammals.

• Korean Journal of Poultry Science
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• v.42 no.3
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• pp.239-245
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• 2015

Transgenic animals have been widely used for developmental biology studies, as disease models, and even in industry such as transgenic bioreactor animals. For transgenic birds, quail has the great advantages of small body size, short generation time, and frequent egg production. To date, retroviral or lentiviral transduction has been used to generate transgenic quail for various purposes. However, the efficiency of transgenic offspring production with these methods is relatively low and viral vector usage has safety issues. Unfortunately, non-viral transgenesis has not been established in quail due to a deficiency of stem cell and germ cell culture systems. In this study, we established a direct in ovo lipofection method that could be used to create transgenic quail without germline-competent cells or viruses. To optimize the injection stage during embryo development, the liposome complex (containing piggyBacCMV-GFP and transposase plasmids) was introduced into an embryonic blood vessel at 50 hr, 55 hr or 60 hr. GFP expression was detected in various tissues (heart, kidney, liver and stomach) on day 12 of incubation under a fluorescence microscope. Additionally, GFP-positive cells were detected in the recipient embryonic gonads. In conclusion, the direct in ovo lipofection method with the piggyBac transposon could be an efficient and useful tool for generating transgenic quail.

• Journal of Embryo Transfer
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• v.24 no.3
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• pp.189-197
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• 2009

Two-pore domain $K^+(K_{2P})$ channels contribute to setting the resting membrane potential in excitable and nonexcitable cells. However, the cellular or tissue distribution and function of $K_{2P}$ channels expressed in mammalian germ cells and reproductive organs have not yet been reviewed by researchers. In this review, we focus on expression, localization and expected properties of $K_{2P}$ channels in germ cells and reproductive organs. The $K_{2P}$ channels are expressed in human cytotrophoblast cells, myometrium, placental vascular system, uterine smooth muscle, and pregnant term tissue, suggesting that $K_{2P}$ channels might be involved in the processes of pregnance. The $K_{2P}$ channels are also expressed in mouse zygotes, monkey sperm, ovary, testis, germ cells, and embryos of Korean cattle. Interestingly, $K_{2P}$ channels are modulated by changes in temperature and oxygen concentration which play an important role in embryonic development. Also, $K_{2P}$ channels are responsible for $K^+$ efflux during apoptotic volume decreases in mouse zygotes. These expression patterns and properties of the $K_{2P}$ channels in reproductive organs and germ cells are likely to help the understanding of ion channel-related function in reproductive physiology.

• Molecules and Cells
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• v.38 no.9
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• pp.743-749
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• 2015

Production of genome-edited animals using germline-competent cells and genetic modification tools has provided opportunities for investigation of biological mechanisms in various organisms. The recently reported programmed genome editing technology that can induce gene modification at a target locus in an efficient and precise manner facilitates establishment of animal models. In this regard, the demand for genome-edited avian species, which are some of the most suitable model animals due to their unique embryonic development, has also increased. Furthermore, germline chimera production through longterm culture of chicken primordial germ cells (PGCs) has facilitated research on production of genome-edited chickens. Thus, use of avian germline modification is promising for development of novel avian models for research of disease control and various biological mechanisms. Here, we discuss recent progress in genome modification technology in avian species and its applications and future strategies.

• Reproductive and Developmental Biology
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• v.29 no.4
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• pp.241-245
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• 2005

Porcine oviduct epithelial cells (POEC) are widely used in co-culture experiments to improve early embryonic development, in vitro fertilization in embryo transfer programs for domestic animals and in vitro maturation of immature germ cells. POEC were mechanically isolated and cultured in tissue culture medium 199. Cells grew continuously, and confluent monolayers were formed after 7 days. After forming confluent monolayer of epithelial cells, supernatant was collected as the condition medium for maturing round spermatids in vitro. Round spermatids were also separated mechanically and cultured in the POEC condition medium. In this study we observed that $20\%$ of round spermatid cultured were matured into elongating spermatid after 24 h, and about $10\%$ of round spermatid cultured showed complete elongation (elongated spermatid) within $24\~48$ h of in vitro culture. No further development was observed within $50\~72$ h and transformed cells lost their viability after 72 h. These preliminary findings suggest that the condition medium from POEC may be possible to overcome the round spermatid block by improving the milieu of culture system.

• Biomedical Science Letters
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• v.10 no.4
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• pp.391-395
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• 2004

The demand for the production of gene-defective mice from embryonic stem (ES) cells is increasing to clarify decisive gene function in vivo. Although blastocyst injection is widely used to generate ES cell-mediated knockout mice, coculture method has been alternatively used because of several advantages, such as low cost and simple procedure. Thus, this experiment was designed to demonstrate the feasibility of the coculture method using J1 ES cells, which are known to be efficient for blastocyst injection. Eight-cell embryos were harvested from 2.5 days post-coitum (dpc), denuded with acid tyrode's solution, and transferred onto trypsinized J1 ES cells. Aggregation was carried out following two typical methods, which are simple coculture method and aggregation in groove prepared by aggregation needle. Successfully aggregated-embryos were developed to blastocysts for 24 h and transferred into uterus of pseudo-pregnant foster mother. Chimeric offspring was judged by coat pigmentation. In this study, we could obtain chimeric mice from all the two aggregation methods, but the chimera production efficiencies in coculture using groove were three times higher at least than those in the other group. In conclusion, these observations suggest that coculture method should be available for production of knockout mice from J1 ES cells. Presently, the germ-line transmission rates of the chimeras produced from the two methods are under investigation.

• Korean Journal of Animal Reproduction
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• v.25 no.1
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• pp.71-77
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• 2001

This experiment was designed to improve the production efficiency of germ-line chimeric mice from phospholipase C (PLC)-$\beta$3 or peroxiredoxin (Prx) E -targeted ($\Delta$) ES cells by the investigating the manipulation conditions and characteristics of Jl ES cells. Four targeting clones were isolated to investigate the karyotypical and morphological stability prior to injection. All clones ($\Delta$PLC$\beta$-3 C3, $\Delta$Prx II C3, C10 and I5) showed more than 80% euploidism, however, most of $\Delta$PLC$\beta$-3 C3 clones were extensively differentiated compared to the other clones. Nine of 13 $\Delta$Prx II chimeras appeared to have at least 80% chimerism, whereas $\Delta$PLC$\beta$-3 C3 chimeras had 20% chimerism at most. Therefore, the morphological stability of ES cells under stable euploidism might mainly affect the production rate of high-coat chimeric mice. To increase the collection rate of injectable blastocysts (IBs), 5 to 10 week -aged C57BL/6J female mice were sacrificed at 3.5 days post-coitum. Ten week-aged mice were the most optimal IB donors by showing the highest collection rate (2.94/mouse) of injectable blastocysts without increase of non-injectable embryos (0.29/mouse). Foster mothers might be another factor because ICR x C57BL/6J F1 foster mother showed more increased productivity in litter size (2.8 vs. 5.6) and chimera (0 vs. 35.3%) than those of ICR foster mothers. In conclusion, the efficient production of germ-line chimeras mainly depends on the maintenance of ES cell morphology during targeting procedure, and the establishment of manipulation conditions might be a key point to maximize it.

• Proceedings of the Korea Society of Poultry Science Conference
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• 2004.11a
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• pp.9-10
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• 2004

We developed a new panel of markers for the characterization of chicken PGCs. The results of immunostaining demonstrated that anti-SSEA-3, anti-SSEA-4, anti-integrin 6, and anti-integrin 1 antibodies. and STA and DBA bound specifically to chicken PGCs. These reagents could be used to characterize chicken PGCs together with conventional marker reagents such as PAS and anti-SSEA-1 antibody. We also showed that double staining of PGCs with the newly developed markers was feasible, which might contribute to rapid detection and accurate characterization of chicken PGCs.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.96-96
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• 2003

Nek2 (NIMA-related protein) is a mammalian cell cycle-regulated kinase that involves in chromosome condensation and centrosome regulation and NuMA (nuclear mitotic apparatus protein) is involved in spindle assembly during a cell cycle. The cellular distribution and organization of the centrosomal components is completely unknown during fertilization and embryonic development. We examined distribution of two well-known centrosomal proteins, Nek2 and NuMA in mouse gametes and embryos to get an insight in the reorganization of centrosomal proteins during germ cell development and early fertilization. Spermatogenic cells, gametes, and embryos were analyzed with anti-Nek2 or -NuMA antibodies by immunological assay, RT-PCR, and overexpression through gene transfection. Mitotically or meiotically active spermatogenic cells were intensively stained with these antibodies in both centrosomes and cytoplasm, whereas the oocytes showed different staining patterns depending on the meiotic stages. During maturation, GV, GVBD, and MI stage were clearly stained with NuMA antibody in the nucleus or cytoplasm at MII. Also, Nek2 was detectable in cytoplasm as scattered spots or chromosome associated at MII. In early developmental embryo, NuMA was detected in nucleus of each blastomere, while Nek2 was detected in cytoplasm. In contrast to previously reported results, Nek2 and NuMA were detected in both decondensing head, and the centriole of demembranated and decondensed sperm or whole body of trypsin-treated sperm for Nek2. During meiotic progress in oocytes, transcripts levels were the highest in MI stage and then downregulated in MII. Also, it shows dramatically change in early developmental embryos, firstly, it was increased until 4 cell stage and reduced in 8 cell stage, and finally, transcript levels were upregulated until blastoscyst. This finding suggests that cnetrosomal component may play an important role in reorganizing of functional centrosome during fertilization process and subsequent development.

• Advances in pediatric surgery
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• v.1 no.2
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• pp.122-132
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• 1995

Teratoma contains elements derived from all three embryonic germ layers and mature teratomas are entirely composed of mature differentiated tissues, while immature types always contain additional embryonic tissues of variable degree of immaturity, especially neuroepithelial elements. Twenty cases of teratoma in infancy and childhood were reviewed and the clinical features and pathologic findings including PCNA expression were studied. Sacrococcygeal teratomas were the most common type(45%), followed by retroperitoneal and ovarian teratomas. There was a predilection of females in a ratio of 4:1 and age distribution was most prevalent below the first year of age(45%). But in ovarian teratomas, the age at diagnosis was above 4 years of age in all cases. Serum alpha-fetoprotein levels were checked in 18 cases. In all mature teratomas and 1 of 5 immature teratomas, the levels were normal. But in 4 of 5 immature teratomas, the serum levels were elevated and progressively declined to normal range after mass excision. Radiologically, calcifications in tumor were found in 60.0% of teratomas and was higher in mature teratomas(69.2%) than immature teratomas(42.9%). Immunohistochemical staining for PCNA(proliferating cell nuclear antigen) was done in 16 cases and PCNA expression was higher in grade III immature teratomas than grade I and II. The operative modes were complete mass excisions. Five immature teratomas were treated with multiagent PEB(Bleomycin, Etoposide, Cisplatin) adjuvant chemotherapy, 3 tolerated well without significant complications, but in one case, severe bone marrow suppression was developed and died of sepsis. In conclusion, grade III immature teratoma showed higher PCNA expression than mature or lower grade immature teratoma, which suggests that chemotherapy after surgical excision may be effective modality for grade III immature teratoma. We think, however, multicenter study is necessary because of low incidence of immature teratoma.