Polycystic ovarian syndrome (PCOS) is a heterogeneous syndrome associated with follicle growth arrest, dysregulated sex hormone profile, hyperthecosis and insulin resistance. Chemerin, a novel adipokine, is associated with obesity and metabolic syndrome. Although obese women and in PCOS subjects have elevated plasma chemerin levels, whether and how chemerin is involved in the regulation of follicular growth/steroidogenesis and pathogenesis of PCOS is unknown. Our objective is to better understand the complex regulatory mechanisms involved in the control of these processes and gain insights in their dysregulation in the pathogenesis of PCOS. We hypothesize that: (a) hyperandrogenism induces small and medium antral follicle growth arrest and ovarian structural changes, resulting from granulosa cell and oocyte apoptosis and theca cell survival, and (b) chemerin regulates follicular growth and steroidogenesis and contributes to the pathogenesis of PCOS. Using immature rats (day 13～15 for follicle culture and day 21～24 for granulosa cells culture) and a chronically androgenized rat model [dihydrotestosterone (DHT); 83 μg daily, day 21～105] which recapitulates the reproductive and metabolic phenotypes of human PCOS, we have examined the granulosa cell expression patterns of chemerin and its receptor CMKLR1 and their steroidogenic and follicle growth capability. DHT treatment resulted in decreased follicle numbers in preantral to preovulatory stages and absence of corpus luteum, but increased numbers of condensed atypical follicles. Atypical follicles, constituted predominantly of theca cells, exhibited high expression of calpain and down‐regulation of the cytoskeletal protein substrates vimentin, fodrin and β‐tubulin. Granulosa cell aromatase expression was significantly down‐regulated, a response accompanied by increased activated caspase‐3 content and DNA fragmentation. While PTEN levels were considerably higher in granulosa cells in the PCOS rats than controls, phospho‐Akt (Ser473) content was lower. In addition, DHT also activated granulosa cell caspase‐3, decreased XIAP, PARP and phospho‐Akt contents and induced apoptosis in vitro, responses that could be attenuated by forced expression of XIAP. These findings are consistent with our hypothesis that dysregulated follicular growth in PCOS is associated with changes in follicular growth dynamics and follicle cell fate, a consequence of dysregulated interactions of pro‐survival (p‐Akt, XIAP, PARP) and proapoptotic (calpain, PTEN, caspase‐3) modulators in a cell‐specific manner. Chemerin and CMKLR1 were expressed in granulosa cells and negatively regulated by gonadotropin in vivo and in vitro. Serum and ovarian chemerin levels in DHT‐treated rats were elevated, and associated with arrested early antral follicular growth, remodeling of the follicle wall and decreased expression of p450 side‐chain cleavage enzyme (p450- scc), aromatase and hydroxysteroid dehydrogenases. Recombinant chemerin inhibited FSH ‐ induced estradiol secretion in granulosa cells from DHT‐treated rats in vitro. Chemerin also suppressed basal and FSH‐ and GDF9‐induced follicle growth and estradiol/ progesterone production in preantral follicle cultures. Moreover, chemerin suppressed FSH‐induced p450scc/aromatase expression and progesterone/estradiol secretion in immature rat granulosa cells in vitro. These studies demonstrate that chemerin is a novel negative regulator in FSH‐induced follicular growth and steroidogenesis and support the notion that the dysregulation of chemerin expression and function contributes to pathogenesis of PCOS. Our observations also suggest that this chronically androgenized rat model may be useful not only for studies on the long term effects of androgen on folliculogenesis, but also on the pathophysiology of PCOS. * This work was supported by grants from the Canadian Institutes of Health Research (CIHR; MOP‐119381) and the World Class University (WCU) program through the Ministry of Education, Science and Technology funded by the National Research Foundation of Korea (R31‐10056).

장기이식분야는 효과적인 면역억제제의 개발과 더불어 비약적으로 발전하여 현재는 다양 한 장기이식이 활발히 시행되고 있다. 그러나 면역억제제의 장기 복용으로 인하여 감염은 물론 장기적으로 암이나 심혈관계 질환 등의 합병증은 여전히 문제가 되고 있다. 이를 극복 하기 위해서 다양한 시도들이 이루어지고 있다. 첫째는 고형 장기이식과 동시에 골수이식을 시행하여 면역관용을 유도하고 면역억제제 복용을 중단함으로써 약제에 의한 장기 부작용을 차단하려는 시도이다. 둘째는 현재도 여 러 장기이식들에서 면역억제제를 끊고도 이식 장기의 기능이 유지되는 임상적으로 면역관용 상태인 환자들이 있다. 면역관용 상태를 감시할 수 있는 검사법을 개발한다면 향후 환자들 에서 이식 후 주기적으로 면역관용 검사를 시행하고 관용 상태로 나타날 경우, 면역억제제 를 끊는 시도를 할 수 있고 면역관용 검사에서 관용으로 갈 확률이 낮을 경우 지속적으로 면역억제제를 사용하는 환자 개인별 맞춤형 면역억제 치료를 할 수 있을 것으로 기대한다. 한편 이런 기술들이 발달하더라도 고령화와 만성병의 증가로 공여 장기의 부족은 여전히 중요한 문제가 될 전망이기 때문에, 이종장기를 이용하여 장기이식의 수급 불균형을 해소 하 고자 하는 시도가 있다. 하지만, 이종이식의 경우 동종 이식 시 보다 격렬한 거부반응이 발 생하는 것으로 알려져 있으며, 돼지에 다량 존재하는 알파갈에 대한 인간의 자연항체 반응 에 의한 초급성 거부반응이 발생하여 이식된 장기가 수분 또는 수시간 내 급속히 파괴된다. 현재 초급성거부반응은 알파갈 적중 돼지의 개발과 인간의 혈청보체 조절인자를 발현하는 형질전환돼지의 개발로 어느 정도 극복이 되었지만, 초급성거부반응 극복 이후에도 여전히 급성혈관성 거부반응이 발생하며, 혈관내피세포가 2차적으로 활성화되어 선천성 면역세포 의 침윤과 함께 혈액응고 현상이 나타난다. 따라서 요즈음은 초급성거부반응 이후의 다양한 급성혈관성 거부반응을 조절하기 위한 다중 형질전환 무균돼지를 생산하는 기술이 개발되었다. 알파갈이 결손된 돼지의 장기를 영 장류에 이식한 경우에 혈관 내피세포가 활성화되어 섬유소가 침착되며, 선천성 면역반응계 세포의 침윤이 일어나는 것이 보고된 바 있다. 이러한 거부반응을 극복하기 위해서 선천성 면역반응 조절과 함께 항응고 유전자를 과발현 시키거나, 섬유소 분해를 촉진하는 유전자 를 도입한 형질전환돼지의 개발이 시도되고 있으며 우리나라에서도 이 분야 연구가 진행 중이 다. 장기이식에 비해 췌도 이식은 혈관을 연결하는 과정이 없기 때문에 이종이식거부반응의 강도가 약하고 시술의 위험이 적어서 전 세계적으로 이종췌도이식에 대한 연구가 활발히 진행되고 있다. 특히, 2009년 데이비드 쿠퍼 그룹에서는 혈청보체 조절인자인 hCD46를 발 현하는 형질전환돼지의 췌도를 영장류에 이식하였을 때 최장 1년까지 인슐린 비의존성을 보인바 있으며 벨기에 듀프렌은 돼지의 췌도를 생체 친화적 소재로 캡슐레이션하여 영장류 에 이식하였고 1년 가까운 생존율을 보고하였다. 이와 같은 이종장기이식은 장기이식 분야 의 중요한 미래 기술로서 장기 기증에 제한적인 문화배경을 가지고 있으며, 고령화 사회에 급속히 들어서는 우리나라에 필수적인 분야로 생각되며, 췌도, 파킨슨병, 각막 이식 등으로 부터 시작하여 고형장기로 그 시도가 확대될 것으로 전망된다. 한편, 다른 방향으로 줄기세포를 이용하여 분화시킴으로써 세포이식에 이용하려는 노력도 계속되고 있다. 최근 역분화 줄기세포 유도 기술이 발달됨에 따라 향후 세포이식 분야와의 접목이 기대되고 있다.

Animal genomics and breeding center works for development of livestock industry through development of breeding technologies based on genomes. Through analysis technology of genomic information with commercialization of DNA chip and development of NGS technique at present, we can select and improve superior breeding stock. DNA chip technique using microarray can analyze millions of SNP genotypes in a short period and we are studying these techniques to make a tool for genomic selection. In the United States, they made a guideline for genomic selection in dairy cattle and this guideline is utilized. In addition Semex company and CRV center use genomic selection for Holstein dairy cattle. Semex says genomic selection reduce two years compared to the existing selection, cost will be shortened 50% and improving speed will be more than 30% accelerated. In Australia, the case of using genomic information has more 10% accuracy than the case of using parent's breeding value without phenotype information. Recently development of NGS technology leads to reduction of analysis costs, increase in analysis data quantity and shorten time of analysis genome. NGS technology is innovative tool in life science. With development of NGS technology, we can expect to increase the efficiency of genomic analysis. Development of NGS technology leads us to expand whole genome study from limited gene study. Human and rodential genome is researched over the past five years, but only recently lots of livestock's genomes like cattle and pig are researched. Also for domestic, studies on livestock genome and genomic information are accomplished but we have a poor infrastructure of genomic analysis. Thus, through the application technology using SNP chip data and NGS, new breeding technology is very important for prior occupation. Animal genomics and breeding center has four strategies and these are divided by application technology. 1. Development of animal breeding and statistical genetics based on genomic information. 2. Development of genomic analysis and application technology through analysis of genetic diversity and structure. 3. Registration of traditional breeds and securing intellectual property rights based on the genome of the unique genetic resources. 4. Development of technologies for improvement of disease resistance and economic traits.

Animal biotechnology is the use of science and engineering to modify living organisms. Examples of animal biotechnology include creating transgenic animals (animals with one or more genes introduced by human intervention), using gene knock out technology to make animals with a specific inactivated gene and producing nearly identical animals by somatic cell nuclear transfer (or cloning). Animal biotechnology in use today is based on the science of genetic engineering. Under the umbrella of genetic engineering exist other technologies, such as transgenics and cloning, that also are used in animal biotechnology. Th main purpose of production of transgenic animals are to produce pharmaceutical proteins for human use. Scientists use reproductive cloning techniques to produce multiple copies of mammals that are nearly identical copies of other animals, including transgenic animals, genetically superior animals and animals that produce high quantities of milk or have some other desirable trait. To date, cattle, sheep, pigs, goats, horses, mules, cats, rats and mice have been cloned, beginning with the first cloned animal, a sheep named Dolly, in 1996. In addition to the use of transgenics and cloning, scientists can use gene knock out technology to inactivate, or “knock out,” a specific gene. It is this technology that creates a possible source of replacement organs called xenotransplantastion. In this talk I deal with current and future of animal biotechnolgy. In addition I would like to introduce Center for the Animal Bioreactors and Xenotranplantaion supported by Rural Development Administration, Korea.

Genomic reprogramming factors in the GV cytoplasm improved cloning efficiency in mice through the pre‐exposure of somatic cell nuclei to a GV cytoplasmic extract prior to nuclear transfer. To overcome difficulties in preparing mice oocyte extract, a pig GV oocyte extract (pGV extract) was developed to investigate the epigenetic reprogramming events in treated somatic cell nuclei. The pGV extract promoted colony formation concomitant with the expression of stem cell markers and repression of differentiated cell markers in treated cells. Using fibroblasts transfected with human Oct‐4 promoter‐ driven enhanced green fluorescent protein (Oct4‐EGFP), pGV extract treatment induced the reactivation of the Oct‐4 promoter in Oct4‐EGFP cells by 10 days post‐treatment. Interestingly, reconstructed embryos with pGV extract‐treated Oct4‐EGFP fibroblast nuclei showed prolonged expression of Oct4 in the ICM of embryos. Using donor nuclei treated with pGV extract, increase the number of high‐quality blastocysts that expressed Me‐H3‐K9, Oct4 and Nanog at levels comparable to in vitro fertilized embryos. The pGV extracttreated fibroblast cells can differentiated into neuronal, pancreas, cardiac, and endothelial lineages that were confirmed by antibodies against specific marker proteins. These data provide evidence for the generation of stem‐like cells from differentiated somatic cells by treatment with GV oocyte extracts in pig. Next, we identified germ line stem cells that supported oogenesis. female germ line stem cells (FGSC) from neonatal pig was established and cultured for more than 6 months. After long‐term culture and many passages, ovarian germ line stem cells maintained their characteristics and telomerase activity, expressed germ cell and stem cell markers and revealed normal karyotype. To further study developmental potential of oocyte‐like cells generated from FGSCs, these cells were aggregated with granulosa cells collected from neonatal pig ovaries. Interestingly after overnight culture in hanging drops, oocyte‐like cells aggregated with granulosa cells and formed structures very similar to primordial follicles containing the oocyte‐like cell in the middle and a layer of granulosa cells around it. Our results demonstrate the presence of a population of germ line stem cells in postnatal pig ovary with the ability to self‐renew and differentiate to oocyte‐like cells that might be useful for follicle engineering and assisted reproductive technologies. However, the functionality of FGSC‐derived oocytes us-ing in vitro maturation, fertilization and embryo development as well as ovarian transplantation is currently under investigation. In conclusion, gene manipulation of FGSCs or iPS cells is a rapid and efficient method of animal transgenesis and may serve as a powerful tool for biomedical science and biotechnology.

SERPINB3 (also known Squamous cell carcinoma antigen 1, SCCA1) is involved in apoptosis, immune response, cell migration and invasiveness of cells. It has been investigated in various types of squamous cell carcinoma. Therefore we investigated the functional role of SERPINB3 gene in human epithelial ovarian cancer (EOC) using laying hens, the most relevant animal model. In 136 laying hens, EOC was found in 10 (7.4%). We compared the expression and localization of SERPINB3 using RT-PCR, quantitative RT-PCR, in situ hybridization and immunohistochemistry, and SERPINB3 activation was detected in chicken and human ovarian cancer cell lines using immunofluorescence microscopy. Thereafter, we examined the prognostic value of SERPINB3 expression in patients with EOC by multivariate linear logistic regression and Cox’ proportional hazard analyses. In present study, SERPINB3 mRNA was induced in cancerous ovaries (p< 0.01), and it was only expressed in the glandular epithelium(GE) of cancerous ovaries of laying hens. SERPINB3 protein was localized predominantly to the nucleus of glandular epithelium in cancerous ovaries of laying hens, and it was abundant in the nucleus of both chicken and human ovarian cancer cell lines. In 109 human patients with EOC, 15 (13.8%), 66 (60.6%) and 28 (25.7%) of those patients showed weak, moderate and strong expression of SERPINB3 protein, respectively. Strong expression of SERPINB3 protein was a prognostic factor for platinum resistance (adjusted OR, 5.94; 95% Confidence Limits, 1.21-29.15). Therefore SERPINB3 may play an important role in ovarian carcinogenesis and be a novel biomarker for predicting platinum resistance and a poor prognosis for survival in patients with EOC. This research was funded by the World Class University (WCU) program (R31-10056), Basic Science Research Program (2010- 0013078) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology and by the Next-Generation BioGreen 21 Program (No.PJ008142), Rural Development Administration, Republic of Korea.

체세포 핵이식은 형질전환 복제 동물 생산과 더불어 그에 따른 바이오 신약의 개발, 장기 생산 등 많은 장점이 있지만, 여전히 체세포 핵이식 동물의 생산성은 임신율이 낮고 비정상 적인 개체의 탄생 등의 문제점이 있다. 그 이유 중 하나로 핵이식에 사용되는 공여세포가 다시 수정란으로 돌아가는 과정에서 후생학적 역분화가 불완전하게 이루어지기 때문이다. 본 연구는 체세포가 유도만능 줄기세포로 역분화하는 과정에서 사용되는 리프로그래밍 전 사인자 (Oct4, Klf4, Sox2와 c-Myc, OKS-M)의 도입과 더불어, 후생학적 변형에 관련된 억 제제 trichostatin A(TSA), 5-aza-20-deoxycytidine(5-aza), GSK-3 inhibitor와 MEK inhibitor (2i)가 복제 수정란에 미치는 영향에 대해서 연구하였다. 젖소 귀 세포에 전사인자 Oct4, Klf4, Sox2와 c-Myc을 도입하였고, 배양 시간이 흐름에 따라 세포크기가 작아짐 (11.72± 3.39, 8.42±4.95, p<0.05)을 볼 수 있었으며, RT-PCR을 통하여 8개의 콜로니 중 4개의 콜 로니에서 외인성 유전자를 발견하였다. 리프로그래밍에 관련된 내인성 유전자의 활성을 증 가시키기 위하여 HDAC 억제제인 trichostatin A (20 nM), DNA methyltransferase 억제제인 5-aza-20-deoxycytidine (10 μM), 줄기세포 분화 경로 억제제인 GSK-3 (3 μM) and MEK (1 μM)를 처리하였다. 4개 중 1개의 콜로니에서 내인성 유전자의 활성이 증가됨을 발견하였 다. H3K9/K14의 acetylation 상태는 큰 차이를 보이지 않았다. 그러나 체세포 핵이식의 분 할률에서는 somatic cells이 85.9±8.98%, OKS-M 처리군이 82.0±4.97%, OKS-M을 도입한 체세포에 TSA, 5-aza, 2i 처리군이 각각 88.4±7.89, 75.3±8.10, 74.2±2.90%로 OKS-M과 TSA를 함께 처리하였을 때 가장 높은 분할률을 보였고, 배반포와 상실배기 까지의 발달률 은 somatic cells이 9.6±3.79%, OKS-M 처리군이 12.6±6.54%, OKS-M을 도입한 체세포에 TSA, 5-aza, 2i를 처리하였을 때 각각 11.1±6.87, 20.1±5.89, 9.5±1.53%로 OKS-M과 5- aza 를 함께 처리하였을 때 유의적으로(p<0.05) 가장 높은 발달률을 보였다. 따라서 전사인자의 도입과 후생학적 변형과 관련된 억제제의 처리는 소 복제 수정란의 발달률 향상에 영향을 주는 것으로 나타남에 따라, 앞으로 다양한 억제제와 처리조건에 따 라 복제수정란의 향상을 위한 최적화된 방법을 유도할 필요가 있다.

In ruminants, Interferon-τ (IFN-τ) has the role of recognizing pregnancy signals produced by the embryo and it may have an important role during the luteolysis. Therefore, the purpose of the present study was to investigate the effect of IFN-τ on prostaglandin synthesis, cyclooxygenase-2 (COX-2) gene expression in vitro and secretion of progesterone (P4) in vivo. The epithelial and stromal cells isolated from bovine endometrium were cultured with different doses of IFN-τ (0, 0.02, 0.2 and 2 μg/ml). Human chorionic gonadotropin (hCG, 1.5 IU/ml) was used as a positive control. Prostaglandin E2 and F2α levels in the culture media were analyzed by enzyme immunoassays, and total RNA was extracted from the cells for RT-PCR. P4 concentrations in blood samples were assayed by chemiluminescent immunoassay system. In epithelial cells, COX-2 gene expression was increased in the presence of IFN-τ (p<0.05), but it was not significantly different in all groups of stromal cells except 2 μg/ml IFN-τ group (p<0.05). Although IFN-τ did not affect PGE2 and PGF2α production in epithelial cells, it decreased PGE2 and PGF2α production significantly in stromal cells (p<0.05). In vivo experiment, the P4 concentrations in blood sample was significantly increased after injection of 1 μg/ml IFN-τ. These results indicate that PG production was mediated by COX-2 expression in the stromal cells but it did not affect in the epithelial cells, and suggest that treatment of IFN-τ was to improve the implantation environment of uterine by maintenance of high P4 concentration. * This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ907008)” Rural Development Administration, Republic of Korea.

Live offspring is obtained from in vitro production of porcine embryos, but the procedure is still associated with great inefficiencies. In mammalian oocytes, acquisition of meiotic competence coincides with a decrease in general transcriptional activity at the end of the oocyte growth phase. In this study, we investigated the expression and sub-cellular localization of positive transcription elongation factor P-TEFb (CDK9/Cyclin T1), a RNA polymerase II CTD kinase during pig oocyte growth and early embryonic development. Localization and expression of components involved in mRNA and rRNA transcription were assessed by immunocytochemistry in growing and fully-grown oocytes. In addition, meiotic resumption, germinal vesicle breakdown, nuclear transcription and embryonic genome activation (EGA) were analyzed in oocytes and embryos cultured in presence of a potent CDK9 inhibitor, flavopiridol. Our analyses, demonstrated that CDK9 became co- localized partially with phosphorylated Pol II CTD and mRNA splicing complexes. Surprisingly, CDK9 was co-localized with Pol I-specific transcription factor, UBF, and gradually localized in nucleolar peripheries at the final steps of oocyte growth. Later, CDK9 became associated with nucleolar structures at 4-cell stage. Treatment with flavopiridol resulted in arrest in meiotic resumption, germinal vesicle breakdown as well as a decline in global transcription. Flavopiridol also inhibited embryo development beyond EGA. All together, these data suggest that CDK9 has a dual role in both Pol I- and Pol II-dependent transcription in pig oocyte growth and embryonic development.

XIST has been known to long-non coding RNA which regulate X-chromosome inactivation in female mammal and the gene has been suggested to having important role in early embryo development and embryonic stem cell. However, its coding region has been unclear in pig. To determine the coding region of XIST in pig, we have examined candidate site of XIST coding region in pig by BLAST, PCR, and sequencing. By comparing pig whole genome sequence (Sus scrofa 10.2) with human, murine, and bovine XIST transcript sequence using BLAST, we selected candidate coding region of XIST in pig. The result showed XIST is coded on the minus strand of NW_003612825 contig and its length was nearly 32kb which was similar to the length of human and bovine XIST gene. With the candidate model, we performed RT-PCR to confirm the coding region of XIST with 24 primer pairs and they were expressed only female porcine embryonic fibroblast (PEF) but not in male PEF. By designing candidate intron spaning primer we could confirmed candidate intron is present between first and last exon (distance, 9.2kb vs product size, 2kb). The seqeucne of amplicon was analyzed and we could confirmed there were 5 small exons (less than 400 bp) like XIST coding region of other species which have 4 to 5 small exon between first and last exon. To confirm coding strand in pig, we conducted strand specific reverse transcription. We confirmed candidate XIST was coded on the negative strand of contig on X-chromosome as the result of homology analysis by BLAST. With the candidate pig XIST sequence, we aligned the sequence with XIST sequences of 3 species, human, mouse, and bull by clustalW. These result showed candidate sequence of pig XIST is most similar to that of bovine and the homology between pig and human was higher than result between mouse and human. These results could support for X chromosome inactivation analysis and the function of XIST in pig preimplantation embryos. * This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2012006276)

The objective of the current study was to describe in vitro embryo production in Hanwoo, analyzing oocytes yield and embryo production. The effects of oocytes production and the number of OPU procedures per animal on embryo production were also evaluated. OPU was done every 3～4 days during experimental period and collected oocytes were fertilized in vitro in both OPU and needle puncture groups. First, we compared the recovery rate of oocytes based on OPU session (Experiment 1). The average of collected oocytes was calculated from every 10 session. The average number of total oocytes recovered per animalonsessionwas 5.16 (mean). Second, we compared the recovery rate base on collection period of OPU (Experiment 2). The following results show the difference of the number of recovered oocytes in every month during the procedure between the months of session. Every animal shows the constant number of recovered oocytes for the first 5 months. However, the recovery rate of oocytes was decreased from month 6 to 8. Third, we compared the developmental rate to blastocyst in two groups (Experiment 3). Oocytes by needle puncture were fertilized with frozen-thawing semen; the cleavage rate 24～48 h after in vitro fertilization (IVF) was 75.8% and blastocyst development rate was 18.8% in needle puncture group. Even though there is lower cleavage rate after IVF in OPU group (61.1%), blastocyst development rate was higher compared with needle puncture group (28.4%). In conclusion, Blastocyst developmental rate could be increased by OPU than classical method of needle puncture. Improvement of bio- technique in collecting oocytes could be applied to understand the reproductive physiology in cattle, expecially Hanwoo. Therefore, further investigation should be done to clarify the efficiency and advantage of OPU involved in reproduction in animals and human being.

유즙 내에 들어있는 유용 단백질을 분리, 정제하였을 때 예상되는 경제적 가치 때문에 우 리는 유즙 내에 들어있는 재조합 hEPO 단백질을 추출하고 정제하려는 많은 시도를 하고 있다. 이러한 노력에도 불구하고 유즙 내에 들어있는 target 단백질의 정제가 매우 어렵고, 그 순도 역시 20%에 지나지 않는다. 우리는 hEPO 유전자를 가지고 있으며 유선에서 hEPO를 분비하는 형질전환 돼지를 생산 보유하고 있다. 그러나, 이들 형질전환 돼지들은 체내에 도입된 유전자의 발현에 의해 야기되는 적혈구 과다증 그리고 혈소판 감소증과 같 은 순환기의 문제가 유발되고 있다. 명백하게, 이러한 종류의 혈액학적 변화는 장기의 정상 적인 기능을 방해하며 형질전환 돼지의 갑작스런 죽음을 초래한다. 그러므로, 앞서 언급한 문제점들을 해결하기 위한 alternative한 방법이 필요하다. 현재 연구에서, 우리는 외인성 hEPO를 발현하는 돼지에서 비정상의 생리학적 증후에 관해서 실험하였으며 hEPO를 생산 하기 위한 alternative choice로서 형질전환 돼지 유래 세포의 배양 system을 제안하였다. 5마리의 F6 hEPO 형질전환 돼지와 4마리와 대조군으로 일반돼지(랜드레이스)를 국립축 산과학원 동물바이오공학과에서 사육 도축하고 순환 장기를 채취하였다. 형질전환 돼지 순 환기 조직(유선, 신장, 폐, 비장)을 계대 배양 후 hEPO mRNA의 발현을 RT-PCR 방법으로 분석하였다. 또한, 면역조직화학 염색법을 통해서 hEPO 형질전환돼지 순환 장기조직에서 발현되는 hEPO의 발현 양상을 분석한 결과로서 유선, 신장 및 폐 조직에서 hEPO의 발현 이 확인되었으나, hEPO 발현 양상이 모든 형질전환동물 조직에서 발현되는 경향을 보이지 는 않았다. 한편, 세포면역화학 분석법에서는 형질전환돼지 유래 세포의 배양 시 hEPO가 발현되고 있는 양상을 확인하였다. 또한, 형질전환 돼지와 일반돼지의 세포 증식률과 증식 속도는 신장 세포에서는 빠른 증식을 나타낸 반면, 폐와 비장 세포에서는 느린 증식률이 확 인되었다. 배양 후 지속적인 세포 증식과 세포의 생존성을 분석하기 위하여 Apoptosis를 TUNEL과 Annexin V를 이용한 방법으로 조사한 결과, 형질전환돼지 유래의 세포와 일반돼 지의 세포 사이에 유의적 차이는 발견하지 못했다. 이들의 결과들로서 본 연구에서는 형질 전환돼지를 생산하여 유용 단백질을 이용하고자 할 경우, 목적으로 하는 세포 뿐 만 아니라 형질전환가축 유래의 다양한 세포를 이용 할 수 있는 가능성을 시사하고 있다.

Acteoside acts as an anti-oxidative activity and anti-apoptosis in the cells. But, it has been not studied on maturation and development of porcine oocytes. The aims of the present study were to examine the effects of acteoside on the morphological progress of meiosis, developmental competence, and ROS in porcine oocytes. Oocytes were matured in tissue culture medium-199, supplemented with acteoside at various concentrations: 0 (control), 10, 30 and 50 μM. The oocytes maturation rates of groups supplemented with acteoside were no significantly different (81.13, 85.96, 82.95 and 83.68%, respectively). Level of ROS was significantly decreased in acteoside treated group. Furthermore, the parthenogenetic blastocyst rate was significantly improved in 10 μM acteoside treated group compared with control group (44.83 vs. 27.75%). And we investigated effect of acteoside on the oocytes condition represented by cytoplasmic maturation by homogeneous distribution and formation of cytoplasmic organelles and regulation of apoptosis-related genes. In the results. during IVM, 10 μM acteoside treated oocytes showed that the mitochondria and lipid droplet were smaller and homogeneous distribution in cytoplasm compare with control oocytes. And reverse transcription polymerase chain reaction (RTPCR) of parthenogenetic blstocysts revealed that acteoside increased the anti-apoptotic genes (Mcl-1, Bcl-2 and Bcl-xL), whereas reduced the expression of pro-apoptotic genes (Bax and Bak). In conclusion, based on the results, the effect of acteoside on IVM was not attractive. However, in acteoside treated group, cytoplasmic maturation seemed to be improved with morphologically uniform distribution of cytoplasmic organelles. Furthermore, embryonic development in acteoside treated group was significantly highly increased than that of non-treated group. Our results represents that addition of acteoside to the IVM medium has a beneficial effect in physiology of porcine oocytes, providing a improved method for porcine oocytes in vitro. * This work was supported by a grant (Code# PJ008148) from BioGreen21 Program, Rural Development Administration, Republic of Korea.

돼지는 인간과 생리적으로 유사하기 때문에 다양한 목적으로 연구되고 있다. 최근에는 돼 지를 이용한 이종장기이식 관련 연구가 큰 주목 받고 있으며, 치료용 단백질을 생산하기 위 한 생체반응기로써 이용되고 있다. 이러한 연구에 있어서 당 사슬의 역할은 매우 중요하다. 당 사슬은 치료용 단백질의 체내 안정성에 큰 영향을 미치며 다양한 방법으로 면역을 조절 한다고 알려져 있다. 하지만 돼지에서의 당 사슬 관련 연구는 미비하며, 많은 당 전이효소 들의 서열이나 기능이 정확히 분석되지 않고 있다. 따라서 이번 연구에서는 당 전이효소 중 하나인 β‐1,3‐N‐acetylglucosaminyltransferase 1 (B3GNT1)을 돼지로부터 동정 후 PK‐15 세 포주를 이용하여 기능분석을 하였다. 이 유전자는 다양한 glycan epitope를 형성하는데 있 어서 중요한 기능을 한다. 먼저 간 조직으로부터 획득된 cDNA를 주형으로 degenerated PCR을 수행하여 유전자를 동정하였다. 동정된 유전자는 368개의 아미노산을 encoding하 는 1227개의 nucleotide로 구성되어 있으며, 다른 종에서 보고된 B3GNT1과 높은 상동성을 가 지고 있는 것을 확인하였다. 또한, 기능 분석을 위하여 돼지 신장세포인 PK‐15에서 B3- GNT1의 과 발현을 유도하였다. RT‐PCR과 Western blot을 통하여 유전자의 과발현을 확인 하고, Lycopersicon esculentum lectin (LEA)를 이용한 ELISA 분석 방법을 통해 효소의 기 능을 확인하였다. B3GNT1은 poly N‐acetylactosamine (polyLacNAc) 형성에 중요한 역할 을 하기 때문에 이를 특이적으로 인지하는 LEA를 사용하였다. 이를 통해 B3GNT1의 과발현이 유도된 세포에서 더 많은 polyLacNAc이 합성되었음을 확인할 수 있었다. 또한, Gal(β1‐ 3)GalNAc structure를 이용한 기질반응을 통해 효소의 기능을 확인하였다. 과발현이 유도 된 세포에서 약 3배 이상의 높은 기질 반응성을 보였으며, 이를 통해 돼지로부터 클로닝 된 B3GNT1의 기능을 확인할 수 있었다. 돼지로부터 당 전이효소를 동정하고 분석하는 연구는 생체반응기로써 돼지를 이용하는 다른 연구에 중요한 기반이 될 것이라고 생각한다

A recent study has reported that pluripotent stem cells can be categorized according to their pluripotent state. The first is a “naïve” state, which is characterized by small, round or dome-shaped colony morphologies, LIF and BMP4 signaling pathways and two active X chromosomes in female; mouse ES cells (mESCs) represent this type. A second “primed” state has also been described and is possible in mouse epiblast stem cells (mEpiSCs) or human ES cells (hESCs). These primed state pluripotent stem cells display flattened monolayer colony morphologies, FGF and Nodal/Activin signaling pathways and X chromosome inactivation in female. It has been suggested that, as a non-permissive species, the porcine species undergoes reprogramming into a primed state during the establishment of pluripotent stem cell lines. Meanwhile, a few studies have reported that primed pluripotent stem cell lines could be reverted to a naïve pluripotent state using various exogenous factors including GSK3β and MEK inhibitors, LIF, hypoxic conditions and up-regulation of Oct3 or klf4. Therefore, the purpose of this study was to investigate whether a LIF-dependent naïve pluripotent stem cell line could be derived from porcine embryonic fibroblasts(PEFs) via doxycycline (dox)-inducible reprogramming factors and LIF. In this study, we have been able to successfully induce PEFs into a LIF-dependent naïve pluripotent-like cell line showing a mESC-like morphology and the expression of pluripotent markers. Our results suggest the possibility of reprogramming to naive pluripotent- like stem cells from PEFs in porcine species. * This work was supported by the BioGreen 21 Program (PJ0081382011), Rural Development Administration, Republic of Korea.

The generation and application of porcine iPSCs (piPSCs) as a large animal model may enable the test for the efficacy and safety of the therapy in the field of human regenerative medicine. Here, we report the generation of piPSC from wild (a 10-day-old Massachusetts General Hospital miniature pig; MGH minipig) and genetically modified pig, alpha1,3-Galactosyltransferase knock-out (—/—) (GalT KO homo) and human CD46 (membrane cofactor protein) knock-in (hCD46 KI) MGH minipig (a 10-day-old). Fibroblasts were isolated from the ear skin of wild and MGH minipigs, respectively. After 2 passages, each of fibroblasts was transduced with cocktail of 6 human factors (POU5F1, NANOG, SOX2, C-MYC, KLF4, and LIN28) and cultured on a mitotically inactive mouse embryonic fibroblast (MEF) monolayer. Both of reprogrammed somatic cells expressed the classical pluripotency markers (POU5F1, NANOG, and SOX2) and surface marker (SSEA1). Similar to mouse ESCs, both piPSCs from wild and transgenic minipigs were negative for SSEA3, Tra-1-60, and Tra-1-81. Further these cells could form embryoid body (EB) and differentiate into 3 germ layers in vitro (ectoderm: FOXJ3 and PAX6, mesoderm: HAND2, and endoderm: SOX17 and GATA6). Our piPSCs may provide useful source as a large animal model for studying approaches that can reduce an immune- rejection of cell or organ transplantation.

S-adenosylhomocysteine hydrolase-like protein 1 (AHCYL1), also known as IP3 receptor- binding protein released with IP3 (IRBIT), regulates IP3-induced Ca2+ release in the cytoplasm of cells and, therefore, is likely to be an important gene regulating various biological processes in the oviduct of chickens. However, the identification of the AHCYL1 gene in chickens has not been investigated. Therefore, the objectives of this study were to examine the tissue- and cell-specific expression of AHCYL1 gene in chicken organs, especially in reproductive organ, and determine functional actions of AHCYL1 in chicken oviduct development via estrogen. The results indicated that AHCYL1 mRNA is expressed in chicken reproductive organs and DES(diethylstilbesterol, a synthetic estrogen agonist) stimulates the cell specific expression of AHCYL1 in immature chicken oviduct. These results suggest that AHCYL1 is a novel estrogen-stimulated gene associated with development of the chicken oviduct. Next, in the present study, we show that inhibition of Erk1/2 can block DES-induced AHCYL1 expression. Also, we found that knockdown of AHCYL1 expression down-regulates expression of oviduct specific genes and AHCYL1 expression is regulated at the post-transcriptional level by specific miRNAs. These results strongly suggest that estrogen-mediated AHCYL1 gene expression plays a crucial role in growth, differentiation and function of the hen oviduct. Also, our results will be useful for understanding the fundamental mechanism(s) of estrogen action responsible for development of hen oviduct. This research was funded by the World Class University (WCU) program (R31-10056), Basic Science Research Program (2010-0013078) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology and by the Next-Generation BioGreen 21 Program (No.PJ008142), Rural Development Administration, Republic of Korea.

The aim of this study was to examine the effect of acteoside (the cyclin-dependent kinase inhibitor) on the SCNT efficiency with adult fibroblasts in dog. Canine adult fibroblasts were obtained from muscle and cell cycle of fibroblasts was synchronized by culturing to confluency, serum starvation and treating with 30 μM acteoside for 48 h. Cell cycle stages, cell cytotoxicity (apoptosis) and, prduction of reactive oxygen species (ROS) were analyzed using flow cytometry. The canine cells, prepared by confluent-cell culture or treating with 30 μM acteoside for 48 h, were injected into enucleated in vivo matured oocytes, the couplets were electrical fused and activated by calcium ionomycin. SCNT embryos using acteoside-treated fibroblasts were surgically transferred into oviducts of estrus cycle synchronized recipient dogs. In cell cycle synchronization (G0/G1), there was no significant difference between serum starvations (83.9%) and acteoside treated groups (81.3%) that were higher than confluent group (78.5%). In production of apoptosis, confluent and acteoside treated groups (4.3 and 4.5%, respectively) were generated less than serum starvation group (21.8%). In case of ROS, serum starvation group was induced a significantly higher than other groups. After synchronization of the donor cell cycle, either confluent or acteoside treated, cells were placed with enucleated in vivo-matured dog oocytes, fused by electric stimulation, activated, and transferred into naturally estrus-synchronized surrogates. Fusion and cleavage rate of acteoside treated group were 64.1 and 41.5%, which were higher than those of confluent group (53.9 and 20.6%, respectively). The reconstructed embryo development rates to 4-cell and 8-cell in acteoside treated group were 29.5 and 14.8%, respectively, while confluent group showed 11.1 and 3.2%, respectively. Total 54 SCNT embryos using acteoside-treated fibroblasts were transferred into oviducts of 2 recipient dogs and one recipient finally delivered one puppy, whereas din`t detected pregnancy on transfer of cloned embryos reconstructed with confluent cells in 6 surrogate dogs. In conclusion, the results of the current study demonstrated that canine fibroblasts could be successfully arrested at the G0/G1 stage with reduced the formation of ROS and apoptosis after acteoside treatment. This results may contribute to improve the effi-ciency of canine SCNT. * This research was supported by iPET (Grants 110056-3), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

Na+/K+-ATPase, an energy-transducing ion pump, is responsible for maintenance of relatively high concentrations of potassium ions but low concentrations of sodium ions in the cell by transport of these ions across the plasma membrane and participates in transport of various nutrients including glucose, amino acids. and ions. Na+/K+-ATPase consists of α, β, and FXYD subunits, but only α and β subunits are needed for basic functions. FXYD subunit is an auxiliary protein for αβ complex of Na+/K+-ATPase. Our recent study has shown that α (ATP1A1-4) and β (ATP1B1-3) subunits of Na+/K+-ATPase are expressed in the uterine endometrium during the estrous cycle and pregnancy in pigs. In this study, we further determined expression of FXYD (FXYD1-7) subunits of Na+/K+-ATPase in the uterine endometrium during the estrous cycle and pregnancy in pigs. Real-time RT-PCR analysis showed that mRNAs for all subtypes of FXYD subunit were expressed in the uterine endometrium during the estrous cycle and pregnancy in a pregnancy status- and stage-specific fashion. In situ hybridization analysis exhibited that transcripts of all subtypes of FXYD subunit were primarily localized to luminal (LE) and glandular epithelia (GE) during the estrous cycle and early pregnancy and to chorionic membrane (CM) during mid to term pregnancy. RT-PCR analysis showed that FXYD subunits were expressed in conceptuses on D12 and D15 of pregnancy. These results indicate that all subtypes of FXYD subunit are expressed in the uterine endometrium and conceptuses during the estrous cycle and pregnancy in a pregnancy status- and stagespecific manner. These suggest that FXYD may be involved in the establishment and maintenance of pregnancy by regulating the activity of Na+/K+-ATPase in nutrient transport at the maternal-fetal interface in pigs. * This work was supported by the Next Generation BioGreen 21 program (#PJ007997), RDA and the National Research Foundation (NRF #2010-0012304) funded by the Korean Government, Republic of Korea.

There are diverse methods of cryopreservation of mammalian embryos with variable degrees of success. Although cryopreservation technique of mammalian embryos has been advanced, freezing stress affect to cellular event such as apoptosis and autophage in embryos. The objective of the study is to investigate the affection of to survival, development, live offspring, apoptosis and autophagy on embryo. Mouse embryos were vitrified and thawed using normal straw and modified cut standard straw (M-CSS), then in vitro cultured until blastocyst stage and transferred to recipient. Recovery rates (100 vs 99.2%), survival rates (99.2 vs 78.6%), developmental rates (18.4 vs 10.7%), total cell numbers (45 vs 37), preganacy rates (34.5 vs 25%) and offspring numbers (10.1 vs 4.9 %) of M-CSS group are significantly higher than those of normal straw vitrified group. Also, rate of apoptosis in blastocysts developed using M-CSS (1.9%) was significantly lower than using normal straw vitrification (2.7%). Apoptosis-related gene, caspase 3, was expressed at the highest level in blastocysts derived from normal straw group. However, no differences of autophagy related gene, Atg6 and expression of LC3 between normal straw and M-CSS groups were observed. In conclusion, the standard vitrification procedure induces mitochondrial apoptosis in zygotes in an autophagy-independent manner, whereas the novel M-CSS procedure may improve embryo vitrification.