칡소의 고유유전자표현형 방식은 E+/E+와 E+/e 2개의 유전자형만이 출현하고, e/e 유 전자형을 가지는 개체는 전혀 출현하지 않아 제주재래흑우에서와 같이 흑색 호반모가 발 현되기 위해서는 기본적으로 E+ allele이 필요한 것으로 보고됨에 따라 본 연구는 수정란 이식 기법을 이용한 울릉칡소 특화단지 조성 연구용역에 의해 생산되어진 칡소의 모색발 현의 현황에 대해 조사하여 칡소의 우량유전자원의 선발과 증식을 통하여 칡소의 고정과 복원을 위해 실시되었다. 조사에 공시한 칡소는 2007년 수정란 이식 기법을 이용한 칡소 울릉특화단지 조성 연 구용역에 의해서 생산되어진 칡소를 대상으로 실시하였으며, 수정란 이식 후 임신 5-6개 월에 울릉관내 농가에 입식된 한우에서 생산된 칡소(이하 입식우) 159두와 울릉도 관내 의 한우에 칡소 수정란을 이식하여 생산된 칡소(이하 관내이식) 117두를 조사 대상으로 공시하였다. 먼저 울릉칡소의 모색 분포는 황모 24.3%(67/276두), 흑모 13.0%(36/276두) 와 호반모 62.7%(173/276두)의 분포를 보였으며, 입식우의 호반모 출현율이 66%(105/ 159두)로 관내이식에 의해 생산된 칡소의 호반모 비율 58.1%(68/117두)보다 다소 높은 경향이었으며, 성별에 따른 모색의 분포를 조사한 결과 수컷의 경우 황모의 비율이 18.1 %(25/ 138두)로 암컷의, 31.4%(43/137두)에 비해 출현율이 낮은 반면 호반모의 비율은 68.1% (94/138두)로 암컷의 56.9%(78/137두)에 비해 다소 높게 나타났다. 을릉도 칡소 모색 분포에 따른 조사를 바탕으로 비경의 흑반점의 강약에 따른 호반모 의 발현 양상을 분석한 결과 비경에 흑반점이 강한 개체들 중 호반모의 발현 정도를 분 석한 결과 호반모 1, 호반모 2, 호반모 3, 호반모 4, 호반모 5의 비율이 각각 2.3% (3/ 109), 24.8%(27/109), 43.1%(47/109두), 19.3%(21/109두), 10.1%(11/109두)로 나타났으며 흑반점이 중인 개체는 각각 7.9%(3/38), 52.6%(20/38), 34.2%(13/38두), 5.3%(2/38두), 0 %(0/38두)로 나타났다. 또한, 흑반점이 약한 개체는 각각 42.5%(17/40두), 45%(18/40두), 7.5%(3/40두), 0%(0/40두), 5%(2/40두)로 나타났다. 이 결과로 미루어 볼 때 비경에 있는 흑반점의 강약이 호반모 발현에서 황모와 흑모의 비율에 영향을 미치는 것으로 판단된다.

Autophagy, the process of bulk degradation and recycling of long-lived proteins, macromolecular aggregates, and damaged intracellular organelles, has recently been shown to be important for pre-implantation development and cavitation in mouse embryos. This study investigated the occurrence of autophagy and its importance in determining the in vitro development of pig embryos produced by in vitro fertilization (IVF) or parthenogenetic activation (PA). Western blot analysis for autophagy marker, microtubule associated protein light chain 3 (MAP-LC3), revealed the temporal pattern of LC3-conversion with intense changes during 10 20 h post-insemination and at morula-blastocyst transition in pig embryos. Specific inhibition of autophagy in 2 4 cell stage pig embryos, by treatment with 3-methyladenine (3MA), did not affect their embryonic development up to morula stage (p>0.05) but completely blocked their progression to the blastocyst stage (0.0±0.0 vs. 28.5±1.7% p<0.05). On the other hand, autophagy-inhibition in morula stage embryos significantly inhibited the formation of blastocoel (14.9±3.6 vs. 37.5±7.2%) and reduced the proportion of expanded blastocysts (5.6±2.6 vs. 29.6± 4.6% p<0.05). TUNEL assay revealed that autophagy-inhibited embryos had significantly increased indices of apoptosis (10.2±0.4 vs. 2.3±0.2) and DNA fragmentation (0.8± 0.1 vs. 0.3±0.1) than those of controls (p<0.05). Interestingly, while anti-oxidants reduced (p<0.05) the apoptosis and improved the blastocyst formation rate in pig embryos, it had no influence (p>0.05) on the expression of MAP-LC3. These data therefore, suggest that autophagy may have essential role during blastocyst formation in pig embryos.

Culture of preantral follicles has important biotechnological implications through its potential to produce large quantities of oocytes for embryo production and transfer. The objective of this study was to determine the comparison of different isolation method of mouse preantral follicles, and to examine in vitro development of mouse preantral follicles isolated by different method. Preantral follicles were mechanically or enzymatically extracted from mouse ovaries. Mechanical isolation method used fine gauge needles and enzymatic method of isolating follicles used 1 mg/ml collagenage (Type IA) and 0.2 mg/ml DNase Ⅰ in Leibovitz L-15 medium. The solution containing Leibovitz L-15 medium, enzyme and ovary fragments was incubated at 37℃ for 30 min. The selection criteria are as follows: primary follicle of 75 to 99 μm, early secondary follicle of 100 to 125 μm and late secondary follicle of 126 to 150 μm in diameter. The recovered preantral follicles were cultured for 10 days in alpha-minimal essential medium (α-MEM) + 5% FBS + ITS + 100 mIU/ ml FSH. The collected primary follicles by enzymatic treatment were higher than mechanical method. Others stage preantral follicle by mechanical isolation were higher than enzymatic method. After 10 days of culture, no statistical differences were shown in survival rates of preantral follicle among the 2 culture groups. The metaphase Ⅱ rates of the oocytes were significantly higher (p<0.05) in mechanical method (17.8%) than in enzymatic method (5.1%). These results suggest that the isolation method of choice depends on the target stage preantral follicles and mechanical isolation is an optimal method of preantral follicles in a culture of mouse preantral follicle.

The necessity of conditional gene expression in pigs for transgenic models is raised. Thus, in this study, Cre-loxP conditional expression in porcine fetal fibroblasts was investigated and the transformed fibroblasts were reprogrammed in enucleated oocytes for further early embryonic development. Fetal fibroblasts from miniature pigs were used for transfection with pCALNL-DsRed including floxed neomycin resistant gene and selected with 750 ug/mL neomycin for two weeks. The transfected cells did not express DsRed under fluorescence microscope. After transient transfection of plasmid DNA expressing Cre, the fibroblasts began to express DsRed. The cells expressing Ds- Red were employed into somatic cell nuclear transfer (SCNT). A total of 121 oocytes were used for SCNT and 76 cloned embryos (62.8%)were cleaved. Six blastocysts were grown up after SCNT and expressed DsRed. Deletion of floxed neomycin resistant gene was confirmed by RT-PCR in cloned blastocysts. Taken together, this study demonstrated that Cre-loxP recombination in miniature pig fibroblasts were successfully worked and those sequential transformed cells were developed into pre-implantation stage via SCNT.

Preservation of sperm is essential for long-term storage of valuable animal genetic resources and for the conservation of threatened mammalian species undergoing progressive extinction. In this study, using pig as a model system, we evaluated the feasibility of germ-plasm preservation via sperm cell lyophilization. We show that, pig sperm can be successfully lyophilized and stored in a liquid nitrogen-free condition for at least 6 months. Intracytoplasmic injection of lyophilized sperm (ICSI), stored at 4℃ for four months, into in vitro matured pig oocytes could successfully develop up to blastocyst stage (13.0±3.0%). Lyophilized sperm could also be stored at room temperature for at least three weeks without further compromising their in vitro development up to the blastocyst stage (14.6±3.2 vs. 16.6±5.1%; p>0.05). Blastocysts produced from ICSI of lyophilized sperm stored at 4℃ or room temperature contained similar number of cells per blastocyst (44.9±3.2 vs. 44.0±4.3; p>0.05) but was significantly lower than those produced from non-lyophilized fresh sperm (52.1±5.8 p>0.05). Interestingly, use of a custom-designed HEPES-buffered, calcium-free, defined medium for the lyophilization resulted in normal post-ICSI embryonic development up to blastula stage (23.4±2.8 vs. 24.0±2.9%) and, the resultant blastocysts contained similar number of cells per blastocyst (47.9±4.3 vs. 50.6±7.0) compared to those generated from non-lyophilized fresh sperm (p>0.05). These lyophilized sperm could also be stored at room temperature for at least three weeks with slight reduction in post-ICSI embryonic development (19.6±1.4%). Therefore, these results suggest that, pig sperm could be successfully and efficiently lyophilized for their long-term storage at 4℃. Lyophilization of sperm could be a practical option for long-term storage of mammalian germ-plasm.

한우(韓牛, Korean Cattle, Bos taurus coreanae)는 모색에 따라 황소, 칡소, 흑소로 나 누어지고 있다. 칡소는 현재 1,700여두가 전국에 사육되고 있는 것으로 추정하고 있다. 그러나 사육기반이 취약하여 멸종위험종으로 분류하고 있다. 따라서 본 연구에서는 칡소 대량 증식에 필요한 수정란이식의 효율성 제고를 위하여 FSH 투여 용량에 따른 과배란, 수정란회수, 이식후 임신율 및 송아지 생시체중을 조사하였다. 공란우는 각각 시험군에 10두씩 배치하였고, FSH 용량은 280 mg 4일 투여 및 240 mg 3일투여법을 사용하였다. 과배란 처리 및 수정란회수는 일반적인 수정란 채란 프로그 램을 준용하였다. FSH 240 mg 및 280 mg 투여시 발정유기는 87.5, 85.3%, 과배란은 81.3, 79.4%로서 차이가 없었다. 수정란 회수 성적이 평균 황체수는 240 mg 및 280 mg 처리군에서 각각 16.5개, 13.6개, 총 수정란수는 각각 7.5±1.8, 6.9±1.8, 이식 가능한 수정란수는 각각 4.1± 1.2, 3.5±1.0, 변성란 1.4±0.6, 2.3±1.1, 미수정란 2.0±0.9, 1.1±0.5개로 유사한 경향이었 다. 용량별 처리군에서 회수된 1등급과 2등급의 수정란을 이식한 후 임신율은 240 mg 군이 38.7%, 280 mg 군은 46%로 280 mg 군이 차이가 없었다. 칡소의 수정란이식 및 인공수정으로 태어난 송아지의 생시체중과 성비를 조사한 결과, 수정란이식으로 태어난 송아지의 생시체중은 24.8±2.9, 인공수정으로 태어난 송아지가 25.6±3.3 kg으로 다소 높 았지만 유의차는 없었다. 본 연구를 통하여 칡소에서 수정란 생산시 FSH 용량에 따른 과배란 및 수정란회수 성 적은 차이가 없었다. 그러나 FSH의 용량이 감소하여도 유사한 결과로서 경제적으로 효 과가 있을 것으로 생각된다.

Plasma glutathione peroxidase (pGPx) is an extracellular antioxidative selenoenzyme which has been detected in various adult tissues, but little is known about the expression and distribution of pGPx during embryogenesis. To investigate the expression patterns of pGPx during embryogenesis, we performed quantitative real-time PCR, in situ hybridization, Western blot, and immunohistochemistry analyses in whole embryos or each developing organ of mice on embryonic days (E)7.5–18.5. In whole embryos of E7.5–8.5, pGPx mRNA was more typically expressed in extra-embryonic tissues including ectoplacental cone, trophectoderm, and decidual cells than in embryos. However, after E9.5, pGPx mRNA and protein levels were increased in the embryos with differentiation and growth, but trended to gradually decrease in the extra-embryonic tissues until E18.5. In sectioned embryonic tissues on E13.5–18.5, pGPx mRNA and protein were mainly expressed in the developing nervous tissues, the sensory organs, and the epithelia of lung, skin, and intestine, the heart and artery, and the kidney. In particular, pGPx immunoreactivity was very strong in the developing liver. These results indicate that pGPx is spatio-temporally expressed in various embryonic organs as well as extra-embryonic tissues, suggesting that pGPx may function to protect the embryos against endogenous and exogenous reactive oxygen species during organogenesis.

The objective of this study was to evaluate the comparison of production efficiency of oocytes and OPU (ovum pick‐up) derived embryos of Hanwoo with Holstein. The OPU session of each species (6 cows) was carried out from the Hanwoo (106 sessions) and Holstein (114 sessions) at intervals of 3 4 days (2 times per week) for 3 months. Cumulus‐oocyte‐complexes (COCs) retrieved were classified into 4 grades by the status of oocyte cytoplasm and cumulus cells. The COCs collected were matured in vitro in TCM‐199 supplemented with 10% FBS, 10 mg/ml FSH and 1 mg/ml estradiol‐ 17β in 5% CO2 and over 99% humidity for 24 h. After 24 h co‐incubation with post‐thaw sperm, the presumed zygotes were cultured in CR1aa medium with 4 mg/ml BSA for 3 days and then changed CR1aa medium with 10% of FBS for another 3 4 days. The Mean number of aspirated follicles and collected oocytes were not significantly different between Hanwoo and Holstein spacies (10.4±0.42 vs. 11.4±0.41 and 7.5±0.38 vs. 6.1±0.37 per session). But the collection rate of oocytes from aspirated follicles were significantly higher in Hanwoo (72.8%) than that in Holstein (53.6%) (p< 0.05). Furthermore, the average number of good quality oocytes (Grade I and II) was 5.9±0.28 and 4.1±0.27 (Mean±SD), and the cleavage rate and the development rate to blastocysts was significantly (p<0.05) higher in Hanwoo (40.0%) than Holstein (21.6%). The OPU derived embryos of Hanwoo were transferred 83 times into 52 recipients and then 42 calves were produced from 44 pregnancy recipients. In conclusion, the efficiency of OPU derived embryo was significantly different between Hanwoo and Holstein species. In vitro culture system for OPU derived embryo production should be optimized for industrialization and the improvement of livestock.

번식우에서 저영양이 지속 될 경우 GnRH 분비이상 등으로 번식장애가 발생하나, 배란 에 앞서 일시적인 영양제한은 과배란처리 할 경우 난포수의 증가, 이식가능수정란의 증 가, 수정란의 질을 개선시키고 임신효율을 향상시킨다는 보고가 있다. 본 연구는 공란우 채란을 위한 과배란 처리시 CIDR 삽입 1주일 전부터 채란일 까지 23일간 일시적인 저영 양, 고영양, 대조구로 구분하여 체내수정란 채란효율을 검토하였다. 가축유전자원시험장 번식우 사양관행에 맞추어 대조구(10두)는 배합사료 1 kg, 고영양구 (8두)는 2.5 kg, 저영 양구(8두)는 0 kg을 제한급여를 하였고 건초는 자유채식을 하였다. 공란우 과배란처리는 공란우의 발정주기에 관계없이 CIDR를 질내 삽입하고 4일째부터 FSH를 4일에 걸쳐 28AU 근육주사 하였으며, CIDR 삽입 후 7일째 PGF2α를 오전 25 mg, 오후 15 mg을 근 육 주사, CIDR를 제거하였다. 인공수정은 PGF2α 주사 후 48시간 전후 발정을 확인하고 12시간 간격으로 정액 2 straw 2회 실시하였으며, 1차 인공수정 후 100 μg GnRH를 근 육주사 후 인공수정 후 7일째 자궁관류에 의하여 수정란 회수하였다. 시험결과로서 공란 우의 황체수에 따른 이식가능 수정란수는 황체수가 10개 미만에서 총 회수난수 3.8±1.46 개, 이식가능난수 1.0±0.77이었으나, 10개 이상에서는 총 회수난수가 12.9±2.30개, 이식 가능난수는 8.3±1.88개로 높게 나타났다. 영양수준에 따른 체내수정란 총 회수난수 및 이 식가능난수는 대조구에서 5.88±1.88, 4.25±1.61, 고영양에서 10.50±3.01, 4.50±3.07개로 회수효율이 낮았으나, 저영양에서는 16.33±4.04, 10.33±3.45개로 2배 이상 이식가능 수정 란수가 많았다. 이들 결과는 과배란 처리시 일시적인 저영양이 배란수 및 질좋은 이식가 능 수정란의 증가에 영향을 미침을 시사하고 있다. 금후 공시두수를 늘려 수정란의 질평 가와 호르몬의 변화, 혈액학적 분석, 이식 후 수태율의 검정 등 좀 더 세부적인 분석이 필요할 것이다.

Unstable Epigenetic reprogramming was DNA methylation, imprinting, RNA silencing, co-valent modifications of histones and remodelling by other chromatin-associated complexes. After fusion with an enucleated oocyte, a differentiated somatic cell can restructure its genetic program and acquire totipotent characteristics. However, these cases happen only with low frequency. primordial germ cells (PGC) was effectively remove of epigenetic modifications in the genetic totipotency which is necessary for the development. The present study was in vitro development of reconstruct PGC NT embryos compared with somatic cell NT embryos. The rate of cleavage did not differ between NT embryos from PGC and somatic cells (87.26 vs 91.36%). However, the rate of development to the blastocyst stage was significantly higher in PGC cell NT than somatic cell NT (31.03 vs 19.27%). PGC from a slightly younger stage of development have succeed to promote normal development of recipient eggs. This difference in results between germ cell and somatic cell nuclear transfers could only be a reflection of intimate differences in their reprograming. These results suggest that PGC NT embryos are significantly higher for the in vitro development. Furthermore, These study may represent an approach towards achieving better production of transgenic animal.

5‐aza‐2’‐deoxyctidine (5‐aza‐dC) is DNA methylation inhibitor and Trichostatin A (TSA) is histone deacytlase inhibitor, both of them can alter the level of the epigenetic modification of cells. The objective of this study was to investigate the effects of treatment with 5‐aza‐dC and TSA into fetal fibroblasts on the development of porcine nuclear transfer (NT) embryos. In this study, experiments were performed in order to modify epigenetic status in donor cells and evaluate developmental potential of NT embryos. 5‐ aza‐dC or TSA or combining treatment of TSA and 5‐aza‐dC was treated into growing donor cells for 1 h exposure and development of NT embryos was evaluated. Experiment was performed with 3 groups: control group (donor cells without treatment); TSA group (donor cell treated with 50 nM TSA for 1 h); TSA + 5‐aza‐dC group (donor cells were treated with 50 nM TSA and 5 nM 5‐aza‐dC for 1 h); TSA+1/2(5‐aza‐dC) group (donor cells were treated with 50 nM TSA for 1h and subsequently treated with 2.5 nM 5‐aza‐dC for another 1h). When donor cells were individually treated with 5 nM 5‐aza‐dC or 50 nM TSA for 1h, the blastocyst rate of NT embryos increased significantly compared with control group [18.8% vs 13.4% (5 nM 5‐aza‐dC group vs control group), and 26.2% vs 11.8% (50 nM TSA group vs control group), p<0.05]. However, the blastocyst rate in combining treatment group (50 nM TSA + 5 nM 5‐aza‐dC) did not increase compare with control group (12.3% vs 11.8%, p>0.05). When the donor cell were individually treated with 50nM TSA for 1 h firstly and then treated with 2.5 nM 5‐aza‐dC for another 1h, the blastocyst rate was significantly improved compared with control and TSA group (28% vs 10.2% and 23.7%, p<0.05). The present study suggested that donor cells treated with TSA or low concentration of TSA+5‐azadC in short time exposure may enhance the development of porcine NT embryo.

In vitro production of porcine embryos, including in vitro maturation of oocytes followed by in vitro fertilization and in vitro culture, may result in live offspring, but it 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 CDK9, a RNA polymerase II CTD kinase during pig oocyte growth. 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 and nuclear transcription were analyzed in oocytes 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 Ispecific transcription factor, UBF, and gradually localized in nucleolar peripheries at the final steps of oocyte growth. Treatment with flavopiridol resulted in arrest in meiotic resumption, germinal vesicle breakdown as well as a decline in global transcription. All together, this data suggest that CDK9 has a dual role in both Pol I‐ and Pol II‐dependent transcription in pig oocyte growth.

In all the studies of mammalian species, chromatin in the germinal vesicle (GV) is initially decondensed with the nucleolus not surrounded by heterochromatin (the NSN configurations). During oocyte growth, the GV chromatin condenses into perinucleolar rings (the SN configurations) or other corresponding configurations with or without the perinucleolar rings, depending on species. During oocyte maturation, the GV chromatin is synchronized in a less condensed state before germinal vesicle breakdown (GVBD) in species that has been minutely studied. As not all the species show the SN configuration and gene transcription always stops at the late stage of oocyte growth, it is suggested that a thorough condensation of GV chromatin is essential for transcriptional repression. Because the GV chromatin status is highly correlated with oocyte competence, oocytes must end the NSN configuration before they gain the full meiotic competence and they must take on the SN/corresponding configurations and stop gene transcription before they acquire the competence for early embryonic development. In this study, we firstly investigated whether the layer of cumulus cells and size of oocytes could determine chromatin configurations in porcine oocytes. Using Hoechst3342 staining, the GV nucleolus and chromatin of porcine oocytes was classified into SN and NSN configurations. Next, we examined the changes in GV chromatin configurations during growth and maturation of porcine oocytes. In addition, the maturation and parthenogenetic development abilities in vitro were significant different between the SN and NSN configurations oocytes. These results indicated that chromatin changes in GV oocytes affect the development potential of parthenogenetic embryos.

In this study, synergic effects of MEM vitamins (MEMv) and beta-mercaptoethanol (bME) supplemented to porcine maturation medium on reactive oxygen species (ROS) of oocytes and embryos, and apoptosis of blastocysts were determined. Cumulus-oocyte- complexes (COCs) were allocated into four treatment groups: 0.05X MEMv, 25 uM bME, 0.05X MEMv + 25 uM bME or a group without MEMv + bME. In experiment 1, COCs were cultured in four respective treatment groups based on NCSU-23 medium for 44 h at 39℃ in a 5% CO2 atmosphere. We evaluated ROS of oocytes. In experiment 2, COCs were cultured in four respective treatment groups and then were fertilized in vitro (IVF) or activated by chemical or electrical method. We determined ROS of early stage embryos (2 cell-4 cells) and apoptosis of blastocysts. DCHFDA for ROS level and TdT-mediated dUTP nick end labelling (TUNEL) for apoptosis were used. As a result, ROS level of oocytes was not significant difference among experimental groups. In early stage embryos produced by IVF, MEMv + bME group showed significantly lower ROS level than that of control group (p<0.05). Level of apoptosis in blastocysts of the MEMv + bME group was significantly lower than that of the control group (p<0.05). In early stage embryos produced by chemical activation, ROS level of MEMv + bME group was significantly lower than that of bME group (p<0.05) without significant difference with those of control and MEMv group. Level of apoptosis in blastocysts in the MEMv + bME group was significantly lower than that of the control group (p<0.05). In early stage embryos produced by electrical activation, ROS level of MEMv + bME group was significantly lower than that of control (p<0.05). However, apoptosis level of blastocyst was not significant difference among experimental groups. In conclusion, the present study indicates that the addition of MEM vitamins and betamercaptoethanol during in vitro maturation is able to alleviate the production of ROS and apoptosis.

Although somatic cell nuclear transfer (SCNT) has successfully been produced cloned animals in several species, the cloning efficiency is extremely low. It is generally believed that the low cloning efficiency is mainly attributed to faulty epigenetic modifications underlying the aberrant reprogramming of donor cell nuclei in recipient cytoplasm after SCNT. The nuclear reprogramming process involves epigenetic modifications, such as DNA demethylation and histone acetylation, which may be a key factor in improving the cloning efficiency. Recently, the histone deacetylase inhibitors (HDACi), such as trichosatin A (TSA) and m-carboxycinnamic acid bishydroxamide (CBHA), to increase histone acetylation have been used to improve the developmental competence of SCNT embryos. Therefore, we compared the effects of TSA with CBHA on the in vitro developmental competence and pluripotency-related gene expressions (Nanog, Oct3/4 and Sox2) in porcine cloned blastocysts. The porcine cloned embryos were treated with a 50 nM concentration of TSA or a 100 μm concentration of CBHA during the in vitro early culture (10h) after cell fusion and then were assessed to cleavage rate, development to the blastocyst stage and pluripotency-related gene expressions in NT blastocysts. All data was analyzed by chi-square. Following 4-5 replicates (245, 200 and 222 for NT, TSA and CBHA treated NT embryos respectively) there was no difference between normal NT and CBHA treated NT embryos, whereas TSA treated NT embryos was significantly decreased for cleavage rate (p<0.05). The developmental competence to the blastocyst stage in CBHA treated NT embryos (18.9%) significantly increased than that of normal NT and TSA treated NT embryos (9.4% and 11.5%) (p<0.05). In addition, all of pluripotent transcription factors (Nanog, Oct3/4 and Sox2) were highly expressed in the CBHA treated NT embryos, however, Sox2 and Oct3/4 were expressed in TSA treated NT embryos and Sox2 was only expressed in normal NT embryos (p<0.05). In conclusion, the treatment of CBHA as a histone deacetylase inhibitor significantly increased the developmental competence of porcine NT embryos and pluripotency- related gene expressions (Nanog, Oct3/4 and Sox2) in NT blastocysts.

쇠고기의 육질 등급을 좌우하는 요인으론 육색, 지방색, 조직감 및 상강도(마블링, 근내 지방도) 등이 있는데 그중 근육 내 지방 축적도는 고기의 연도 및 다즙성에 영향을 미치 며 등급을 결정하는 중요한 요인 중 하나이다. 최근 들어 근육 내 지방 합성에 관련된 유전자 연구 및 SNP 탐색을 통한 DNA maker 개발 등 분자생물학적으로 많은 연구보고 가 있으나 등급 간 유의성에 대한 정확한 증명에 관한 연구는 미비하다. 따라서 본 연구는 한우등급 판정소에서 공인하여 선정된 1++의 등급을 판정받은 한우 6두와 2등급으로 판정받은 한우 6두에서 근내지방도와 관련된 유전자로 알려져 있는 GPAT, ACSS2, MGAT, EXT1, FABP4, BCO2, LPL, DGAT, PLIN2, ADSF, TG와 연도와 관련된 유전자인 CAST와 CAPN3의 mRNA 발현양상을 분석하고 유의적 차이점을 확인 하여 경제 형질 선발 마커의 사용 가능성을 선발하기 위하여 등심조직 내 mRNA를 추출 하였고, 각 유전자를 탐침으로 이용하여 sqRT-PCR 및 양적 분석을 위한 nano-spector mate fluorescence system으로 발현양상을 측정하였다. 이후 유의적 평가를 검증 위하여 SAS program (V9.1)의 t-test 검정을 실시하였다. 각 경제 형질 유용 유전자의 발현양상을 분석한 결과 ADSF와 LPL의 경우 평균 근내 지방도가 8±1인 1++등급보다 2±1인 2등급에서 높은 발현양상을 보이고 있었으며, 그 값 이 유의적 차이를 보이지 않았다. 이 외 근내지방도 및 연도와 관련된 유전자 그룹의 경 우 1++등급이 2등급보다 발현양상이 높게 측정되었다. 근내지방도 및 연도에 관련된 유 전자로 선발한 GPAT, ACSS2, DGAT, PLIN2의 경우 유의성 분석결과 유의적 차이점이 없는 것으로 나타났으며, MGAT, EXT1, CAPN3, FABP4, BCO2, CAST, TG의 경우 유의 성(p<0.05)이 있었다. 따라서 본 연구 결과에 의하여 근내지방도 선발 표지 마커로 사용이 가능한 MGAT, EXT1, TG, FABP4, BCO2와 연도 측정에 관련한 CAST, CAPN3의 총 7개의 유전자를 선발 할 수 있었다.

Sialic acid는 9탄당의 단당류로 당단백질이나 당지질의 당사슬 말단에 존재한다. Sialyltransferase의 활성에 따른 sialic acid의 함량의 변화는 세포의 생존, 분화, 증식등 다양한 방면으로 영향을 미치며 대표적인 당단백질 의약품인 EPO의 경우 말단 sialic acid의 함량은 체내활성이나 반감기와 밀접한 관련이 있다. 따라서 sialytransferase는 다 양한 방면의 연구에서 중요한 효소 중 하나이다. 하지만 돼지에서는 당사슬 관련 연구가 미흡하며 관련 효소들도 아직까지 그 염기서열이나 세포내 기능이 명확하게 알려지지 않 았다. 따라서 이번 연구에서는 기존에 클로닝 된 ST6Gal1을 돼지유래 세포주 PK-15세포 를 이용하여 세포내 기능을 분석하였고, ST6Gal1 단백질의 발현량을 각 조직별로 비교분 석하였다. N-terminal 구역에 Flag-tagging된 pig ST6Gal1을 pcDNA3.1(+) vector에 cloning하여 PK-15 cell에 trasfection하였다. Rea-time PCR과 Western blot을 이용해 효소의 발현을 확인한 후, α2-6 sialic acid를 특이적으로 인지하는 Sambucus nigra agglutinin (SNA)을 사용하여 immunofluoresescence microscopy analysis를 수행하였다. 그 결과, ST6Gal1-transfected cell에서 α2-6 sialic acid의 증가를 간접적으로 확인할 수 있었다. 또 한, adhesion assay를 통해 ECM 기질의 일종인 fibronectin에 대한 부착력이 증가함을 알 수 있었다. 이는 PK-15 cell의 β-integrin에 존재하는 당사슬의 α2-6 sialic acid 함량의 증가와 연관이 있는 것으로 생각되어 진다. 조직별 발현량 분석을 통해서는 간에서 특이 적으로 높은 발현을 보였으며 이는 human, mouse 등의 다른 종들에서의 결과와도 일치 한다. 또한, 자돈(5일)보다는 성돈(24개월령)에서 ST6Gal1의 발현량이 전체적으로 높게 관찰되었다.

Atopic dermatitis (AD) is a chronically relapsing, non‐contagious pruritic skin disease with two phases: acute and chronic. Previous studies have shown that cathepsin S (CTSS) is a cysteine protease linked to inflammatory processes, including atherosclerosis and asthma. The possibility that this or other cysteine proteases might evoke itch or be part of a classical ligand‐receptor signaling cascade has not been considered previously. Recently, CTSS was known as a ligand for proteinase‐activated receptor 2 (PAR2) associated with itching. In the present study, we showed that CTSS‐overexpressing transgenic (TG) mice spontaneously developed a skin disorder similar to chronic AD under conventional conditions. This study suggest that CTSS overexpression triggers PAR2 activation in dendritic cells (DCs), resulting in promotion of CD4+ differentiation involved in MHC class II expression. In addition, we investigated mast cells and macrophages and found significantly higher mean levels of T‐helper type 1 (Th1) cell‐associated cytokines than of T‐helper type 2 (Th2) cell‐associated cytokines in CTSS‐overexpressing TG mice. These results suggest that increasing of PAR2 expression in DCs mediated by CTSS overexpression induces scratching behavior and Th 1 cell‐associated cytokines, and can trigger chronic AD symtoms.

This study was conducted to detect the specific fragment genes by using RAPD-PCR and RFLP method in the Korean Tiger cattle and Korean Native cow. And then, the specific fragment gene was investigated by the analysis of the genes for detection significance according to the expressing pattern. We found the specific expression gene by the RAPD-PCR analysis in Korean Tiger cattle. It were a detected the differences of the species in the colour and external section. The Korean Tiger cattle were vary low compare to the Korean Native cattle by analysis result of polymorphism and distribution. And the polymorphism over 500bp into the size classification detected was highly pattern and it could be utilize by the resource of the specific gene. There was a found the specific gene by sequencing in the 1855bp gene fragment of Korean Tiger cattle. And the sequencing result of the R9B was different between Korean Native cow and Holstein cattle. Thus, this gene can be apply as the specific gene in the Korean Tiger cattle.

Oct4 and Nanog are well-known transcription factors related with self renewal of embryonic stem cell. In low-dose of Nanog, transcription of oct4 is increased; however, oct4 is down-regulated upon high-dose of Nanog. There is a negative feedback loop between oct4 and Nanog. To identify this regulation, we generated 4 nested sets for mouse oct4 promoter. Luciferase activities of oct4 were declined upon high-dose Nanog in all constructs. The declined effects of oct4 upon high-dose Nanog were moderated with DNMT and HDAC inhibitors (5-AZA-cytidine and trichostatin A) in 3 constructs (1867, 1346, 754). But, one construct (2179) was only sensitive to TSA. Taken together, these effects were also represented in semi-quantitative RT-PCR and Western blotting data. These data suggest that negative regulation of oct4 gene upon high-dose Nanog would be accomplished by DNMT and HDAC. Further, it will be studied whether these constraining molecules bind to CR1-4 region of oct4 promoter upon low- and high-dose of Nanog.