Pluripotent stem cells could self-renew and differentiate into various cells. In particular, porcine pluripotent stem cells are useful for preclinical therapy, transgenic animals, and agricultural usage. These stem cells have naïve and primed pluripotent states. Naïve pluripotent stem cells represented by mouse embryonic stem cells form chimeras after blastocyst injection. Primed pluripotent stem cells represented by mouse epiblast stem cells and human embryonic stem cells. They could not produce chimeras after blastocyst injection. Populations of embryonic stem cells are not homogenous; therefore, reporter systems are used to clarify the status of stem cells and to isolate the cells. For this reason, studies of the OCT4 reporter system have been conducted for decades. This review will discuss the naïve and primed pluripotent states and recent progress in the development of porcine OCT4 reporter systems.

The transcription factor POU5F1, also known as OCT4 plays critical roles in maintaining pluripotency during early mammalian embryonic development and in embryonic stem cells. It is important to establish an OCT4 promoter region-based reporter system to study pluripotency. However, there is still a lack of information about the porcine OCT4 upstream region. To improve our understanding of the porcine OCT4 regulatory region, we identified conserved regions in the porcine OCT4 promoter upstream region by sequence-based comparative analysis using various mammalian genome sequences. The similarity of nucleotide sequences in the 5' upstream region was low among mammalian species. However, the OCT4 promoter and four regulatory regions, including distal and proximal enhancer elements, had high similarity. The putative transcription factor binding sites in the Oct4 5' upstream region nucleotide sequences from mice and pigs also differed. Some of these genes are related to pluripotency, and further research will allow us to better understand the differences in species-specific pluripotency. Next, a functional analysis of the porcine OCT4 promoter region was conducted. Luciferase reporter assay results indicated that the porcine OCT4 distal enhancer and proximal enhancer were highly activated in mouse embryonic stem cells and embryonic carcinoma cells, respectively. Similar to OCT4 upstream-based reporter systems derived from other species, the porcine OCT4 upstream region-based reporter constructs showed exclusive expression patterns depending on the state of pluripotency. This work provides basic information about the porcine OCT4 upstream region and various porcine OCT4 fluorescence reporter constructs, which can be applied to study species-specific pluripotency in early embryo development and the establishment of embryonic stem cells in pigs. This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education (NRF-2017R1D1A1B03032256).

The embryonic genome activation (EGA) is genetically activated states that embryos make the materials such as growth factors for using themselves. EGA is various because they have many materials, different site, different stage, also different species. At this time, transcription factors are expressed. Transcription factors bind to specific DNA region, and regulate the gene expression. Thus, we check the expression of transcription factors, we can know that embryo development is very well or not. The development stages of embryos are basically the stages from fertilization to blastocyst. So, we check the embryos oocyte to blastocyst. In our experiments, we focus the early developmental transcription factors such as Cdx2, Oct4, Sox2, Nanog and E-Cadherin. Above antibody factors showed different expression sites, and there were many differentiated parts from other animal species. In addition, we compared the SCNT and parthenogenetic activation (PA) because these are same methods using electrical activation among the embryo production methods. Our results showed not only similar patterns but also different patterns between pig and mouse. Therefore, we have to investigate that different patterns of transcription factors play a role in pigs, and why occur.

Interferon-tau (IFNT) is regarded, generally, to be the conceptus protein that signals maternal recognition of pregnancy in ruminants. Although the discovery was made over two decades ago, the molecular mechanisms that regulate IFNT expression are not well understood. Previous studies demonstrated that transcription factors, caudal-related homeobox- 2 (CDX2), JUN, ETS2 and a transcriptional coactivator CREB binding protein (CREBBP) positively influenced IFNT gene expression, while OCT4 may exhibit negative regulation. We and others have observed that both CDX2 and OCT4 coexist during early stages of conceptus elongation but as development proceeds, OCT4 expression diminishes. The objective of this study was to evaluate the stimulatory and inhibitory effects of CDX2 and OCT4, respectively, on IFNT gene transcription when evaluated with other transcription factors. Human choriocarcinoma JEG3 cells were co-transfected with an ovine IFNT (-654 base pair)-luciferase reporter (-654-oIFNT-Luc) construct and several transcription factor expression plasmids. When the reporter construct was co-transfected with either CDX2, ETS2 or CJUN, transcription of -654-oIFNT-Luc increased about two-fold compared to -654-oIFNT-Luc alone. When -654-oIFNT-Luc was co-transfected with both c-jun and Ets-2, activity of -654-oIFNT-Luc was increased about four-fold; cotransfection with JUN, ETS2 and CDX2 increased -654-oIFNT-Luc expression 12X, indicating that the stimulatory activity of the transcription factors was additive. OCT4, when cotransfected with -654-oIFNT-Luc, reduced expression of the later about 40% when compared to -654-oIFNT-Luc alone. When co-transfected with JUN and/or ETS2, OCT4 abolished the stimulatory effect of these transcription factors. OCT4 also inhibited the stimulatory activity of CDX2 alone, but not when CDX2 was combined with JUN and/or ETS2. Therefore, when combined with the other transcription factors, CDX2 over the transcriptional inhibitory activity of OCT4. Conversely, when cells were transfected initially with OCT4 (0h) followed by transfection with CDX2, ETS2 and JUN 24 h later, -654-oIFNT-Luc expression was reduced to control (-654-oIFNT-Luc, alone) levels. Not surprisingly, 12S E1A, an inhibitor of transcriptional coactivator CREBBP, reduced stimulation of -654- oIFNT-Luc expression by CDX2, ETS2 and JUN, in combination, by about 40%, indicating that proper transcription complex formation is required for maximum expression. In conclusion, it is suggested that prior to conceptus elongation, pre-existing OCT4 may inhibit IFNT expression, but as elongation proceeds, IFNT expression increases, resulting from incremental increases in CDX2 expression, diminished OCT4 expression, and possibly proper transcription factor complex formation. Key words) Interferon-tau, CDX2, OCT4, transcription

The Oct-4 (octamer-4), a member of the POU family transcription factor, is expressed in early mouse embryogenesis and in pluripotent embryonic stem (ES) lines. Oct-4 expression is thought to remain confined to the germline after gastrulation in the embryo. Therefore, the study was designed to, study the location of Oct-4 protein in the ovaries, placenta and testis of Korean native cattle (Hanwoo). Expression of Oct-4 mRNA in the ovaries and placenta of bovine was confirmed by RT-PCR and immunohistochemical analysis. Oct-4 was expressed in granulosa, thecal cells irrespective of the shape and size of follicles and endometerium of Korean native cattle (Hanwoo). Expression of Oct-4 was profound in all the tissues of Korean native cattle (Hanwoo) suggestung their role in them. Oct-4 localization and expression could contribute to further developmental studies in Korean native cattle (Hanwoo).

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.

DNA methylation is one of the reasons for poor survival of clone animals. The OCT-4 gene is essential for maintaining pluripotency of embryonic stem (ES) cells and early embryos. We previously reported that the 5'-promoter region of Oct-4 gene was a target of DNA methylation and the methylation status was changed variously during embryonic development in bovine. The study conducted to examine the expression and methylation pattern of tissue-dependent differentially methylated region (T-DMR) of Oct-4 gene in bovine somatic cell nuclear transfer (SCNT) and in vitro fertilization (IVF) blastocysts. The Oct-4 gene expression was evaluated by RT-PCR and fluorescence immunocytochemistry. The methylation pattern of T-DMR was analyzed using restriction mapping and bisulfite sequencing methods. The Oct-4 transcripts were highly expressed in IVF, while they were not expressed in SCNT. The Oct-4 protein was not detected or expressed at very low level in SCNT, the intensity of Oct-4 protein, however, was strong in IVF. On the other hand, the T-DMR of Oct-4 gene was hypermethylated in SCNT compared to that of IVF. These results suggested that expression and the failure of demethylation of Oct-4 gene was closely associated with incomplete development of SCNT embryos.

Recent studies on nuclear transfer and induced pluripotent stem cells have demonstrated that differentiated somatic cells can be returned to the undifferentiated state by reversing their developmental process. These epigenetically reprogrammed somatic cells may again be differentiated into various cell types, and used for cell replacement therapies through autologous transplantation to treat many degenerative diseases. To date, however, reprogramming of somatic cells into undifferentiated cells has been extremely inefficient. Hence, reliable markers to identify the event of reprogramming would assist effective selection of reprogrammed cells. In this study, a transgene construct encoding enhanced green fluorescent protein (EGFP) under the regulation of human Oct4 promoter was developed as a reporter for the reprogramming of somatic cells. Microinjection of the transgene construct into pronuclei of fertilized mouse eggs resulted in the emission of green fluorescence, suggesting that the undifferentiated cytoplasmic environment provided by fertilized eggs induces the expression of EGFP. Next, the transgene construct was introduced into human embryonic fibroblasts, and the nuclei from these cells were transferred into enucleated porcine oocytes. Along with their in vitro development, nuclear transfer embryos emitted green fluorescence, suggesting the reprogramming of donor nuclei in nuclear transfer embryos. The results of the present study demonstrate that expression of the transgene under the regulation of human Oct4 promoter coincides with epigenetic reprogramming, and may be used as a convenient marker that non-invasively reflects reprogramming of somatic cells.

DNA 메틸화는 조직특이적인 유전자 조절에 관여하고, 정상적인 배 발달에 필수적이다. POU5F1은 octamer-binding transcription factor 4 (Oct-4)를 encode하며, 초기 분화에 중요한 전사인자이다. 본 실험에서 소의 Oct-4가 조직특이적이고 발달의존적인 epigenetic 표지 인지를 검토하고자, 착상 전 수정란에서 Oct-4 전사산물과 상류 promoter 영역의 CpGs의 메틸화를 조사하였다. Oct-4 전사산물은 정자 그리고 2-cell에서 8-cell 수정란까지 낮은 수준으로 존재하지만, 상실배와 배반포에서 높게 검출되었다. 이러한 결과는 배 발달 과정의 상실배 단계에서 Oct-4의 de novo 발현이 시작됨을 의미한다. Oct-4 상류 promoter 영역에는 메틸화 가변 영역 (tissue-dependent differentially methylated region, T-DMR)이 존재한다. Oct-4 메틸화 가변 영역의 메틸화 상태는 정자, 성체 체조직과 난자에서 서로 다르고, 수정란으로부터 배반포 단계까지 변화하였는데, 이는 착상 전 초기 배 발달 과정에 active 메틸화와 탈메틸화가 일어남을 의미한다. 이상의 결과, Oct-4 유전자 상류 promoter 영역은 DNA 메틸화의 타깃이고, 그 메틸화 상태는 소 수정란 발달 동안에 다양하게 변화한다.

During early embryo development, Oct-4 is an important transcription factor for the early differentiation. The present study was first examined methylation status in distal enhancer and promoter region of Oct-4 during mouse pre-implantation embryo development. In oocyte and sperm, high methylation was observed in both distal and proximal of promoter in Oct-4. Following fertilization, relatively high methylation level remained until 8-cell stage embryos, but decreased at the morula and blastocyst stage. Specific gene knock down of Oct-4 by siRNA injection into zygote induced higher methylation rates of both distal and proximal region ofpromoter of Oct-4. These results suggest a functional link between the DNA methylation status of distal and promoter region in the Oct-4 gene and the gene sequence-specific transcriptional silencing by exogenous siRNA injection during mouse pre- implantation embryos.

배아 줄기세포는 미분화상태에서 자가 재생을 유지할 수 있다. 자가 재생은 OCT4, SOX2와 NANOG와 같은 많은 인자들이 작용한다. 생쥐 배아 줄기세포에서 OCT4와 SOX2가 Nanog 프로모터에 결합하여 Nanog 유전자의 발현을 촉진한다는 사실은 생쥐 promoter에 관한 정밀분석으로 알려져 있다. 본 연구에서는 인간 Nanog promoter를 정밀 분석하기 위해 연속적인 결손 돌연변이를 가진 promoter-reporter constru