This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
1. Introduction
Proliferation and differentiation are characteristics of stem cells, which are valuable for their use in human regenerative medicine. Economical animals, especially pigs, are useful for the production of disease models and transgenic animals. There are two states of stem cells: naïve and primed [1, 2]. Mouse epiblast stem cells are in the primed state and do not produce chimera after blastocyst injection analysis. However, mouse embryonic stem cells can form chimera after blastocyst injection analysis; therefore, they are naïve [3, 4]. This is a higher pluripotent state, but it has never been reported in animals other than mouse. Although research on pluripotent stem cells is underway in pigs [5–9], no naïve pig embryonic stem cells have been reported.
Because embryonic stem cell populations are not homogenous, reporter systems are used to classify the status of stem cells and isolate cells in certain conditions. Although it is one of the most necessary parts of studying stem cells and pluripotency, the lack of such research remains a problem. OCT4, a pluripotent marker, is one of many pluripotency-related genes that have been studied as a reporter gene since it is only expressed in pluripotent cells [10]. The transcription factor OCT4 is an important marker of undifferentiated status in early mammalian embryonic development and embryonic stem cells. It has a critical role as a central regulator in maintaining pluripotency and self-renewal. There are four conserved regions (CR1, CR2, CR3, and CR4) in the 5
The Oct4-based reporter system has already been developed in other species and is used to distinguish, separate, and identify pluripotent stem cells [19–22]. However, there has been a lack of information on the porcine OCT4 upstream reporter system until now. Porcine OCT4 upstream region-based GFP reporter systems were reported in 2011 [23, 24], and a dual-reporter system using GFP and RFP was reported in 2016 [25]. However, a luciferase assay, which is used for promoter analysis, was not conducted [17, 19, 20]. Thus, these studies were limited by the fact that the classification of cells using the reporter system was only based on cell morphology analysis.
In this study, we first conducted an analysis of the porcine OCT4 upstream region. A luciferase assay was performed for functional analysis of the promoter region. Next, we used a porcine OCT4 upstream-based dual-reporter system to determine the mRNA expression of cells that were separated by fluorescence-activated cell sorting (FACS). Through the above experiments, we confirmed that the cells could be separated by our reporter system.
2. Materials and Methods
2.1. Animal Welfare
The authors assert that all procedures that contributed to this work complied with the ethical standards of the relevant national and institutional guides on the care and use of laboratory animals. The care and experimental use of pigs were approved by the Institute of Laboratory Animal Resources, Seoul National University (SNU-140328-2).
2.2. Sequence Alignment of Oct4 URS in Animals and Sequence-Based Analysis
A 3.5 kb porcine OCT4 nucleotide sequence was aligned with the Oct4 sequences of human, goat, rabbit, mouse, bovine, and vole from the University of California Santa Cruz (https://genome.ucsc.edu/). Computational analysis of DNA sequences was conducted using the DNA sequence-based analysis programs ALIGN Query, Clustal, and BlastN. Putative transcription factor binding sites (TFBS) from mouse and pig DNA sequences were identified and compared.
2.3. Construction of Porcine Oct4 Upstream Region-Derived Reporter
To generate the longest 3.2 kb OCT4 upstream region-based GFP reporter system (OCT4-GFP), 3.2 kb of the Oct4 regulatory region was inserted into a peGFP vector [23]. A 188,961 bp BAC clone (CH242-102G9) was used as sample DNA. It was used to construct a pOCT4-∆PE-eGFP vector (DE-GFP) containing CR1 and CR4 and a pOCT4-∆DE-DsRed2 vector (PE-RFP) containing CR1, CR2, and CR3 (Figure 1(a)). Based on this, four vectors were constructed to conduct luciferase analyses. The longest pOCT4-pGL3 basic vector (OCT4-Luc) contained all conserved regions, the pOCT4-∆PE-pGL3 basic vector (DE-Luc) contained CR1 and CR4, and the pOCT4-∆DE-pGL3 vector (PE-Luc) contained CR1, CR2, and CR3. Lastly, the pOCT4-CP-pGL3 basic vector (CP-Luc) contained only CR1 (Figure 1(b)).
[figures omitted; refer to PDF]
2.4. Embryonic Stem Cell, Embryonic Carcinoma Cell, and Embryonic Fibroblast Culture
Embryonic stem cell culture media consisted of Dulbecco’s minimum Eagle’s medium (DMEM) (WELGENE) supplemented with 15% fetal bovine serum (FBS), 2 mM GlutaMAX, 0.1 mM β-mercaptoethanol, 1× minimum essential medium (MEM) nonessential amino acids (Gibco), 1× antibiotic/antimycotic, and 1000 units/mL of leukemia inhibitory factor (LIF; Millipore, MA, USA). Embryonic carcinoma culture and embryonic fibroblast media consisted of DMEM (WELGENE) supplemented with 10% FBS, 2 mM GlutaMAX, 0.1 mM β-mercaptoethanol, 1× MEM nonessential amino acids (Gibco), and 1× antibiotic/antimycotic. The media were changed every day, and all cells were cultured under humidified conditions with 5% CO2 at 37°C. When colonies of mouse embryonic stem cells and mouse embryonic carcinoma cells were ready for passaging, the cells were subcultured using trypsin.
2.5. Luciferase Activity Assay
ES-E14TG2a mouse embryonic stem cells, P19 mouse embryonic carcinoma cells, and porcine embryo fibroblast cells were plated into six-well plates (Nunc) (Figure 2). Each reporter construct was transfected into cells using the Lipofectamine 3000 reagent (Invitrogen) according to the manufacturer’s protocol. Cell lysate was prepared 48 h after transfection. Luciferase activity was detected using a Luciferase Assay System (Promega). Transfection of the pGL3-basic vector (Promega), which does not contain inserted DNA, was used as a control to determine the background level of luciferase activity.
[figures omitted; refer to PDF]
2.6. Fluorescence-Activated Cell Sorting
ES-E14TG2a mouse embryonic stem cells were plated into six-well plates (Nunc). Each reporter construct was transfected into cells using the Lipofectamine 3000 reagent (Invitrogen) according to the manufacturer’s protocol. After 48 h, embryonic stem cells were suspended in fluid by trypsin treatment. The cells were separated according to the degree of fluorescence signal using a FACSAria II (BD Biosciences).
2.7. Analysis of mRNA Expression by Quantitative Real-Time PCR
To verify the gene expression levels in pluripotent cells, we performed quantitative real-time PCR (qPCR). Total RNA from individual samples was extracted using a Dynabeads mRNA DIRECT kit (Life Technologies) according to the manufacturer’s instructions. cDNA synthesis was performed using a High-Capacity RNA-to-cDNA (cDNA reverse transcription) kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. Briefly, at a final volume of 20 μL, cDNA synthesis was performed at 37.5°C for 60 min, and samples were subsequently incubated at 95°C for 5 min to activate the reverse transcription reaction. Synthesized cDNA samples were stored at −80°C until subsequent use. qPCR was performed using an ABI 7300 Real-Time PCR System (Applied Biosystems). A DyNAmo HS SYBR Green qPCR Kit (Thermo Scientific, Rockford, IL, USA) was used for real-time quantification of the PCR products. For amplification, 0.1 μM of each primer listed in Supporting Information Table 1 and 0.5 μL of cDNA were added to a 10 μL reaction mixture. The reactions were performed under the following conditions: 1 cycle at 95°C for 10 min, 40 cycles at 95°C for 15 s, and 60 s at the annealing temperature. The dissociation curve was analyzed to confirm the specificity of the PCR product. ACTB was used as a control gene to determine the relative quantity. All genes were measured in triplicate, and the relative expression ratios were analyzed using the 2-ΔΔCt (threshold cycle) method [26].
2.8. Statistical Analysis
All data were analyzed using the GraphPad Prism statistical program (GraphPad Software, San Diego, CA, USA). qPCR data were analyzed using analysis of variance and Fisher’s least significant difference. All data are reported as the
3. Results
3.1. Sequence Analysis of the Oct4 5
In a comparative analysis of the Oct4 5
[figures omitted; refer to PDF]
Table 1
Alignment of the nucleotide sequences from porcine OCT4 promoter regions with those of six mammalian species (%).
Regions | Pig-mouse | Pig-vole | Pig-rabbit | Pig-cow | Pig-goat | Pig-human |
---|---|---|---|---|---|---|
CR1 | 76.8 | 76.0 | 83.5 | 66.7 | 94.5 | 89.1 |
CR2 | 83.3 | 78.7 | 77.4 | 90.1 | 88.1 | 94.2 |
CR3 | 93.5 | 88.4 | 94.9 | 98.5 | 97.9 | 96.4 |
CR4 | 80.8 | 79.7 | 87.0 | 91.5 | 91.6 | 89.2 |
5 |
54.8 | 54.1 | 63.8 | 62.0 |
70.7 | 60.8 |
The regions with the highest homology are bolded. CR1: conserved region 1; CR2: conserved region 2; CR3: conserved region 3; CR4: conserved region 4; 5
[figures omitted; refer to PDF]
3.2. Functional Analysis of the Porcine OCT4 Upstream Regulatory Region
To analyze the role of the transcription regulatory elements of porcine OCT4, we performed a luciferase reporter assay (Figure 5). Pluripotent embryonic stem cell lines have not been derived from pig species; therefore, we transiently transfected luciferase reporter vectors containing various elements of porcine OCT4 upstream regulatory regions into ES-E14TG2a mouse embryonic stem cells, P19 mouse embryonic carcinoma cells, and mouse embryonic fibroblasts. OCT4-Luc (pOCT4-pGL3) had the longest porcine OCT4 upstream region and included all of the conserved regions (CR1, CR2, CR3, and CR4). Luciferase expression in mouse embryonic stem cells and mouse embryonic carcinoma cells expressing OCT4-Luc was significantly higher than that in mouse embryonic fibroblasts. DE-Luc (pOCT4-∆PE-pGL3) included CR1 and CR4. In this case, only luciferase expression in mouse embryonic stem cells expressing DE-Luc was significantly higher than that in control. In contrast, only luciferase expression in mouse carcinoma cells expressing PE-Luc (pOCT4-∆DE-pGL3) was significantly higher than that in mouse embryonic fibroblasts. PE-Luc contained CR1, CR2, and CR3. CP-Luc (pOCT4-CP-pGL3) only contained CR1, and cells transfected with this vector were not significantly different (Figure 5(a)).
[figures omitted; refer to PDF]
Mouse embryonic stem cells expressing OCT4-Luc and DE-Luc had significantly higher luciferase expressions than cells expressing the control pGL3 vector. In contrast, mouse embryonic carcinoma cells expressing OCT4-Luc and PE-Luc had significantly higher luciferase expressions than cells expressing the control pGL3 vector. In mouse embryonic fibroblasts, luciferase expression from all constructs was not significantly different (Figure 5(b)).
3.3. Gene Expression Patterns in Embryonic Stem Cells and Embryonic Carcinoma Cells Separated by the Porcine OCT4 Reporter System
ES-E14TG2a mouse embryonic stem cells and P19 embryonic carcinoma cells expressing DE-GFP (pOCT4-∆PE-eGFP) and PE-RFP (pOCT4-∆DE-DsRed2) were separated by FACS into groups with high and low GFP or RFP fluorescence signals. Then, the mRNA expression in each of the groups was analyzed by qPCR. In mouse embryonic stem cells, a comparison of naïve (Tbx3, Nr0b1, Rex1, Esrrb, Nanog, and Klf2) and primed (Gata6, Mixl1, Fgf5, and Otx2) state marker gene expression using a dual-reporter assay consisting of DE-GFP (pOCT4-∆PE-eGFP) and PE-RFP (pOCT4-∆DE-DsRed2) showed that naïve and primed marker expression differed in fluorescence signal between high-expressing cells and low-expressing cells. Specifically, when the expression of pluripotency genes in GFP-low-expressing cells was divided by that in GFP-high-expressing cells, GFP-low-expressing cells had relatively higher primed marker gene expressions than GFP-high-expressing cells, whereas naïve marker genes were expressed with a similar level in both cells (Figure 6(a)). It is indicated that a loss of activity of a distal enhancer gradually induces conversion of naïve pluripotency to primed pluripotency in mouse ESCs. In contrast, RFP-low-expressing cells had relatively higher expressions of several naïve marker genes and lower expressions of some primed marker genes (e.g., mixl1 and gata6) than RFP-high-expressing cells. Interestingly, some primed marker genes, such as otx2 and fgf5, were more highly expressed in RFP-low-expressing cells than in RFP-high-expressing cells (Figure 6(b)). There were no significant differences in the expression of all genes when P19 was divided into a DE-based GFP reporter system (Figure 6(c)). The PE-based RFP reporter system also showed irregular expression regardless of whether the genes were markers of naïve or primed states (Figure 6(d)).
[figures omitted; refer to PDF]
4. Discussion
In this study, we investigated the porcine OCT4 upstream region, provided examples of various reporter systems derived from porcine OCT4, and performed functional tests of the reporter system. The porcine OCT4 upstream region has four conserved regions, two separate enhancers, several main conserved elements (e.g., the core promoter), and orthologs in other mammals [11, 17, 19, 20]. The porcine OCT4 upstream sequences were more similar to those of human and goat than to those of mouse. This may partially explain why porcine OCT4 expression is similar to that of human and different from that of mouse during early embryo development [27]. However, the two enhancers of porcine OCT4 showed mechanisms similar to those previously reported in mouse. A large gap was found in the Oct4 upstream region nucleotide sequence in both species; as such, differences in expression can occur when Oct4 upstream region-based reporter systems constructed from one species are inserted into another species [23]. Therefore, a porcine-specific reporter system is essential for porcine stem cell research.
Enhancers with multiple cognate binding sites for various transcription factors can increase the expression of genes [28]. In particular, Oct4 has two enhancers, a DE and a PE. They are used to produce OCT4 in various pluripotent cells, working simultaneously or sequentially. Because different factors exist depending on the pluripotent state, OCT4 has two enhancers to form OCT4 in different environments and is configured to operate in two different environments. The DE is a key element of the Oct4 gene in embryonic stem cells [12, 29] and is also the most important factor in the embryonic stem cell reporter system based on the results from a previous qPCR experiment. For instance, there were ambiguities in the case of mouse embryonic stem cells separated by PE-based reporter vectors, but in the case of mouse embryonic stem cells separated by DE-based reporter vectors, the division of cell status was clear based on the intensity of the fluorescence signal. Embryonic stem cells exhibit heterogeneity during culture [30–32]; therefore, a reporter system can be used to separate more naïve cells in such mixed populations.
Embryonic carcinoma cells are primed pluripotent stem cells, and the Oct4 PE is epiblast- and embryonic carcinoma-specific [12, 14]. Because P19 cells are known to produce Oct4 using the PE, it is meaningless to classify P19 as a DE-based GFP signal. In fact, there was no significant difference between embryonic carcinoma cells sorted by GFP. The PE-based RFP reporter system also had problems separating embryonic carcinoma cells. In particular, the expression of genes associated with naïve and primed states in embryonic carcinoma cells sorted by fluorescent signal randomly changed. As previously reported [25], it is possible to distinguish between fluorescence patterns, but the results from the analysis of sorted cells showed that it was difficult to separate primed pluripotent cells. These results are likely due to the fact that PEs differ greatly among species [23]. Therefore, this system should be reexamined in pigs.
In summary, we conducted an analysis of the porcine OCT4 upstream region, from which we suggested reporter systems. These reporter systems were used to distinguish and isolate mouse embryonic stem cells based on pluripotent status. Many studies of pluripotency have been conducted in mouse using Oct4-based reporter systems, which have been used to identify and purify pluripotent cells, as well as observe the reprogramming process [33–37]. In future experiments, we plan to introduce this reporter system into cells of porcine origin and conduct the various aforementioned experiments. Such reporter systems enable the nondestructive classification of the condition of live pluripotent cells and can be used to find a naïve-state porcine stem cell line to study species-specific pluripotency.
Disclosure
Jae Yeon Hwang’s present address is Department of Cellular & Molecular Physiology, Yale School of Medicine, New Haven, CT 06510, USA.
Conflicts of Interest
All authors declare no competing financial interests.
Authors’ Contributions
Seung-Hun Kim is responsible for the conception and design, provision of study material, collection and assembly of data, data analysis and interpretation, and manuscript writing. Kwang-Hwan Choi is responsible for the conception and design, data analysis and interpretation, and manuscript writing. Dong-Kyung Lee is responsible for the data analysis and interpretation. Mingyun Lee is responsible for the data analysis and interpretation. Jae Yeon Hwang is responsible for the conception and design and provision of study material. Chang-Kyu Lee is responsible for the conception and design, data analysis and interpretation, manuscript writing, and final approval of the manuscript.
[1] J. Nichols, A. Smith, "Naive and primed pluripotent states," Cell Stem Cell, vol. 4 no. 6, pp. 487-492, DOI: 10.1016/j.stem.2009.05.015, 2009.
[2] J. H. Hanna, K. Saha, R. Jaenisch, "Pluripotency and cellular reprogramming: facts, hypotheses, unresolved issues," Cell, vol. 143 no. 4, pp. 508-525, DOI: 10.1016/j.cell.2010.10.008, 2010.
[3] J. Hanna, A. W. Cheng, K. Saha, J. Kim, C. J. Lengner, F. Soldner, J. P. Cassady, J. Muffat, B. W. Carey, R. Jaenisch, "Human embryonic stem cells with biological and epigenetic characteristics similar to those of mouse ESCs," Proceedings of the National Academy of Sciences of the United States of America, vol. 107 no. 20, pp. 9222-9227, DOI: 10.1073/pnas.1004584107, 2010.
[4] C. Buecker, H. H. Chen, J. M. Polo, L. Daheron, L. Bu, T. S. Barakat, P. Okwieka, A. Porter, J. Gribnau, K. Hochedlinger, N. Geijsen, "A murine ESC-like state facilitates transgenesis and homologous recombination in human pluripotent stem cells," Cell Stem Cell, vol. 6 no. 6, pp. 535-546, DOI: 10.1016/j.stem.2010.05.003, 2010.
[5] B. P. V. L. Telugu, T. Ezashi, S. Sinha, A. P. Alexenko, L. Spate, R. S. Prather, R. M. Roberts, "Leukemia inhibitory factor (LIF)-dependent, pluripotent stem cells established from inner cell mass of porcine embryos," The Journal of Biological Chemistry, vol. 286 no. 33, pp. 28948-28953, DOI: 10.1074/jbc.M111.229468, 2011.
[6] S. H. Fujishiro, K. Nakano, Y. Mizukami, T. Azami, Y. Arai, H. Matsunari, R. Ishino, T. Nishimura, M. Watanabe, T. Abe, Y. Furukawa, K. Umeyama, S. Yamanaka, M. Ema, H. Nagashima, Y. Hanazono, "Generation of naive-like porcine-induced pluripotent stem cells capable of contributing to embryonic and fetal development," Stem Cells and Development, vol. 22 no. 3, pp. 473-482, DOI: 10.1089/scd.2012.0173, 2013.
[7] D. J. Kwon, H. Jeon, K. B. Oh, S. A. Ock, G. S. Im, S. S. Lee, S. K. Im, J. W. Lee, S. J. Oh, J. K. Park, S. Hwang, "Generation of leukemia inhibitory factor-dependent induced pluripotent stem cells from the Massachusetts General Hospital miniature pig," BioMed Research International, vol. 2013,DOI: 10.1155/2013/140639, 2013.
[8] W. Zhang, Y. Pei, L. Zhong, B. Wen, S. Cao, J. Han, "Pluripotent and metabolic features of two types of porcine iPSCs derived from defined mouse and human ES cell culture conditions," PLoS One, vol. 10 no. 4, article e0124562,DOI: 10.1371/journal.pone.0124562, 2015.
[9] J. K. Park, H. S. Kim, K. J. Uh, K. H. Choi, H. M. Kim, T. Lee, B. C. Yang, H. J. Kim, H. H. Ka, H. Kim, C. K. Lee, "Primed pluripotent cell lines derived from various embryonic origins and somatic cells in pig," PLoS One, vol. 8 no. 1, article e52481,DOI: 10.1371/journal.pone.0052481, 2013.
[10] S. Jerabek, F. Merino, H. R. Schöler, V. Cojocaru, "OCT4: dynamic DNA binding pioneers stem cell pluripotency," Biochimica et Biophysica Acta (BBA) – Gene Regulatory Mechanisms, vol. 1839 no. 3, pp. 138-154, DOI: 10.1016/j.bbagrm.2013.10.001, 2014.
[11] V. Nordhoff, K. Hübner, A. Bauer, I. Orlova, A. Malapetsa, H. R. Schöler, "Comparative analysis of human, bovine, and murine Oct-4 upstream promoter sequences," Mammalian Genome, vol. 12 no. 4, pp. 309-317, DOI: 10.1007/s003350010279, 2001.
[12] Y. I. Yeom, G. Fuhrmann, C. E. Ovitt, A. Brehm, K. Ohbo, M. Gross, K. Hübner, H. R. Schöler, "Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells," Development, vol. 122, pp. 881-894, 1996.
[13] T. Yoshimizu, N. Sugiyama, M. de Felice, Y. I. Yeom, K. Ohbo, K. Masuko, M. Obinata, K. Abe, H. R. Scholer, Y. Matsui, "Germline-specific expression of the Oct-4/green fluorescent protein (GFP) transgene in mice," Development, Growth and Differentiation, vol. 41 no. 6, pp. 675-684, DOI: 10.1046/j.1440-169x.1999.00474.x, 1999.
[14] A. Gillich, S. Bao, N. Grabole, K. Hayashi, M. W. B. Trotter, V. Pasque, E. Magnúsdóttir, M. A. Surani, "Epiblast stem cell-based system reveals reprogramming synergy of germline factors," Cell Stem Cell, vol. 10 no. 4, pp. 425-439, DOI: 10.1016/j.stem.2012.01.020, 2012.
[15] H. Okazawa, K. Okamoto, F. Ishino, T. Ishino-Kaneko, S. Takeda, Y. Toyoda, M. Muramatsu, H. Hamada, "The oct3 gene, a gene for an embryonic transcription factor, is controlled by a retinoic acid repressible enhancer," The EMBO Journal, vol. 10 no. 10, pp. 2997-3005, DOI: 10.1002/j.1460-2075.1991.tb07850.x, 1991.
[16] S. Minucci, V. Botquin, Y. I. Yeom, A. Dey, I. Sylvester, D. J. Zand, K. Ohbo, K. Ozato, H. R. Scholer, "Retinoic acid-mediated down-regulation of Oct3/4 coincides with the loss of promoter occupancy in vivo," The EMBO Journal, vol. 15 no. 4, pp. 888-899, DOI: 10.1002/j.1460-2075.1996.tb00423.x, 1996.
[17] H. M. Yang, H. J. Do, J. H. Oh, J. H. Kim, S. Y. Choi, K. Y. Cha, H. M. Chung, J. H. Kim, "Characterization of putative cis-regulatory elements that control the transcriptional activity of the human Oct4 promoter," Journal of Cellular Biochemistry, vol. 96 no. 4, pp. 821-830, DOI: 10.1002/jcb.20588, 2005.
[18] N. Hattori, K. Nishino, Y. G. Ko, N. Hattori, J. Ohgane, S. Tanaka, K. Shiota, "Epigenetic control of mouse Oct-4 gene expression in embryonic stem cells and trophoblast stem cells," The Journal of Biological Chemistry, vol. 279 no. 17, pp. 17063-17069, DOI: 10.1074/jbc.M309002200, 2004.
[19] X. Cheng, S. Meng, J. Deng, W. Lai, H. Wang, "Identification and characterization of the Oct4 extended transcriptional regulatory region in Guanzhong dairy goat," Genome, vol. 54 no. 10, pp. 812-818, DOI: 10.1139/g11-047, 2011.
[20] S. P. Medvedev, A. I. Shevchenko, E. A. Elisaphenko, T. B. Nesterova, N. Brockdorff, S. M. Zakian, "Structure and expression pattern of Oct4 gene are conserved in vole Microtus rossiaemeridionalis," BMC Genomics, vol. 9 no. 1,DOI: 10.1186/1471-2164-9-162, 2008.
[21] L. Gerrard, D. Zhao, A. J. Clark, W. Cui, "Stably transfected human embryonic stem cell clones express OCT4-specific green fluorescent protein and maintain self-renewal and pluripotency," Stem Cells, vol. 23 no. 1, pp. 124-133, DOI: 10.1634/stemcells.2004-0102, 2005.
[22] Y. Li, Q. Zhang, X. Yin, W. Yang, Y. du, P. Hou, J. Ge, C. Liu, W. Zhang, X. Zhang, Y. Wu, H. Li, K. Liu, C. Wu, Z. Song, Y. Zhao, Y. Shi, H. Deng, "Generation of iPSCs from mouse fibroblasts with a single gene, Oct4, and small molecules," Cell Research, vol. 21 no. 1, pp. 196-204, DOI: 10.1038/cr.2010.142, 2011.
[23] L. Huang, N. Fan, J. Cai, D. Yang, B. Zhao, Z. Ouyang, W. Gu, L. Lai, "Establishment of a porcine Oct-4 promoter-driven EGFP reporter system for monitoring pluripotency of porcine stem cells," Cellular Reprogramming, vol. 13 no. 2, pp. 93-98, DOI: 10.1089/cell.2010.0069, 2011.
[24] M. Nowak-Imialek, W. A. Kues, B. Petersen, A. Lucas-Hahn, D. Herrmann, S. Haridoss, M. Oropeza, E. Lemme, H. R. Schöler, J. W. Carnwath, H. Niemann, "Oct4-enhanced green fluorescent protein transgenic pigs: a new large animal model for reprogramming studies," Stem Cells and Development, vol. 20 no. 9, pp. 1563-1575, DOI: 10.1089/scd.2010.0399, 2011.
[25] W. S. Sun, J. L. Chun, J. T. Do, D. H. Kim, J. S. Ahn, M. K. Kim, I. S. Hwang, D. J. Kwon, S. S. Hwang, J. W. Lee, "Construction of a dual-fluorescence reporter system to monitor the dynamic progression of pluripotent cell differentiation," Stem Cells International, vol. 2016,DOI: 10.1155/2016/1390284, 2016.
[26] K. J. Livak, T. D. Schmittgen, "Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method," Methods, vol. 25 no. 4, pp. 402-408, DOI: 10.1006/meth.2001.1262, 2001.
[27] N. Kirchhof, J. W. Carnwath, E. Lemme, K. Anastassiadis, H. Schöler, H. Niemann, "Expression pattern of Oct-4 in preimplantation embryos of different species," Biology of Reproduction, vol. 63 no. 6, pp. 1698-1705, DOI: 10.1095/biolreprod63.6.1698, 2000.
[28] M. Bulger, M. Groudine, "Functional and mechanistic diversity of distal transcription enhancers," Cell, vol. 144 no. 3, pp. 327-339, DOI: 10.1016/j.cell.2011.01.024, 2011.
[29] G. Shi, Y. Jin, "Role of Oct4 in maintaining and regaining stem cell pluripotency," Stem Cell Research & Therapy, vol. 1 no. 5,DOI: 10.1186/scrt39, 2010.
[30] A. M. V. Guedes, D. Henrique, E. Abranches, "Dissecting transcriptional heterogeneity in pluripotency: single cell analysis of mouse embryonic stem cells," Methods in Molecular Biology, vol. 1516, pp. 101-119, DOI: 10.1007/7651_2016_356, 2016.
[31] T. S. Tanaka, "Transcriptional heterogeneity in mouse embryonic stem cells," Reproduction, Fertility, and Development, vol. 21 no. 1, pp. 67-75, DOI: 10.1071/RD08219, 2009.
[32] Z. S. Singer, J. Yong, J. Tischler, J. A. Hackett, A. Altinok, M. A. Surani, L. Cai, M. B. Elowitz, "Dynamic heterogeneity and DNA methylation in embryonic stem cells," Molecular Cell, vol. 55 no. 2, pp. 319-331, DOI: 10.1016/j.molcel.2014.06.029, 2014.
[33] J. T. Do, H. R. Scholer, "Nuclei of embryonic stem cells reprogram somatic cells," Stem Cells, vol. 22 no. 6, pp. 941-949, DOI: 10.1634/stemcells.22-6-941, 2004.
[34] D. W. Han, J. T. Do, L. Gentile, M. Stehling, H. T. Lee, H. R. Schöler, "Pluripotential reprogramming of the somatic genome in hybrid cells occurs with the first cell cycle," Stem Cells, vol. 26 no. 2, pp. 445-454, DOI: 10.1634/stemcells.2007-0553, 2008.
[35] J. B. Kim, V. Sebastiano, G. Wu, M. J. Araúzo-Bravo, P. Sasse, L. Gentile, K. Ko, D. Ruau, M. Ehrich, D. van den Boom, J. Meyer, K. Hübner, C. Bernemann, C. Ortmeier, M. Zenke, B. K. Fleischmann, H. Zaehres, H. R. Schöler, "Oct4-induced pluripotency in adult neural stem cells," Cell, vol. 136 no. 3, pp. 411-419, DOI: 10.1016/j.cell.2009.01.023, 2009.
[36] J. B. Kim, H. Zaehres, G. Wu, L. Gentile, K. Ko, V. Sebastiano, M. J. Araúzo-Bravo, D. Ruau, D. W. Han, M. Zenke, H. R. Schöler, "Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors," Nature, vol. 454 no. 7204, pp. 646-650, DOI: 10.1038/nature07061, 2008.
[37] J. T. Do, D. W. Han, L. Gentile, I. Sobek-Klocke, M. Stehling, H. T. Lee, H. R. Schöler, "Erasure of cellular memory by fusion with pluripotent cells," Stem Cells, vol. 25 no. 4, pp. 1013-1020, DOI: 10.1634/stemcells.2006-0691, 2007.
You have requested "on-the-fly" machine translation of selected content from our databases. This functionality is provided solely for your convenience and is in no way intended to replace human translation. Show full disclaimer
Neither ProQuest nor its licensors make any representations or warranties with respect to the translations. The translations are automatically generated "AS IS" and "AS AVAILABLE" and are not retained in our systems. PROQUEST AND ITS LICENSORS SPECIFICALLY DISCLAIM ANY AND ALL EXPRESS OR IMPLIED WARRANTIES, INCLUDING WITHOUT LIMITATION, ANY WARRANTIES FOR AVAILABILITY, ACCURACY, TIMELINESS, COMPLETENESS, NON-INFRINGMENT, MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. Your use of the translations is subject to all use restrictions contained in your Electronic Products License Agreement and by using the translation functionality you agree to forgo any and all claims against ProQuest or its licensors for your use of the translation functionality and any output derived there from. Hide full disclaimer
Copyright © 2019 Seung-Hun Kim et al. This is an open access article distributed under the Creative Commons Attribution License (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License. http://creativecommons.org/licenses/by/4.0/
Abstract
OCT4 plays pivotal roles in maintaining pluripotency during early mammalian embryonic development and in embryonic stem cells. It is essential to establish a reporter system based on the OCT4 promoter region to study pluripotency. However, there is still a lack of information about the porcine OCT4 upstream reporter system. 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
You have requested "on-the-fly" machine translation of selected content from our databases. This functionality is provided solely for your convenience and is in no way intended to replace human translation. Show full disclaimer
Neither ProQuest nor its licensors make any representations or warranties with respect to the translations. The translations are automatically generated "AS IS" and "AS AVAILABLE" and are not retained in our systems. PROQUEST AND ITS LICENSORS SPECIFICALLY DISCLAIM ANY AND ALL EXPRESS OR IMPLIED WARRANTIES, INCLUDING WITHOUT LIMITATION, ANY WARRANTIES FOR AVAILABILITY, ACCURACY, TIMELINESS, COMPLETENESS, NON-INFRINGMENT, MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. Your use of the translations is subject to all use restrictions contained in your Electronic Products License Agreement and by using the translation functionality you agree to forgo any and all claims against ProQuest or its licensors for your use of the translation functionality and any output derived there from. Hide full disclaimer
Details





1 Department of Agricultural Biotechnology, Animal Biotechnology Major, Research Institute of Agriculture and Life Science, Seoul National University, Seoul 08826, Republic of Korea
2 Department of Agricultural Biotechnology, Animal Biotechnology Major, Research Institute of Agriculture and Life Science, Seoul National University, Seoul 08826, Republic of Korea; Designed Animal & Transplantation Research Institute, Institute of Green Bio Science and Technology, Seoul National University, Gangwon-do 25354, Republic of Korea