Identification and Characterization of the OCT4 Upstream Regulatory Region in Sus scrofa

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′ upstream region was low among mammalian species. However, the OCT4 promoter and four regulatory regions, including distal and proximal enhancer elements, had high similarity. 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. A comparison analysis of naïve and primed state marker gene expression in a dual-reporter assay showed that the expression levels of naïve and primed markers differed in fluorescence signal between high-expressing cells and low-expressing cells. 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.

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124 Functional analysis of porcine OCT4 transcriptional regulatory region-based reporter system

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab124 ◽

2019 ◽

Vol 31 (1) ◽

pp. 187

Author(s):

S.-H. Kim ◽

K.-H. Choi ◽

D.-K. Lee ◽

M. Lee ◽

M.-H. Cho ◽

...

Keyword(s):

Stem Cells ◽

Functional Analysis ◽

Embryonic Stem Cells ◽

Promoter Region ◽

Fluorescent Protein ◽

Regulatory Region ◽

Embryonic Stem ◽

Upstream Region ◽

Reporter Assay ◽

Reporter System

Gene OCT4 plays pivotal roles in maintaining pluripotency of early mammalian embryonic development and embryonic stem cells. It is essential to establish a reporter system based on the OCT4 promoter region for the study of pluripotency. However, there is still a lack of sufficient information about the porcine OCT4 upstream reporter system. To improve our understanding of the porcine OCT4 regulatory region, first, we conducted an investigation to find conserved regions in the porcine OCT4 promoter upstream region by sequence-based comparative analysis using various mammalian genome sequences. A similarity of nucleotide sequences of the 5′ upstream region was low among mammalian species. However, the OCT4 promoter and 4 regulatory regions including distal and proximal enhancer elements have a high similarity. Next, a functional analysis of the porcine OCT4 promoter region was conducted. Luciferase reporter assay indicated that the porcine OCT4 distal enhancer and proximal enhancer are highly activated in mouse embryonic stem cells and embryonic carcinoma cells, respectively (n=3). Comparison analysis of naïve (Tbx3, Nr0b1, Rex1, Esrrb, Nanog, Klf2) or primed (Gata6, Mixl1, Fgf5, Otx2) state marker gene expression in a dual-reporter assay using pOCT4-DE-eGFP and pOCT4-PE-DsRed2 showed that expression of naïve and primed markers were up-regulated in cells with high green fluorescent protein and red fluorescent protein expression, respectively (n=3). Porcine OCT4-upstream region-based reporter constructs showed exclusive expression patterns depending on the state of pluripotency. This work could provide basic information for the porcine OCT4 upstream region and the various porcine OCT4-fluorescence reporter constructs, which can be applied to study species-specific pluripotency in early embryo development and for the establishment of embryonic stem cells in pigs. This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through the Development of High Value-Added Food Technology Program, funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA, 118042-03-1-HD020).

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Abstract 17986: Molecular Beacon-based Purification of Ventricular Cardiomyocytes From Differentiating Embryonic Stem Cells by Targeting Intracellular Mrna

Circulation ◽

10.1161/circ.130.suppl_2.17986 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Kiwon Ban ◽

Brian Wile ◽

Kyu-Won Cho ◽

Sangsung Kim ◽

Jason Singerd ◽

...

Keyword(s):

Stem Cells ◽

Transcription Factor ◽

Embryonic Stem Cells ◽

Cell Sorting ◽

Disease Modeling ◽

Embryonic Stem ◽

Target Cells ◽

Fluorescence Signal ◽

Ventricular Cardiomyocytes ◽

Specificity And Sensitivity

Background: Ventricular cardiomyocytes (CMs) are an ideal cell type for cardiac cell therapy since they are the main cells generating cardiac forces. However, isolating them from differentiating pluripotent stem cells (PSCs) has been challenging due to the lack of specific surface markers. Here we show that ventricular CMs can be purified from differentiating mouse embryonic stem cells (mESCs) using molecular beacons (MBs) targeting specific intracellular mRNAs. MBs are dual-labeled oligonucleotide hairpin probes that emit a fluorescence signal when hybridized to target mRNAs, allowing isolation of specific target cells by fluorescence activated cell sorting (FACS) with high specificity and sensitivity. Methods and Results: We generated three different MBs (IRX4-1, -2, -3) designed to target specific regions of mRNAs of iroquois homeobox protein 4 (Irx4), a specific transcription factor for ventricular CMs. Among three IRX4 MBs, IRX4-2 MB demonstrated the highest sensitivity and specificity, thus IRX4-2 MB was selected to purify mESC-derived ventricular CMs. Subsequently, IRX4-2 MBs were delivered into cardiomyogenically differentiating mESC cultures and cells showing strong signals from IRX4-2 MBs were FACS-sorted. Flow cytometry demonstrated that 92~97% of IRX4-2 MB-positive cells expressed a marker for ventricular CMs myosin light chain 2 ventricular isoform (Myl2) as well as cardiac troponin 2 (Tnnt2). Importantly, higher than 98% of IRX4-2 MB-positive cells displayed ventricular CM-like action potentials during electrophysiological analyses. These IRX4-2 MB-based purified ventricular CMs continuously maintained their CM characteristics verified by synchronous beating, Ca2+ transient, and expression of ventricular CM-specific proteins. Conclusions: We established a novel MB-based cell sorting system targeting a transcription factor that is specific for ventricular CM to generate homogeneous and functional ventricular CMs. This is the first report to show the feasibility of isolating pure ventricular CMs without modifying host genes, and this platform will be useful for therapeutic applications, disease modeling, and drug discovery.

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Targeting of De Novo DNA Methylation Throughout the Oct-4 Gene Regulatory Region in Differentiating Embryonic Stem Cells

PLoS ONE ◽

10.1371/journal.pone.0009937 ◽

2010 ◽

Vol 5 (4) ◽

pp. e9937 ◽

Cited By ~ 51

Author(s):

Rodoniki Athanasiadou ◽

Dina de Sousa ◽

Kevin Myant ◽

Cara Merusi ◽

Irina Stancheva ◽

...

Keyword(s):

Dna Methylation ◽

Stem Cells ◽

Embryonic Stem Cells ◽

De Novo ◽

Regulatory Region ◽

Embryonic Stem ◽

Gene Regulatory

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Development and Validation of a Rex1-RFP Potency Activity Reporter Assay That Quantifies Stress-Forced Potency Loss in Mouse Embryonic Stem Cells

Stem Cells and Development ◽

10.1089/scd.2015.0169 ◽

2016 ◽

Vol 25 (4) ◽

pp. 320-328 ◽

Cited By ~ 9

Author(s):

Quanwen Li ◽

Nardhy Gomez-Lopez ◽

Sascha Drewlo ◽

Elly Sanchez-Rodriguez ◽

Jing Dai ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cells ◽

Reporter Assay ◽

Development And Validation

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Transcription and mRNA splicing of the human lactoferrin gene controlled by the regulatory region of the bovine ? S1 casein gene in the mammary gland of transgenic mice and in mouse embryonic stem cells

Russian Journal of Genetics ◽

10.1007/s11177-005-0077-x ◽

2005 ◽

Vol 41 (3) ◽

pp. 227-232 ◽

Cited By ~ 1

Author(s):

E. S. Zakharova ◽

M. V. Pryzhkova ◽

A. V. Kibardin ◽

T. G. Ermolkevich ◽

S. G. Kadulin ◽

...

Keyword(s):

Stem Cells ◽

Mammary Gland ◽

Embryonic Stem Cells ◽

Transgenic Mice ◽

Regulatory Region ◽

Embryonic Stem ◽

Mrna Splicing ◽

Human Lactoferrin ◽

Casein Gene ◽

Lactoferrin Gene

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Differential Transcriptional Regulation of the NANOG Gene in Chicken Primordial Germ Cells and Embryonic Stem Cells

10.21203/rs.3.rs-81505/v1 ◽

2020 ◽

Author(s):

Hee Jung Choi ◽

So Dam Jin ◽

Deivendran Rengaraj ◽

Jin Hwa Kim ◽

Bertrand Pain ◽

...

Keyword(s):

Stem Cells ◽

Transcriptional Regulation ◽

Embryonic Stem Cells ◽

Germ Cells ◽

Primordial Germ Cells ◽

Embryonic Stem ◽

Regulatory Elements ◽

Luciferase Reporter ◽

Cell Type ◽

Cell Type Specific

Abstract BackgroundNANOG is a core transcription factor (TF) in embryonic stem cells (ESCs) and primordial germ cells (PGCs). Regulation of the NANOG gene by TFs, epigenetic factors, and autoregulatory factors is well characterized in ESCs, and transcriptional regulation of NANOG is well established in these cells. Although NANOG plays a key role in germ cells, the molecular mechanism underlying its transcriptional regulation in PGCs has not been studied. Therefore, we investigated the mechanism that regulates transcription of the chicken NANOG (cNANOG) gene in PGCs and ESCs. ResultsWe first identified the transcription start site of cNANOG by 5’-rapid amplification of cDNA ends PCR analysis. Then, we measured the promoter activity of various 5’ flanking regions of cNANOG in chicken PGCs and ESCs using the luciferase reporter assay. cNANOG expression required transcriptional cis-regulatory elements, which were positively regulated by POU5F3 (OCT4) and SOX2 and negatively regulated by TP53 in PGCs. The proximal region of the cNANOG promoter contains a positive cis-regulatory element (CCAAT/enhancer-binding protein (CEBP)-binding site) in ESCs. Furthermore, small interfering RNA-mediated knockdown demonstrated that POU5F3, SOX2, and CEBP played a role in cell type-specific transcription of cNANOG.ConclusionsWe show for the first time that different cis-regulatory elements control transcription of cNANOG in a cell type-specific manner. This finding might help to elucidate the mechanism that regulates cNANOG expression in PGCs and ESCs.

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210 DEFINING THE MINIMAL PROMOTER REGION OF BOVINE VASA HOMOLOGUE (Bvh) BY IN SILICO ANALYSIS

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab210 ◽

2013 ◽

Vol 25 (1) ◽

pp. 253

Author(s):

L. F. Malaver-Ortega ◽

H. Sumer ◽

P. J. Verma

Keyword(s):

Stem Cells ◽

Germ Cell ◽

Promoter Region ◽

Primordial Germ Cell ◽

Embryonic Stem ◽

Regulatory Elements ◽

Upstream Region ◽

Functional Level ◽

Cell Specification ◽

Germ Cell Specification

The DEAD box polypeptide 4, DDX4 or VASA, is a highly conserved gene that encodes a putative RNA helicase with the motif DEAD (Asp-Glu-Ala-Asp). Although little is known about its role in germ cell genesis, VASA is one of the earliest, specialised markers of primordial germ cell (PGC) specification. Furthermore, this process of specification has been recapitulated to some degree in vitro using embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) in mice and humans, using VASA expression as one of the criteria for differentiation and sorting of the differentiated cells. In order to establish a system for isolation and tracking of bovine iPSC undergoing germ cell specification, we analysed all regulatory elements in the 5-kb upstream region [RC 5083564–5088564 Bos taurus (Hereford) chromosome 20 genomic scaffold: NW_003104511.1] of the bovine VASA homologue (Bvh) locus, which is thought to be the putative promoter region of Bvh, and in the in vivo validated promoter regions, for the corresponding homologous genes in human and mouse. We performed the analysis using 2 different approaches: at the sequence level, by orthologous promoter alignment of transcription factor (TF) binding sites (TFBS) using DiAling®, and at the functional level, by functional unit analysis (complex model) using Frameworker® (Genomatix, Munich, Germany). The initial DiAling® analysis did not produce similarities between the 3 analysed species. In contrast, using the complex analysis of functional units, we identified 85 single elements common to all 3, and 795, 482, and 129 models composed of 2, 3, and 4 elements, respectively. The number of models was reduced to 3 [M1, M2, and M3 (P = 4.8 × 10–11)] by increasing the number of TF (each model composed of 6 different elements). As a result, members of SOX/SRY-sex/testis determining and related HMG box factor family related with germ cell specification, pluripotent-related factors such as members of the octamer binding protein family, and TFs common to numerous vertebrate genes such as homeobox transcription factors were identified (Table 1). Based on these results, we determined a region of approximate 0.6 kb upstream of the Bvh gene, which encloses a core of TFBS conserved at the functional level between species. We propose that this sequence is the best candidate for driving the expression of reporter genes under Bvh promoter control. Table 1.Six element models

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