Diagram of the CpG sites located in the ±2 kb region of the transcription start site of porcine <i>XIST</i> and the methylation status of <i>XIST</i> CpG sites in male and female porcine embryonic fibroblasts.

<p>(A) One of the transcription start sites (TSS) identified by 5`-RACE-PCR was set as a +1 (asterisk, 289233^rd nucleotide of NW_003612825.1). Black and empty bars indicate regions upstream and downstream of the TSS, respectively. Methylation of CpG sites in six regions (BS1-1, BS2-1, BS2-2, BS3, BS4-1, and BS4-2) was analyzed. The diagram is scaled. (B) Profiles of the six target regions. Each circle represents a single CG dinucleotide identified in the amplified region. The length of each amplicon was 250 bp –450 bp and contained between 8–16 CG dinucleotides. The diagram is not scaled. (C) The methylation status of 67 <i>XIST</i> CpG sites were analyzed in male and female embryonic fibroblasts. Each circle indicates a CpG dinucleotide. Filled and empty circles represent methylated and unmethylated CpG dinucleotides, respectively. The horizontal line represents one individual clone.</p

Identification of the Porcine <i>XIST</i> Gene and Its Differential CpG Methylation Status in Male and Female Pig Cells

<div><p><i>XIST</i>, a long non-coding RNA, plays an important role in triggering X chromosome inactivation in eutherians, and is used extensively for qualifying stem cells and cloned embryos. However, a porcine <i>XIST</i> has not yet been thoroughly identified despite its biological importance in a wide variety of research fields. Here, we present a full-length porcine <i>XIST</i> sequence assembled using known sequences (GenBank), RNA-Seq data (NCBI SRA), and PCR/sequencing. The proposed porcine <i>XIST</i> gene model encodes a 25,215-bp transcript consisting of 7 exons, including two conserved and two porcine-specific repeat regions. Transcription covering the entire <i>XIST</i> region was observed specifically in female cells, but not in male cells. We also identified eight transcription starting sites (TSSs) and evaluated CpG methylation patterns in the upstream (+2.0 kb) and downstream (−2.0 kb) regions. Sixty-seven CG di-nucleotides identified in the target region were considered to be candidate CpG sites, and were enriched in the following two regions: −284 to +53 bp (13 sites) and +285 to +1,727 bp (54 sites) from the selected TSS. Male 5` region of <i>XIST</i> (64.5 sites, 96.26%) had a higher level of CpG methylation than female DNA (33.4 sites, 49.85%). Taken together, our results revealed that the porcine <i>XIST</i> gene is expressed exclusively in female cells, which is influenced by the lower level of CpG methylation in the putative promoter region compared with male cells. The porcine <i>XIST</i> presented in this study represents a useful tool for related research areas such as porcine embryology and stem cell biology.</p></div

Analysis of repeat sequences in porcine <i>XIST</i> RNA.

<p>(A) Distribution of repeated sequences in the porcine <i>XIST</i> gene model. Gray rectangles indicate exons, and the yellow boxes in the exons indicate repeat regions. The regions having homology with human, bovine, and mouse <i>XIST/Xist</i> are represented to red (h), green (b), and blue (m) boxes, respectively. The first and second repeat regions showed similarity with all accessed <i>XIST/Xist</i> sequence from other species (dashed lines). The diagram is scaled. (B) The first repeat region in porcine <i>XIST</i>. A monomer of the first repeat region in pigs (left panel) share a consensus sequence and a predicted secondary structure of the mouse <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073677#pone.0073677-Wutz2" target="_blank">[28]</a>. Gray text indicates non-conserved sequences. Eight repeated monomers identified in the porcine <i>XIST</i> first repeat region (+327 to +695) were aligned with a consensus sequence (Right panel). Red character indicates different sequence compared to the consensus sequence in conserved stem-loop region. (C) The second repeat region (+2,723 to +2,870) is shown, with non-repeat sequences in gray. (D) The third repeat region (+4,302 to +13,102) fraction. The first five repeats and last repeat were aligned with the consensus sequence. Red characters mean the sequences which were not same to consensus sequence. (E) Alignment of the fourth repeat region (+24,059 to +24,356). Two copies of the monomer were perfectly matched.</p

Diagram of the candidate <i>XIST</i> expressing region in the pig.

<p>(A) Designed <i>XIST</i> model and primer sets. Candidate <i>XIST</i> ncRNA expression regions on the pig X-chromosome scaffold (NW_003612825.1) determined by BLAST searches are represented by black boxes. Empty and filled arrowheads indicate the 289233^rd and 257103^rd nucleotides of the scaffold, which were aligned with the transcription start site (TSS) and the last sequence of human and bovine <i>XIST,</i> respectively. The designed primer sets are represented as black and gray lines: female-expressed primer sets are represented as black lines and regions that were not expressed in either sex (α and β) are represented as gray lines. Restriction enzymes used to digest each amplicon are shown. The diagram is scaled. (B) Diagram of the identified porcine <i>XIST</i> gene model. The porcine <i>XIST</i> expressing region was identified by sequencing. Filled rectangles indicate exons, and boxes with diagonal lines indicate undefined regions. Filled and empty arrowheads indicate candidate 5′ and 3′ end regions identified from alignment with human and bovine <i>XIST</i>, respectively. The diagram is scaled.</p

Epigenetic Changes of Lentiviral Transgenes in Porcine Stem Cells Derived from Embryonic Origin

<div><p>Because of the physiological and immunological similarities that exist between pigs and humans, porcine pluripotent cell lines have been identified as important candidates for preliminary studies on human disease as well as a source for generating transgenic animals. Therefore, the establishment and characterization of porcine embryonic stem cells (pESCs), along with the generation of stable transgenic cell lines, is essential. In this study, we attempted to efficiently introduce transgenes into Epiblast stem cell (EpiSC)-like pESCs. Consequently, a pluripotent cell line could be derived from a porcine-hatched blastocyst. Enhanced green fluorescent protein (EGFP) was successfully introduced into the cells via lentiviral vectors under various multiplicities of infection, with pluripotency and differentiation potential unaffected after transfection. However, EGFP expression gradually declined during extended culture. This silencing effect was recovered by <i>in vitro</i> differentiation and treatment with 5-azadeoxycytidine. This phenomenon was related to DNA methylation as determined by bisulfite sequencing. In conclusion, we were able to successfully derive EpiSC-like pESCs and introduce transgenes into these cells using lentiviral vectors. This cell line could potentially be used as a donor cell source for transgenic pigs and may be a useful tool for studies involving EpiSC-like pESCs as well as aid in the understanding of the epigenetic regulation of transgenes.</p></div

Change in EGFP expression during extended culture and <i>in vitro</i> differentiation.

<p>(A) EGFP expression levels of transduced EpiSC-like pESCs declined during extended culture. The decreased expression recovered during <i>in vitro</i> differentiation. (B) To analyze the effects of the epigenetic state on transgene expression, the DNA methylation patterns of the CMV promoter region in the transgene of each sample were evaluated via bisulfite sequencing. Along with decreases in EGFP expression, the DNA methylation levels of the CMV promoter region increased. Although DNA methylation levels increased during extended culture, they were not altered by <i>in vitro</i> differentiation. (Each circle indicates individual CpG dinucleotides. White and dark circles represent unmethylated and methylated CpGs, respectively. Each row represents one individual clone of amplified PCR products). Values noted by a–h indicate they are significantly different. Data are the mean± S.E.M. (<i>n</i> = 3).</p

Derivation and characterization of EpiSC-like pESCs.

<p>(A) EpiSC-like pESCs derived from <i>in vitro</i>-produced embryos represented typical morphologies of mouse epiblast stem cells and human embryonic stem cells, and have alkaline phosphatase activity (left panel: no-stained colony; right panel: AP stained colony). (B) EpiSC-like pESCs have a normal karyotype (36+ XX) and (C) expressed genes related to pluripotency, as determined by RT-PCR (NT: non-transfected EpiSC-like pESCs passage 14, T: transfected EpiSC-like pESCs passage 14, W.B.: water blank). (D) Expression of pluripotent markers was detected using immunocytochemistry (passage number: 13). (E) Embryoid bodies were generated in suspension culture and spontaneously differentiated onto culture dishes. (F, G) The differentiated cells expressed differentiation marker genes at the mRNA and protein levels (Ectoderm: <i>CRABP2</i>, neurofilament, Mesoderm: <i>DES</i>, vimentin, Endoderm: <i>AFP</i>, cytokeratin 17; passage number: 17). Scale bars = 100 µm, except for A and G (scale bar = A: 200 µm, G: 50 µm).</p

Primer sets for bisulfite sequencing.

<p>Primer sets for bisulfite sequencing.</p

Characterization of EGFP-transduced EpiSC-like pESCs.

<p>(A) EGFP-transduced EpiSC-like pESCs have a normal karyotype (36+ XX). (B) The proportion of EGFP-expressing cells increased to 70–80% via selection for the EGFP-expressing part of colonies. (C) Embryoid bodies formed from EGFP-expressing EpiSC-like pESCs and expressed EGFP after aggregation (left panel: bright field, right panel: EGFP; scale bar = 100 µm). (D) EGFP-expressing EpiSC-like pESCs expressed pluripotent genes at the protein level (scale bar = 100 µm; passage number: 13) and (E) could be differentiated into three germ layers (scale bar = 50 µm; passage number: 23).</p

Silenced transgenes can be reactivated by treatment with the DNA methylase inhibitor, 5′-aza-2′-deoxycitidine (5-AzadC).

<p>(A) 5-AzadC treatment reactivates the silenced transgene, EGFP. (B) The recovered expression level was measured by flow cytometry. (C) 5-AzadC demethylated the methylated CMV promoter region. Values noted by a–h indicate they are significantly different. Data are the means± S.E.M. (<i>n</i> = 3).</p