Aggregation of pnbiPSCs into <i>in vitro</i> fertilized embryos, and their development <i>in vitro</i>.

<p>*RFP vector was introduced</p><p>Aggregation of pnbiPSCs into <i>in vitro</i> fertilized embryos, and their development <i>in vitro</i>.</p

Phase-contrast images of biPSCs established in two different culture conditions.

<p>(A) bADCs. (B) Primary colonies appearing in primed cell-culture medium. (C) Established primed-type biPSCs. (D) Colonies converted from the primed to naïve state. (E) Primary colonies appearing in niPSCs medium. (F) Established naïve-type biPSCs. (A)–(C), (E), scale bars = 500 μm. (D), (F), scale bars = 100 μm.</p

Naïve-type features of iPSCs.

<p>(A) pnbiPSCs cultured for 4 days in niPSC medium in the presence of JAK inhibitor. (B) pnbiPSCs cultured in the presence of DMSO. (C) The number of cells cultured in the presence of JAK inhibitor or DMSO (*p < 0.05). (D) <i>XIST</i> expression evaluated in pbiPSCs, but not in pnbiPSCs. Immunocytochemistry images of methylation status at H3K27me3 sites (E, pbiPSCs; F, Hoechst staining; G, pnbiPSCs; H, Hoechst staining). Arrowheads indicate puncta of H3K27me3. (A), (B), scale bars = 500 μm.</p

Endogenous and exogenous expression of genes specific to undifferentiated ESCs in biPSCs.

<p>mRNA expression was evaluated by reverse-transcription polymerase chain reaction (RT-PCR). pbiPSCs (P5), primed-type iPSCs at passage 5; pbiPSCs (P50), primed-type iPSCs at passage 50; pnbiPSCs, naïve-type iPSCs at passage 3 converted from primed-type iPSCs at passage 48; nbiPSCs, naïve-type iPSCs cultured under naïve medium from primary culture; bADCs, bovine amnion-derived cells; SNL feeder, SNL feeder cells; vector, plasmid DNA of PB vectors; bACT, bovine β-ACTIN specific for cattle; Uni-ACT, universal β-ACTIN that reacts with both cattle and mice.</p

Production of chimeric fetuses from bovine embryos using the aggregation method.

<p>(A) Naïve-type biPSCs expressing Tag-RFP were aggregated with host at the 8- to 16-cell stage of <i>in vitro</i> fertilized embryos. (B) Chimeric fetuses at day 90 of gestation derived from aggregated embryos. (C) PCR analysis using transgene-specific primers for genomically integrated Oct3/4-2A-Klf4 sequences in 14 tissues. Genomic DNA isolated from pnbiPSCs was used as a positive control. H₂O was used as a negative control (buffer alone for RT-PCR). Immunofluorescence analysis showing the distribution of pnbiPSC-derived cells (RFP-positive with red signals) in the small intestine (D), placenta (E), gonad (F, VASA-positive cells with green signals; arrowheads indicate the portion that is double-positive for RFP and VASA), and kidney (G) of the chimeric fetus. Nuclei were stained with DAPI (blue).</p

Differentiation potential of biPSCs in culture.

<p>(A) Embryoid body formation of pbiPSCs grown for 6 days in low cell-adhesion dishes. Immunocytochemical staining for markers for the three germ-layer in differentiated cells derived from pbiPSCs. α-fetoprotein (B, endoderm), actin smooth muscle (C, mesoderm), and glial fibrillary acidic protein (D, ectoderm) were used as markers. (E) Embryoid body formation by pnbiPSCs. Immunocytochemical staining for α-fetoprotein (F), actin smooth muscle (G), glial fibrillary acidic protein (H). (A), (E), scale bars = 500 μm. (B)–(D), (F)–(H), scale bars = 100 μm.</p

Reprogramming of bovine amnion-derived cells (bADCs) into iPSCs using Dox-inducible PB vectors.

<p>(A) Timeline for the establishment of primed-type biPSC lines. (B) Timeline for the establishment of naïve-type biPSC lines.</p

Characterization of biPSCs.

<p>(A) Alkaline phosphatase activity in primed-type iPSCs (pbiPSCs). (B) Alkaline phosphatase activity in naïve-type iPSCs derived from pbiPSCs (pnbiPSCs). (C) Karyotyping image of pbiPSCs at passage 65. (D) Karyotyping image of nbiPSCs at passage 10. (E) Proportion of cells with the indicated number of chromosomes (n = 20). (F)–(I) OCT3⁄4 (F, OCT3⁄4 staining; G, Hoechst staining) and NANOG (H, NANOG staining; I, Hoechst staining) expression in pbiPSCs. (J)–(M) OCT3⁄4 (J, OCT3⁄4 staining; K, Hoechst staining) and NANOG (L, NANOG staining; M, Hoechst staining) expression in pnbiPSCs. (A), (B), (J)–(M), scale bars = 100 μm. (F)–(G), scale bars = 500 μm.</p

The Promoter of the Oocyte-Specific Gene, <i>Oog1</i>, Functions in Both Male and Female Meiotic Germ Cells in Transgenic Mice

<div><p><i>Oog1</i> is an oocyte-specific gene whose expression is turned on in mouse oocytes at embryonic day (E) 15.5, concomitant with the time when most of the female germ cells stop proliferating and enter meiotic prophase. Here, we characterize the <i>Oog1</i> promoter, and show that transgenic <i>GFP</i> reporter expression driven by the 2.7 kb and 3.9 kb regions upstream of the <i>Oog1</i> transcription start site recapitulates the intrinsic <i>Oog1</i> expression pattern. In addition, the 3.9 kb upstream region exhibits stronger transcriptional activity than does the 2.7 kb region, suggesting that regulatory functions might be conserved in the additional 1.2 kb region found within the 3.9 kb promoter. Interestingly, the longer promoter (3.9 kb) also showed strong activity in male germ cells, from late pachytene spermatocytes to elongated spermatids. This is likely due to the aberrant demethylation of two CpG sites in the proximal promoter region. One was highly methylated in the tissues in which <i>GFP</i> expression was suppressed, and another was completely demethylated only in Oog1pro3.9 male and female germ cells. These results suggest that aberrant demethylation of the proximal promoter region induced ectopic expression in male germ cells under the control of 3.9 kb <i>Oog1</i> promoter. This is the first report indicating that sex-dependent gene expression is altered according to the length and the methylation status of the promoter region. Additionally, our results show that individual CpG sites are differentially methylated and play different roles in regulating promoter activity and gene transcription.</p> </div

19th International Conference on Interactive Collaborative Learning

This book presents the proceedings of the 19th International Conference on Interactive Collaborative Learning, held 21-23 September 2016 at Clayton Hotel in Belfast, UK. We are currently witnessing a significant transformation in the development of education. The impact of globalisation on all areas of human life, the exponential acceleration of developments in both technology and the global markets, and the growing need for flexibility and agility are essential and challenging elements of this process that have to be addressed in general, but especially in the context of engineering education. To face these topical and very real challenges, higher education is called upon to find innovative responses. Since being founded in 1998, this conference has consistently been devoted to finding new approaches to learning, with a focus on collaborative learning. Today the ICL conferences have established themselves as a vital forum for the exchange of information on key trends and findings, and of practical lessons learned while developing and testing elements of new technologies and pedagogies in learning