XIST has been known to long-non coding RNA which regulate X-chromosome inactivation in female mammal and the gene has been suggested to having important role in early embryo development and embryonic stem cell. However, its coding region has been unclear in pig. To determine the coding region of XIST in pig, we have examined candidate site of XIST coding region in pig by BLAST, PCR, and sequencing. By comparing pig whole genome sequence (Sus scrofa 10.2) with human, murine, and bovine XIST transcript sequence using BLAST, we selected candidate coding region of XIST in pig. The result showed XIST is coded on the minus strand of NW_003612825 contig and its length was nearly 32kb which was similar to the length of human and bovine XIST gene. With the candidate model, we performed RT-PCR to confirm the coding region of XIST with 24 primer pairs and they were expressed only female porcine embryonic fibroblast (PEF) but not in male PEF. By designing candidate intron spaning primer we could confirmed candidate intron is present between first and last exon (distance, 9.2kb vs product size, 2kb). The seqeucne of amplicon was analyzed and we could confirmed there were 5 small exons (less than 400 bp) like XIST coding region of other species which have 4 to 5 small exon between first and last exon. To confirm coding strand in pig, we conducted strand specific reverse transcription. We confirmed candidate XIST was coded on the negative strand of contig on X-chromosome as the result of homology analysis by BLAST. With the candidate pig XIST sequence, we aligned the sequence with XIST sequences of 3 species, human, mouse, and bull by clustalW. These result showed candidate sequence of pig XIST is most similar to that of bovine and the homology between pig and human was higher than result between mouse and human. These results could support for X chromosome inactivation analysis and the function of XIST in pig preimplantation embryos. * This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2012006276)

Several studies have been conducted with the aim of establishing embryonic stem cell lines from porcine embryos. However, most researchers to date have found it difficult to maintain an ES-like state in derived cell lines, with the cells showing a strong tendency to differentiate into an epithelial or EpiSC-like state. We have also been able to derive cell lines of an EpiSC-like state and a differentiated non-ES-like state from porcine embryos of various origins, including invitro fertilized(IVF), in vivo derived, IVF aggregated and parthenogenetic embryos. In addition, we have generated induced pluripotent stem cells(piPSCs) via plasmid transfection of reprogramming factors (Oct4, Sox2, Klf4 and c-Myc) into porcine fibroblast cells. X chromosome inactivation (XCI) have recently been addressed as a hallmark to determine whether pluripotent cell is naïve or primed state. In this study, we could confirm the X chromosome inactivation status in female cell lines as well as marker expression, pluripotency and of our Epi- SC-like pESC lines along with our piPSC line. All of our cell lines showed AP activity and expressions of the genes Oct4, Sox2, Nanog, Rex, TDGF1, bFGF, FGFR1, FGFR2, Nodal and Activin-A involved in pluripotency and signaling pathways, XCI in female cell lines, in vitro differentiation potential and a normal karyotype, thus displaying similarities to epiblast stem cells or hES cells. Therefore, it may be inferred that, as a non-permissive species, the porcine species undergoes reprogramming into a primed state during the establishment of pluripotent stem cell lines.

자연상태(自然狀態)에서 발현(發現)된 병반(病斑)을 이용(利用), 도열병균(稻熱病菌)의 포자형성량(胞子形成量)과 이탈량(離脫量)을 년(年)에 조사(調査)하였다. 절취(切取)한 병반(病斑)의 포자형성량(胞子形成量)과 자연상태하(自然狀態下)에서의 포자이탈량(胞子離脫量)은 병반발현후(病斑發現後) 일(日) 사이에 최고치(最高値)에 달(達)했으며 그 수(數)는 각각(各各) 16,200개(個)와 15,900개(個)였다. 자연상태하(自然狀態下)에서의 포자이탈(胞子離脫)은 30일간(日間) 지속(持續)되었다.

The four transcription factors Oct4, Sox2, Klf4 and c-Myc have been used for making induced pluripotent stem cells. Many efforts have focused on reducing the number of transcription factors, especially c-Myc and Klf4 known as oncogene, for making induced pluripotent stem cells. Recently it have been demonstrated that Oct4 and Sox2 are able to reprogram human fibroblasts or cord blood cells to induced pluripotent stem cells and Oct4 has the ability to reprogram mouse and human neural stem cell to induced pluripotent stem cells. These researches imply cell types for reprogramming experiments have great influence on selection of reprogramming factors. Here we report that pig kidney cortex fibroblasts need only c-Myc factor when they are used for making induced pluripotent stem cells. We used two vector system including drug-inducible vector system and constitutive expression vector system. The two systems generate induced pluripotent stem cells from pig kidney fibroblasts successfully. These one-factor induced pluripotent stem cells are not only similar but also different to pig embryonic stem-like cells. These two one-factor induced pluripotent stem cell lines can express pluripotency related genes and be differentiated into all three germ layers in vitro. However, these two cell lines can be sub-cultured as a single cell by trypsin. Our results support that single factor, c-Myc, is sufficient to converting pig kidney cortex fibroblasts into induced pluripotent stem cells.

Chrysanthemum (Chrysanthemum morifolium) is one of the most popular ornamental species in the world due to the great diversity of inflorescence form and color. There has been increasing demands for various types of chrysanthemums, such as cut flowers, potted plants and bedding plants. However, the genomic studies of this species have been not extensively conducted relative to other ornamental species due to high levels of polyploidy (2n = 4x =36 or 2n = 6x = 54) and heterozygosity as well as large genome size. In this work, we developed a molecular tool for cultivar identification using simple sequence repeats (SSRs) and investigated genetic diversity in 127 chrysanthemum cultivars. Of the 150 SSR primer pairs tested in this study, 62 primers were obtained from previous studies, while 88 primers were designed using the unigene sequences of C. nankingense and the Expressed Sequence Tag (EST) sequences of C. morifolium in the NCBI database. Thirty SSR primers were selected based on polymorphism and banding patterns in a subset of 8 cultivars and used to amplify the DNA of 127 chrysanthemum cultivars. The UPGMA dendrogram based on these 30 SSR markers showed that most of chrysanthemum cultivars were divided into five clusters. These results will benefit chrysanthemum research community to develop elite cultivars.

Aromatase is an enzyme that converts testosterone to estrogen. This enzyme, present in the sperm as well as various tissue and cells, has been considered to be related to the fertility of human and mouse sperm. Therefore, we examined effect of aromatase inhibitor on viability and fertility of sperm, and quantity of aromatase in sperm groups with different density in pig. To analyze the effect of aromatase on sperm viability, we treated aromatase inhibitor to the sperm with different concentrations (0, 10, 20, 50, 100, 200, 500 μM) at different time (0.5, 1, 2, 4, 8 hours). After the treatment, the sperm viability was calculated by hypo-osmotic swelling test. We selected 0, 50, 100 μM concentration during 0.5 hour as inhibitor treatment condition before in vitro fertilization. Next, we examined fertility and quantified aromatase protein in sperms with different density. In the first experiment, viability of sperm was decreased following the increasement of inhibitor concentration. The aromatase inhibited sperm showed lower penetration rate and cleavage rate than those of non-treated sperm. Concentration of 50 μM inhibitor had no significant effect on the sperm viability, but it significantly reduced sperm fertility. Second, sperms with low density showed higher penetration rate, but no significant difference between sperms with high density. In conclusion, aromatase is responsible for viability and fertility of porcine sperm similar to mouse and human, however, density of sperm has no correlation with quantity of aromatase protein.