CRISPRs(clustered regularly interspaced short palindromic repeats) / CRISPR - associated(CAS) system has been used genome editing technology. Genome stage modification using CRISPR/CAS9 system can be used to wide research for the gene functional study and therapeutics. However, improving of CRISPR/CAS9 system in efficiency is essential for application in various fields. Here, we treated various chemicals during the procine early embryo development to increase the mutation of target site by NHEJ(non-homologous end joining). Firstly, we confirmed the chemical toxicity after parthenogenetic activation and then check embryo puality using by counting of total cell number and TUNEL Assay in blastocyst satge. To check any improvement on mutation rate by NHEJ pathway. AZT(3′-Azido-3′-deoxythymidine, antiretroviral drug – 0.1 μM) was treated after injection of cas9 and sgRNA target to OCT4 exon 5 during the zygote stage, followed by PCR sequencing. As a result, AZT treated group shows a significantly increased in knock-out efficiency as a consequence of NHEJ. Nocodazole(anti-neoplastic agent – 200ng/ml), RO-3306 (specific inhibitor of CDK1 - 10 μM) and NU-7026(PKC signalling inhibitor - 50 μM) was treated after injection of cas9 and sgRNA with eGFP vector during the zygote stage(hpa8~hpa20) and checked a efficiency of knock-in by PCR sequencing. Interestingly, nocodazole treatment groups increased of insertion of eGFP sequence in blastocyst stage compared with non-treat group(control : 8.33%, nocodazole treatment : 16.67%). However, RO-3306 and NU-7026 made a no impact. In summary, CRISPR/CAS9 system with treatment of chemicals during porcine embryogenesis can be improving of site-specific mutation and enhancement of CRISPR genome editing.

Unlike somatic cells mitosis, germ cell meiosis consists of two consecutive rounds of divisions that segregate homologous chromosomes and sister chromatids, respectively. The meiotic oocyte is characterized by an absence of centrioles and asymmetric divisions. Centriolin is a relatively novel centriolar protein that functions in mitotic cell cycle progression and cytokinesis. Here, we explored the function of centriolin in meiosis and showed that it was localized to meiotic spindles, and concentrated at the spindle poles and midbody during oocyte meiotic maturation. Unexpectedly, knockdown of centriolin in oocytes with either siRNA or Morpholino micro-injection, did not affect meiotic spindle organization, cell cycle progression, or cytokinesis (as indicated by polar body emission), but led to a failure of peripheral meiotic spindle migration; and symmetric division or large polar body emission. These data suggest that, unlike in mitotic cells, the centriolar protein centriolin does not regulate cytokinesis, but plays an important role in regulating asymmetric division of meiotic oocytes.

Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) have a important role in influence of pre-messenger RNA (pre-mRNA) processing and mRNA metabolism and transportation in cells. Recently, hnRNP A2/B1 can recognize m6A modifications on pre-mRNA or pre-miRNA and affect alternative splicing and miRNA processing in HeLa Cells. However, roles of hnRNP A2/B1 in various cells and tissues, especially in elary embryo development, are unclear. Here, we investigated the temporal and spatial expression patterns of hnRNPA2B13 during mammalian early embryo development. In mouse, hnRNPA2B1 was localized at the nucleus after 1-cell stage, however, hnRNPA2B1 was expressed after 2-cell stage in pig. Then, knockdown of hnRNP A2/B1 induced by RNA interference (RNAi) was used to analyze the effect of hnRNP A2/B1 in preimplantation develop in pigs. Knockdown of hnRNP A2/B1 delayed embryo development. Interestingly, ICM marker OCT4 and Sox2 was significantly decreased in blastocyst stage. mRNA expression show that transcription factors which is Pou5f1, Sox2, Nanog, Cdx2 and AP2γwas decreased the transcription levels without the changing of junction protein, ZO-1, occludin, and CXADR. Outgrowth results indicated that knock-down of hnRNPA2B1 embryos cannot format the colony. Knock-down of Methyltransferase like 3(METTL3) embryos mislocalized the hnRNPA2/B1 at the nucleus. In summary, the expression patterns of hnRNPA2/B1 differ between mouse and porcine embryos, and these differences may reflect species-specific functions during preimplantation embryo development. Our results suggested that hnRNPA2/B1 is necessary for newly synthesis of mRNA related with transcription factor, and early embryo development by the RNA epigenetic modification.

Interferon tau (IFNT), produced by the mononuclear trophectoderm, signals the process of maternal recognition of pregnancy in ruminants, but its expression in vivo is not well characterized. Objectives of this study were to determine IFNT gene isoforms expressed in the bovine uterus, and to identify differences in promoter sequences of IFNT genes that differ in their expression. Through the RNA-seq analysis of bovine conceptuses on days 17, 20 and 22 (day 0 = day of estrus), the expression of only two IFNT transcripts, IFNT1 and IFNTc1, were found, which were indeed classified into the IFNT gene clade. IFNT mRNAs were highest on day 17, and then decreased on days 20, and 22, which were also supported by the results of quantitative RT-PCR. Bovine ear-derived fibroblast (EF) cells were then cotransfected with luciferase reporter constructs carrying 5‘-upstream (positions -1000 to +51) regions of IFNT1 or IFNTc1 and various transcription factor expression plasmids. CDX2, either alone or with other Ap-1, ETS2 and/or CREBBP transcription factors, was found to increase luciferase activity approximately 10 and 18 fold more than twice of those cotransfected with bIFNT1, c1-Luc construct. Furthermore, The degree of transcriptional activation by a combination of the AP1, ETS2, CREBBP and/or CDX2 expression vectors was similar to that of CDX2 along plasmid. However, expression patterns of these Luc activity differented. Whereas bIFNTc1-Luc showed lowest antivity had than bIFNT1-Luc reports. Although, lowest antivity had of the bIFNTc1 –Luc report, cotransfected with the bIFNTc1-Luc construct and AP1(JUN) or/and ETS2 expression plasmid, Luc activity was enhanced approximately 2 and 4-fold more than the bIFNT1-Luc. Furthermore, along CDX2 expression factor had high effect on activity of bIFNT1-Luc reporter than the c1 gene in EF cells. These results suggest that two forms of IFNT genes are expressed in utero and their transcriptional regulations differ.

Multiple interferon tau (IFNT) genes exist in bovine. An antiluteolytic substance secreted by the bovine conceptus and primarily responsible for maternal recognition of pregnancy is bovine trophoblast protein 1 (bIFNT1), one of new type I interferon tau (IFNT) genes. The objectives of this research were to investigate whether multiple, distinct gene encode bIFNT1 and other type I bIFNT gene in the bovine genome or not and to examine the expression of bIFNT1 and other bIFNTc1 mRNAs during conceptus development. The transcription of these genes could be regulated through caudal-related homeobox-2 (CDX2) and ETS2 and/or AP1(JUN) expression, a transcription factor implicated in the control of cell differentiation in the trophectoderm. The presence of mRNAs encoded by bIFNT1 and type I bIFNTc1 genes were examined quantitatively via reverse transcription-polymerase chain reaction (RT-PCR) analysis of total cellular RNA (tcRNA) extracted from on the days 17, 20 and 22 bovine conceptuses. bIFNT1 was highly expressed on the day 17 and transcripts were gradually and weakly detectable on the days 20 and 22. However, the other bIFNTc1 gene examined transcripts was highly expressed on the day 20 and transcripts were weakly detectable on the days 17 and 22 bovine conceptuses. Furthermore, human choriocarcinoma JEG3 was co-transfected with an -1kb-bIFNT1/c1-Luc constructs and several transcription factor expression plasmids. Compared to each -1kb-bIFNT1/c1-Luc increased when this constructs were co-transfected with, ETS2, AP1(JUN), CREBBP and/or CDX2. Also, bIFNTc1 gene was had higher effect on activity by alone ETS2, and AP1(JUN) expression factors in choriocarcinoma JEG3 cell. However, bIFNT1 gene expression of the upstream region was not idented. These results demonstrate that these genes display differential, tissue-specific expression and developmental regulation during pregnancy.

The number of reported mitochondrial genomes (mitogenomes) from the monotypic Lasiocampoidea has been limited until recently. In this study, we sequenced the complete mitogenome of the lappet moth, Kunugia undans (Lepidoptera: Lasiocampidae), and compared it to those of other lasiocampid species and macroheteroceran superfamilies (59 species in six superfamilies). The 15,570-bp long K. undans genome had the typical set of genes found in animal mitogenomes, with the exception of one additional trnR that are located between trnA and trnN loci. Considering that the two trnR copies are located in tandem with proper secondary structures and identical anticodons, a gene duplication event might be responsible for the presence of the two tRNAs. In summary, the general mitogenome characteristics of Lasiocampoidea did not differ greatly from the remaining macroheteroceran superfamilies, but it did exhibit some unique features.

Currently, only limited number of mitochondrial genomes (mitogenome) is available from Odonata. In order to extend current mitogenome data for comparative biology and phylogeny we sequenced complete mitogenomes of two endangered dragonfly species, Libellula angelina and Nannophya pygmaea (Ododana: Libellulidae). The whole genomes were 15,233 bp in L. angelina and 15,112 bp in N. pygmaea and included a typical set of genes (13 protein-coding genes, two rRNA genes, and 22 tRNA genes) and one major non-coding A+T-rich region. The arrangement of the genomes was identical to typical one found in insects. Phylogenetic reconstruction using concatenated sequences of 13 PCGs and two rRNAs of Odonata (17 species in eight families in three suborders) using both Bayesian Inference (BI) and Maximum Likelihood (ML) methods have shown a strong support for monophyletic Zygoptera (BI, BPP = 1 and ML, 100%). Currently, further scrutinized analysis is under progress.

The spotted-wing drosophila (SWD), Drosophila suzukii (Diptera: Drosophilidae), is an economically damaging pest that feeds on most thin-skinned fruits. In this study, we sequenced portions of the mitochondrial (mt) COI and ND4 genes from a total of 195 individuals collected mainly from Korea. A total of 139 haplotypes were obtained from the concatenated COI and ND4 sequences. A dataset combining GenBank sequences with our own data identified a total of 94 worldwide COI haplotypes with a maximum sequence divergence of 5.433% (32 bp). A rough estimate of genetic diversity in each country showed higher diversity in ancestral distributional ranges, but the invasion over Asian countries seems to have been substantial because haplotype diversity was only 2.35-3.97-fold lower in the USA, Canada, and Italy than that in the populations ancestral ranges.

Cycloamyloses (CA) from high-amylose rice starch (Dodamssal, 50.94% amylose) was produced to form a complex with anthocyanin extracted from black rice (sihtoheukmi). The effects of CA concentration (1, 5, 10 and 30%, w/v), pH (2, 4, 6, and 8), UVB irradiation time (~24h) and thermal treatment time (~24h) on the oxidant capacity of the complex were investigated. Anthocyanin consisting of 95% cyanidin-3-glucoside (C3G) was extracted from the black rice by suspending in 60%(v/v) ethanol containing 0.2%(v/v) hydrochloric acid for 90 min at 50°C. Cycloamyloses (CA) produced by 4-α-glucanotransferase (4αGTase) formed complexes with C3G by shaking the mixture for 48 hours at 30°C. Antioxidant activities (radical scavenging capacity) were determined by using ABTS [2,2'-azinobis-(3-ethylbenzothiazoline- 6-sulfonic acid)]. The antioxidant activities of the complexes increased with pH increasing. Upon UVB irradiation and heat treatment, the antioxidant activities were more retained for the complexes incorporating 10% CA concentration or above compared to the C3G control. Degradation rate constant (Kd) and half-life (T1/2) were obtained from degradation data of CA complex with cyanidin-3-glucoside. The effect of CA complex formation with C3G is significantly observed at pH4. The results suggest that the CA complex formed with C3G indeed has an enhanced antioxidant potential toward UVB and thermal degradation.