• Proceedings of the PSK Conference
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• 2003.10b
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• pp.145.3-146
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• 2003

Transgenic Eleutherococcus senticosus plants were prepared by introducing the genes for squalene synthase (SQS), hygromycin phosphotransferase (HPT) and green fluorescent Protein (GFP) through Agrobacterium-mediated transformation. The enzyme, SQS, represents a putative branch point in the isoprenoid pathway capable of diverting carbon flow specifically to the biosynthesis of phytosterol and oleanolic acid. The full SQS gene was isolated from P. ginseng roots. Early globular embryo clusters developed from embryogenic callus were used as the explant source. (omitted)

• Development and Reproduction
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• v.19 no.4
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• pp.181-187
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• 2015

Early life stage mortality in fish is one of the problems faced by loach aquaculture. However, our understanding of immune system in early life stage fish is still incomplete, and the information available is restricted to a few fish species. In the present work, we investigated the expression of immune-related transcripts in loach during early development. In fishes, recombination-activating gene 1 (RAG-1) and sacsin (SACS) have been considered as immunological function. In this study, the expression of the both genes was assessed throughout the early developmental stages of loach using real-time PCR method. maRAG-1 mRNA was first detected in 0 dph, observed the increased mostly until 40 dph. Significant expression of maRAG-1 was detected in 0 to 40 dph. These patterns of expression may suggest that the loach start to develop its function after hatching. On the other hand, maSACS was detected in unfertilized oocyte to molura stages and 0 to 40 dph. maSACS mRNA transcripts were detected in unfertilized oocytes, suggesting that they are maternally transferred.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.1
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• pp.77-86
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• 1998

The uptake of glucose for metabolism and growth is essential to most animal cells and is mediated by glucose-transporter (GLUT) proteins. The aim of this study was to determine which class of glucose transporter molecules was responsible for uptake of glucose in the mouse early embryo and at which stage the corresponding genes were expressed. In addition, co-culture system with vero cell was used to investigate the effect of the system on GLUT expression. Two-cell stage embryos were collected from the superovulated ICR female and divided into 3 groups. As a control, embryos were cultured in 0.4% BSA-T6 medium which includes glucose. For the experimental groups, embryos were cultured in either co-culture system with vero cells or glucose-free T6 medium supplemented with 0.4% BSA and pyruvate as an energy substrate. 2-cell to blastocyst stage embryos in those groups were respectively collected into microtubes (50 embryos/tube). Total RNA was extracted and RT-PCR was performed. The products were analysed after staining ethidium bromide by 2% agarose gel electrophoresis. Blastocysts were collected from each group at l20hr after hCG injection. They were fixed in 2.5% glutaraldehyde, stained with hoechst, and mounted for observation. In control, GLUT1 was expressed from 4-cell to blastocyst. GLUT2 and GLUT3 were expressed in morula and blastocyst. GLUT4 was expressed in all stages. When embryos were cultured in glucose-free medium, no significant difference was shown in the expression of GLUT1, 2 and 3, compared to control. However GLUT4 was not expressed until morular stage. When embryos were co-cultured with vero cell, there was no significant difference in the expression of GLUT1, 2, 3 and 4 compared to control. To determine cell growth of embryos, the average cell number of blastocyst was counted. The cell number of co-culture ($93.8{\pm}3.1$, n=35) is significantly higher than that of control and glucose-free group ($76.6{\pm}3.8$, n=35 and $68.2{\pm}4.3$, n=30). This study shows that the GLUT genes are expressed differently according to embryo stage. GLUTs were detectable throughout mouse preimplantation development in control and co-culture groups. However, GLUT4 was not detected from 2- to 8-cell stage but detected from morula stage in glucose-free medium, suggested that GLUT genes are expressed autocrinally in the embryo regardless of the presence of glucose as an energy substrate. In addition, co-culture system can increase the cell count of blastocyst but not improve the expression of GLUT. In conclusion, expression of GLUT is dependent on embryo stage in preimplantation embryo development.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.4
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• pp.456-464
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• 2010

RNA interference (RNAi) is an acknowledged useful and effective tool to study gene function in various cells. Here, we suppressed the Connexin 43 (Cx 43) gene expression during in vitro development of ovine pre-implantation embryos using the RNAi method. The 353 bp Cx 43 double-stranded RNA was microinjected into in vitro fertilized ovine zygotes, and the levels of target mRNA and protein were investigated. Control groups included uninjected zygotes or those injected with RNase-free water. The dsRNA injection resulted in the specific reduction of Cx 43 transcripts as analyzed by quantitative real-time RT-PCR and decreased protein levels as shown by Western blot analysis at the blastocyst stage. Microinjection of Cx 43 dsRNA led to 20.3%, 21.7% and 34.5% blastocyst rates and 19.2%, 37.5% and 41.3% hatched blastocyst rates in Cx 43 dsRNA-injected, water-injected and uninjected groups, respectively. Then the RNAi could not significantly affect cell number and cell death rates of blastocysts. Therefore, suppression of Cx 43 dsRNA and proteins did not apparently affect the development potential of ovine pre-implantation embryos but may play a role in embryo quality. RNAi technology is a promising approach to study gene function in early ovine embryogenesis.

• Molecules and Cells
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• v.41 no.12
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• pp.1061-1071
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• 2018

From Xenopus embryo studies, the BMP4/Smad1-targeted gene circuit is a key signaling pathway for specifying the cell fate between the ectoderm and neuro-ectoderm as well as the ventral and dorsal mesoderm. In this context, several BMP4/Smad1 target transcriptional factors have been identified as repressors of the neuro-ectoderm. However, none of these direct target transcription factors in this pathway, including GATA1b, Msx1 and Ventx1.1 have yet been proven as direct repressors of early neuro-ectodermal gene expression. In order to demonstrate that Ventx1.1 is a direct repressor of neuro-ectoderm genes, a genome-wide Xenopus ChIP-Seq of Ventx1.1 was performed. In this study, we demonstrated that Ventx1.1 bound to the Ventx1.1 response cis-acting element 1 and 2 (VRE1 and VRE2) on the promoter for zic3, which is a key early neuro-ectoderm gene, and this Ventx1.1 binding led to repression of zic3 transcription. Site-directed mutagenesis of VRE1 and VRE2 within zic3 promoter completely abolished the repression caused by Ventx1.1. In addition, we found both the positive and negative regulation of zic3 promoter activity by FoxD5b and Xcad2, respectively, and that these occur through the VREs and via modulation of Ventx1.1 levels. Taken together, the results demonstrate that the BMP4/Smad1 target gene, Ventx1.1, is a direct repressor of neuro-ectodermal gene zic3 during early Xenopus embryogenesis.

• Reproductive and Developmental Biology
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• v.30 no.2
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• pp.107-113
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• 2006

Development of placenta is a complex process that is critical for the pregnancy and controlled by many factors including cytokines, hormones, growth factors and apoptotic molecules. Recently, it has been shown that progranulin (PGRN) functions in growth of embryo and trophectoderm as well as cell migration. To initiate understanding the role of PGRN in human placental development, we investigated the expression of PGRN mRNA and protein in early and late gestation human placentas, term cytotrophoblast cells and two choriocarcinoma cell lines, JEG-3 and Jar. Reverse transcriptase polymerase chain reaction identified mRNAs derived from the PGRN gene in all samples. Immunoblot analysis showed that PGRN proteins are present in early and late gestation human placentas with decreasing levels over gestation and that PGRN proteins are present in normal and transformed trophoblast cells. Immunohistochemical analysis using paraformaldehyde-fixed tissue sections taken from early and late stages of pregnancy showed that PGRN proteins are present in cytotrophoblast cells, syncytiotrophoblast and extravillous cytotrophoblast cells and that expression pattern of PGRN differed according to the stage of cell differentiation. The results of this study are consistent with the hypothesis that PGRN proteins have critical roles in placental development and suggest that PGRN may function in trophoblast cell growth and differentiation.

• The Korean Journal of Zoology
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• v.33 no.3
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• pp.365-372
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• 1990

For the effort to produce transgenic amphibians, a plasmid DNA sequence (cytoplasmic actin promoter-linked bacterial $\beta$-galactosidase gene) was microinjected into fertilized Xenopus eggs. It appeared that the injection of 20 nl solution containing 1-2 ng of DNA was not toxic, but over 4 ng was toxic to embryonic development. The translational product of $\beta$-gal gene ($\beta$-galactosidase) had enzyme activity in all three germ layers of the embryo. Expression of the injected $\beta$-gal genes was first detected at mid-gastrula stage, and the activity persisted up to stage 43 (feeding tadpole) with decreased level of retention. However, the level of the expression was various among the injected individuals as well as each experiment. That is, $\beta$-galactosidase activities did not appear in all cells, instead a localized distribution pattern. Although other possibilities could not be omitted, this mosaic distribution of gene expression seemed to arise from unequal partition of the injected DNA into each blastomere during early cleavage.

• Journal of Veterinary Clinics
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• v.24 no.3
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• pp.295-299
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• 2007

The detrimental effects of gene transfection on embryo development and the molecular mechanism behind the differential expression of genes related to early embryo development were assessed in the production of transgenic cow embryos through somatic cell nuclear transfer (NT). Parthenogenetic, IVF, and transgenic NT embryos derived from ${\alpha}_1$-antitrypsin transfected ear fibroblast cells was produced. To investigate the molecular mechanism behind lower developmental competence of transgenic NT embryos, the differential mRNA expression of three genes ($IFN-{\tau}$, Oct4, Fgf4) in the 3 types of embryo (Parthenogenetic, IVF, transgenic NT) was examined. RNA was extracted from ten blastocysts derived from 3 types of embryos and reverse-transcripted for synthesis of the first cDNA. The quantification of 3 gene transcripts ($IFN-{\tau}$, Oct4, and Fgf4) was carried out in three replicate by quantitative real-time reverse transcriptase PCR. Expression level of $IFN-{\tau}$ mRNA was significantly higher in transgenic NT embryos than parthenogenetic and IVF embryos (P<0.05). However, expression level of Oct4 and Fgf4 of transgenic NT embryos was significantly lower than IVF embryos (P<0.05). Altered levels of these three mRNA transcripts may explain some of the embryonic/fetal/neonatal abnormalities observed in offspring from transgenic NT embryos.

• Environmental Analysis Health and Toxicology
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• v.25 no.3
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• pp.241-251
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• 2010

Crucian carp (Carassius auratus) has been used as the sentinel species for POPs (Persistent Organic Pollutants) monitoring in aquatic environment. However, there is little information for dioxin toxicity and especially, early life stage toxicity in crucian carp have been never carried out. In this study, we investigated several toxic effects for 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) in fertilized egg obtained by natural fertilization from crucian carp. The embryos at 3 h post-fertilization (hpf) were treated with 0.039, 0.156, 0.625, and 2.5 (${\mu}g/L$) TCDD by waterborne exposure for 60 minutes and changed with fresh water 2 times per day. Fertilized eggs started hatching at 51 hpf and TCDD exposed embryo showed decrease of hatching rate in a dose-dependent manner at 75 hpf. Pericardial edema was continuously observed in larvae exposed to TCDD from hatching start time (51 hpf), followed by the onset of mortality. In addition, AhR-related gene, CYP1A was clearly increased by TCDD in a dose dependent manner. These results indicated that fertilized eggs obtained from crucian carp have the TCDD related gene regulation and a distinct TCDD developmental toxicity syndrome by TCDD exposure. Therefore, we suggested that early life stage test in crucian carp could be used as test methods on dioxins toxicity.

• Journal of Embryo Transfer
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• v.33 no.4
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• pp.195-203
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• 2018

Interferon-tau (IFNT) is known as a major conceptus protein that signals the process of maternal recognition of pregnancy in ruminants. Also, multiple interferon genes exist in cattle, However, molecular mechanisms of these bovine IFNT (bIFNT) genes whose expressions are limited have not been characterized. We and others have observed that expression levels of bovine subtype IFNT genes in the tissues of ruminants; thus, bIFNT1 and other new type I (bIFNTc1/c2/c3) gene co-exist during the early stages of conceptus development and non-trophoblast cells. Its genes transcription could be regulated through CDX2 and ETS2 and JUN and/or cAMP-response element binding protein (CREB)-binding protein (CREBBP) expression, a transcription factor implicated in the control of cell differentiation in the trophectoderm. Bovine ear-derived fibroblast cells, were co-transfected with luciferase reporter constructs carrying upstream (positions -1000 to +51) regions of bIFNT1 and other new type I gene and various transcription factor expression plasmids. Compared to each - 1kb-bIFNT1/c1/c2/c3-Luc increased when this constructs were co-transfected with CDX2, ETS2, JUN and/or CREBBP. Also, Its genes was had very effect on activity by CDX2, either alone or with the other transcription factors, markedly increased luciferase activity. However, the degree of transcriptional activation of the bIFNTc1 gene was not similar to that bIFNT1/c2/c3 gene by expression plasmid. Furthermore, Sequence analyses also revealed that the expression levels of bIFNT1/c2/c3 gene mRNAs expression were highest on day 17, 20 and 22 trophoblast and, Madin-Darby bovine kidney (MDBK), Bovine ear-derived fibroblast (EF), and endometrium (Endo) non-trophoblast cells. But, bIFNTc1 mRNA had not same expression level, bIFNTc1 lowest levels than those of IFNT1/c2/c3 gene in both trophoblast and non-trophoblast cells. These results demonstrate that bovine subtype bIFNT genes display differential, in the trophoblast and non-trophoblast cells.