• Current Research on Agriculture and Life Sciences
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• v.8
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• pp.51-58
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• 1990

The study was carried out to investigate the viability of mouse embryos bisected at the stages of 8-cell, morula and blastocyst and, also, to find out the feasibility of offspring production by transfer of the bisected blastocysts with Quick-splitting. The results obtained are summarized as follows: 1. The mouse embryos were bisected with bio-cut blade at the stages of 8-cell and morula, and cultured in Medium 2. The bisected embryos were developed to blastocyst stage by 64% and 81%. respectively. 2. The blastocyts which were cultured in Medium 2 after being bisected at the stages of 8-cell and morula were observed normal outgrowth in Ham's F-10 medium by 86% and 90%, respectively. 3. The blastocyts which were Quick-splitted in Medium 2 were observed normal outgrowth by 97%. however, no offspring was obtained by transfer of Quick-splitted blastocysts.

• Molecules and Cells
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• v.19 no.1
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• pp.46-53
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• 2005

Human embryonic stem (hES) cells, unlike most cells derived from adult or fetal human tissues, represent a potentially unlimited source of various cell types for basic clinical research. To meet the increased demand for characterized hES cell lines, we established and characterized nine new lines obtained from frozen-thawed pronucleus-stage embryos. In addition, we improved the derivation efficiency from inner cell masses (to 47.4%) and optimized culture conditions for undifferentiated hES cells. After these cell lines had been maintained for over a year in vitro, they were characterized comprehensively for expression of markers of undifferentiated hES cells, karyotype, and in vitro/in vivo differentiation capacity. All of the cell lines were pluripotent, and one cell line was trisomic for chromosome 3. Improved culture techniques for hES cells should make them a good source for diverse applications in regenerative medicine, but further investigation is needed of their basic biology.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.5
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• pp.2081-2086
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• 2015

Gecko is a kind of traditional Chinese medicine with remarkable antineoplastic activity. However, undefined mechanisms and ambiguity regarding active ingredients limit new drug development from gecko. This study was conducted to assess anti-angiogenic properties of the aqueous extracts of fresh gecko (AG) or macromolecular components separated from AG (M-AG). An enzyme-linked immunosorbent assay (ELISA) approach was applied to detect the vascular endothelial growth factor (VEGF) secretion of the tumor cells treated with AG or M-AG. The effect of AG or M-AG on vascular endothelial cell proliferation and migratory ability was analyzed by tetrazolium dye colorimetric method, transwell and wound-healing assays. Chick embryo chorioallantoic membrane (CAM) assays were used to ensure the anti-angiogenic activity of M-AG in vivo. The results showed that AG or M-AG inhibited the VEGF secretion of tumor cells, the relative inhibition rates of AG and M-AG being 27.2% and 53.2% respectively at a concentration of $20{\mu}L/mL$. AG and M-AG inhibited the vascular endothelial (VE) cell proliferation with IC50 values of $11.5{\pm}0.5{\mu}L/mL$ and $12.9{\pm}0.4{\mu}L/mL$ respectively. The VE cell migration potential was inhibited significantly (p<0.01) by the AG (${\geq}24{\mu}L/mL$) or M-AG (${\geq}12\mu}L/mL$) treatment. In vivo, neovascularization of CAM treated with M-AG was inhibited significantly (p<0.05) at a concentration of ${\geq}0.4{\mu}L/mL$. This study provided evidence that anti-angiogenesis is one of the anti-tumor mechanisms of AG and M-AG, with the latter as a promising active component.

• Journal of Embryo Transfer
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• v.28 no.1
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• pp.19-24
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• 2013

This study assesses of efficiency of oocyte recovery and in vitro development for during the non breeding season in goat. Thirty-four matured goats, maintained in a pen under natural day length and fed hay ad libitum, were pretreated with progestagen implanted CIDR for 10 days. Superovulation treatment of the goats received twice daily intramuscular injections of a total of 70 mg FSH for 3 days from Day 8 of CIDR. All the gonadotropin treated goats were injected with 10 mg $PGF_2{\alpha}$ on Day 8 and 400~600 IU hCG in the afternoon on Day 10. Oocytes were recovered by follicle aspiration or oviduct flushing at 35 to 40 h after hCG injection through mid-ventral incision. The in vivo matured oocytes were activated by ionomycin (5 min) and 6-DMAP (3.5~4 h). The activated oocytes were cultured in mSOF medium containing 0.8% BSA at $38.5^{\circ}C$ in an atmosphere of 5% $CO_2$, 5% $O_2$, 90% $N_2$ for 7~8 days. There was no significant difference in the mean number of CL and in vivo matured and follicular oocytes recovered. But, quality of I+II grade follicular oocytes was lower (p<0.05) in the prepubertal goat (25.0%) than the adults (52.4%). The same results were also observed in the cleavage and blastocyst rate of activated oocytes. The clavage and blastocyst rate from prepubertal derived oocytes were lower (p<0.05) in the prepubertal goat (54.5%, 23.3%) than the adult goat (86.8%, 46.6%). Considering overall these results, we suggest that maturation of donor goats is a major factor affecting recovered oocytes quality and in vitro development of activated goat oocytes. There was no significant difference in oocyte quality between seasonal treatments.

• Journal of Life Science
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• v.16 no.4
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• pp.571-578
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• 2006

HtrA2/Omi, a mitochondrial trypsin-like serine protease, is pivotal in regulating apoptotic cell death. Several lines of recent evidence suggest that HtrA2 is associated with the pathogenesis of neurodegenerative disorders; however, the physiological function of HtrA2 still remains elusive. For studying physiological function of HtrA2 in depth, it is necessary to develop a suitable expression system in the model animal. We therefore utilized the zebrafish as a model animal to establish expression of human HtrA2 (hHtrA2) in vivo. For expression of mature HtrA2 as GFP fusion in zebrafish embryos, the HtrA2 (WT) or (S306A) cDNAs with the C-terminal GFP tag were inserted into the pCS2+ plasmid. Expression patterns of HtrA2 in HEK293 cells were first monitored by immunofluorescence staining and immunoblot assays, showing approximately 64 kDa of the HtrA2-GFP fusion proteins. Subsequently, the hHtrA2 plasmid DNA or in vitro transcribed mRNA was microinjected into zebrafish embryos. The expression patterns of HtrA2 in Zebrafish embryos were monitored by GFP fluorescence in 24 hours-post-fertilization (hpf). Although expression patterns of HtrA2-GFP in developing embryos were different between the injected DNA and mRNA, both nucleic acids revealed good expression levels to further study the physiological role of HtrA2 in vivo. This study provides a suitable condition for expressing hHtrA2 in the zebrafish embryos as well as a method for generating useful system to investigate physiological properties of the specific human genes.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.121-121
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• 2003

The standard protocol for the production of transgenic mouse from ES-injected embryo has to process via chimera producing and several times breeding steps, In contrast, tetraploid-ES cell complementation method allows the immediate generation of targeted murine mutants from genetically modified ES cell clones. The advantage of this advanced technique is a simple and efficient without chimeric intermediates. Recently, this method has been significantly improved through the discovery that ES cells derived from hybrid strains support the development of viable ES mice more efficiently than inbred ES cells do. Therefore, the objective of this study was to generate transgenic mice overexpressing human resistin gene by using tetrapioid-ES cell complementation method. Human resistin gene was amplified from human fetal liver cDNA library by PCR and cloned into pCR 2.1 TOPO T-vector and constructed in pCMV-Tag4C vector. Human resistin mammalian expression plasmid was transfected into D3-GL ES cells by lipofectamine 2000, and then after 8~10 days of transfection, the human resistin-expressing cells were selected with G418. In order to produce tetraploid embryos, blastomeres of diploid embryos at the two-cell stage were fused with two times of electric pulse using 60 V 30 $\mu$sec. (fusion rate : 93.5%) and cultured upto the blastocyst stage (development rate : 94.6%). The 15~20 previously G418-selected ES cells were injected into tetraploid blastocysts, and then transferred into the uterus of E2.5d pseudopregnant recipient mice. To investigate the gestation progress, two El9.5d fetus were recovered by Casarean section and one fetus was confirmed to contain human resistin gene by genomic DNA-PCR. Therefore, this finding demonstrates that tetraploid-ES mouse technology can be considered as a useful tool to produce transgenic mouse for the rapid analysis of gene function in vivo.

• Macromolecular Research
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• v.16 no.6
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• pp.510-516
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• 2008

New antitumor active polymers, poly(methacryloyl-2-oxy-1,2,3-propanetricarboxylic acid-co-exo-3,6-epoxy-l,2,3,6-tetrahydrophthalic acid) [poly(MTCA-co-ETAc)], poly(methacryloyl-2-oxy-l,2,3-propanetricarboxylic acid-co-hydrogen ethyl-exo-3,6-epoxy-l,2,3,6-tetrahydrophthalate) [poly(MTCA-co-HEET)], and poly(methacryloyl-2-oxy-l,2,3-propanetricarboxylic acid-co-a-ethoxy-exo-3,6-epoxy-1,2,3,6-tetrahydrophthaloyl-5-fluorouracil) [poly(MTCA-co-EETFU)] were synthesized and characterized. Their antitumor activity, inhibition of DNA replication and antiangiogenesis were examined. The structures of the polymers were identified by FT-IR, $^1H$ and $^{13}C$-NMR spectroscopy. The number average molecular weights of the fractionated polymers determined by GPC ranged from 9,400 to 14,900, and polydispersity indices were less than 1.7. The in vitro cytotoxicity of these polymers was determined and their antitumor activity was evaluated. The $IC_{50}$ values (the drug concentration at inhibition of 50% tumor growth) indicated that the synthesized polymers were much better inhibitors of cancer cells and showed lower cytotoxicity than the free 5-FU. The in vivo antitumor activity of the conjugates was examined using mice bearing the sarcoma 180 tumor cell line. The life spans (TIC) of the mice treated with the conjugates were higher than those treated with the free 5-FU. In addition, the synthesized conjugates showed excellent antiangiogenic activity based on an embryo chorioallantoic membrane assay.

• Korean Journal of Ecology and Environment
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• v.36 no.1 s.102
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• pp.83-94
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• 2003

Concentrated releases of zinc into water usually results from discharges associated with industrial purpose. The released zinc into soil is corroded and released into water. In aquatic environment, exess zinc is toxic to the organisms and causes the growth inhibition and malformation of them as a heavy metal. In this study, excess zinc toxicity was tested by FETAX (frog embryo teratogenetic assay with Xenopus)as in vivo system. Xenopus embryos at st.9 were exposed to $100{\sim}900\;{\mu}M$ of zinc for 7 days and 81% of individuals were survived in 100 ${\mu}M$, and 25% were survived in 1000M of zinc solution. In external malformations, swelled belly and intestinal dysplasia were common, and all of tested individuals showed these malformations in 200 ${\mu}M$ or higher concentration of zinc. In 400 ${\mu}M$ or higher concentration, all of tested tadpoles showed faded heart. Also, hypo-pigmentation, lens hernia and loose digestive track were very frequently found in 100 ${\mu}M$ of zinc. The histological study with paraffin section of zinc treated tadpoles showed following abnormalities; regeneration of photoreceptor on retina, reduced vitreous chamber in eye, reduction of red blood cells in heart, abnormal liver, swelling of pronephric cell, muscle dysplasia and palatal papilloma. These abnormalities may be caused by the degeneration of mitochondria, inhibition of cell adhesion, and the formation of leghemoglobin by zinc due to the substitution of $Ca^{2+}$ by $Zn^{2+}$. The body length was reduced due to the excess zinc. From a statistical result, body lengths of 300 ${\mu}M$ or higher concentrative g개ups was significantly reduced comparing that of control group. Recently, many spontaneous malformations and reduction of amphibians are reported, From the results of present study, excess zinc mi호t be a factor of amphibian reduction, and the control of zinc discharges is very important.

• Journal of Embryo Transfer
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• v.26 no.3
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• pp.147-152
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• 2011

This study was conducted to examine whether efficiency of oocyte production from superovulated prepubertal goats. Fifteen prepubertal and twenty adult goats, maintained in a pen under natural day length and fed hay ad libitum, were pretreated with progestagen implanted CIDR for 10 days. Superovulation treatment of the goats received twice daily intramuscular injection of a total of 70 mg FSH for 3 days from Day 8 of CIDR. All the gonadotrophin treated goats were injected with 10 mg $PGF_2{\alpha}$ on Day 8 and 400~600 IU hCG in the afternoon on Day 10. Oocytes were recovered by follicle aspiration or oviduct flushing at 35 to 40 h after hCG injection through mid-ventral incision. The in vivo matured oocytes was activated by ionomycin (5 min) and 6-DMAP (3.5~4 h). The activated oocytes were cultured in mSOF medium containing 0.8% BSA at 38.5$^{\circ}C$ in an atmosphere of 5% CO$_2$, 5% O$_2$, 90% N$_2$ for 7~8 days. There was no significant difference in the mean number of CL and in vivo matured and follicular oocytes recovered. But, quality of I + II grade follicular oocytes was lower (p<0.05) in the prepubertal goat (25.0%) than the adults (52.4%). The same results were also observed in the cleavage and blastocyst rate of activated oocytcs. The cleavage and blastocyst rate from prepubertal derived oocytes were lower (p<0.05) in the prepubertal goat (54.5%, 23.3%) than the adult goat (86.8%, 46.6%). Considering overall these results, we suggest that maturation of donor goats is a major factor affecting recovered oocytes quality and in vitro development of activated goat oocytes.

• Korean Journal of Animal Reproduction
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• v.22 no.1
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• pp.11-17
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• 1998

This study was carried out to investigate the in vivo developmental potential of mouse zona-hatched blastocysts (HBs). The HBs were cultured in vitro until day 5 and day 6 from zygotes produced in vivo and classified to small (S-HBs), medium (M-HBs) and large (L-HBs) on the basis of embryo diameters. The results obtained in these experiments were summarized as follows ; 1) when the blastocysts at day 4 were further cultured for $24\sim48hr$, HBs obtained at day 5 and day 6 culture in vitro were 29.1% and 22.8%, respectively. 2) Also, when the total cell number of HBs were counted, cell numbers of classified HBs on day 5 and day 6 to small ($77.3\pm5.3$, $59.6\pm4.4$), medium ($83.7\pm4.0$, $66.8\pm3.5$) and large ($100.7\pm2.6$, $88.9\pm3.8$) were increased as their size increases. Especially, there were significantly different between S-HBs and L-HBs (p<0.01). 3) In addition, when the classified HBs were transferred into when the classified HBs were transferred into day 3 pseudopregnant recipients, the pregnancy and implantation rates of S-HBs (28.6%, 15.7%), M-HBs (44.4%, 30.9%) and L-HBs (62.5%, 49.1%) at day 5 were increased as their size increases. However, this pattern was not showed in embryo transfer of day 6 HBs. But, when the live fetuses formation against total implantation rates were observed, the result (87.5%) of S-HBs of day 5 was significantly higher than that of the others (p<0.01). Therefore, this study demonstrates that in vitro cultured healthy HBs can not only be developed normally with good pregnancy rates, implantation rates and live fetuses formation, but also served as a fundamental data for utility of supernumerary HBs in human blastocyst transfer.