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Effect of Defined KSOM Medium on the Development of 1-antitrypsin Transgenic Nuclear Transfer Bovine Embryos

  • M.M.U. Bhuiyan;J.K. Cho;G. Jang;Park, E.S.;S.K. Kang;Lee, B.C.;W.S. Hwang
    • Proceedings of the Korean Society of Embryo Transfer Conference
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    • 2002.11a
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    • pp.74-74
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    • 2002
  • Production of u 1-antitrypsin ($\alpha$AT) in transgenic cows has a great value in the field of medicine. The present study was conducted to determine the effect of chemically defined KSOM media on in vitro development of bovine transgenic nuclear transfer (NT) embryos. An expression plasmid for human $\alpha$AT was constructed by inserting a bovine beta-casein promoter, a green fluorescent protein (GFP) marker gene, and a human $\alpha$AT target gene into a pcDNA3 plasmid. Cumulus cells as donor nuclei in NT were collected from a Holstein cow and transfected by lipid-mediated method using FuGene6 (Roche Molecular Biochemicals, USA) as reagent. GFP expressed cumulus cells were introduced into recipient oocytes under DIC microscopy equipped with FITC filter set. After electrical fusion and chemical activation, reconstructed embryos were cultured in 1) SOF + 0.8% BSA, 2) KSOM + 0.8% BSA, 3) KSOM + 10% FBS and 4) KSOM +0.01% PVA for 192 h at 39$^{\circ}C$ with 5% $CO_2$, 5% $O_2$ and 90% $N_2$in humidified condition. The development of the embryos was recorded and the GFP expression in blastocyst was determined under FITC filter. The average fusion rate was 73.8% (251/340; n=8). The development rates to 2-4 cells, morula, blastocysts and expression rates in blastocysts varied from 70.3 to 76.5%, 30.2 to 33.8%, 25.4 to 33.8% and 11.8 to 15.6%, respectively. The difference in development and expression rates of embryos among 4 culture groups was not significant (P>0.05). This study indicates that chemically defined KSOM medium is also able to support development of bovine transgenic NT embryos at similar rate of SOF or KSOM supplemented with BSA or serum.

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SURFACE ROUGHNESS OF EXPERIMENTAL COMPOSITE RESINS USING CONFOCAL LASER SCANNING MICROSCOPE (공초점 레이저 주사 현미경을 이용한 실험적 레진의 표면 조도에 대한 연구)

  • Bae, J.H.;Lee, M.A.;Cho, B.H.
    • Restorative Dentistry and Endodontics
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    • v.33 no.1
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    • pp.1-8
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    • 2008
  • The purpose of this study was to evaluate the effect of a new resin monomer, filler size and polishing technique on the surface roughness of composite resin restorations using confocal laser scanning microscopy. By adding new methoxylated Bis-GMA (Bis-M-GMA, 2,2-bis[4-(2-methoxy-3-methacryloyloxy propoxy) phenyl] propane) having low viscosity, the content of TEGDMA might be decreased. Three experimental composite resins were made: EX1 (Bis-M-GMA/TEGDMA = 95/5 wt%, 40 nm nanofillers); EX2 (Bis-M-GMA/TEGDMA = 95/5 wt%, 20 nm nanofillers); EX3 (Bis-GMA/TEGDMA = 70/30 wt%, 40 nm nanofillers). Filtek Z250 was used as a reference. Nine specimens (6 mm in diameter and 2 mm in thickness) for each experimental composite resin and Filtek Z250 were fabricated in a teflon mold and assigned to three groups. In Mylar strip group, specimens were left undisturbed. In Sof-lex group, specimens were ground with #1000 SiC paper and polished with Sof-lex discs. In DiaPolisher group, specimens were ground with #1000 SiC paper and polished with DiaPolisher polishing points. The Ra (Average roughness), Rq (Root mean square roughness), Rv (Valley roughness), Rp (Peak roughness), Rc (2D roughness) and Sc (3D roughness) values were determined using confocal laser scanning microscopy. The data were statistically analyzed by Two-way ANOVA and Tukey multiple comparisons test (p = 0.05). The type of composite resin and polishing technique significantly affected the surface roughness of the composite resin restorations (p < 0.001). EX3 showed the smoothest surface compared to the other composite resins (p < 0.05). Mylar strip resulted in smoother surface than other polishing techniques (p < 0.05). Bis-M-GMA. a new resin monomer having low viscosity, might reduce the amount of diluent, but showed adverse effect on the surface roughness of composite resin restorations.

INFLUENCE OF THE SURFACE ROUGHNESS ON TRANSLUCENCY AND SURFACE COLOR OF THE DENTAL COMPOSITE RESINS (복합레진의 표면 거칠기에 따른 투명도와 표면 색상의 차이에 관한 연구)

  • Cho Kyu-Jeong;Park Su-Jung;Cho Hyun-Gu;Kim Dong-Jun;Hwang Yun-Chan;Oh Won-Mann;Hwang In-Nam
    • Restorative Dentistry and Endodontics
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    • v.31 no.4
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    • pp.312-322
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    • 2006
  • The objectives of this study were to evaluate the effect of surface roughness on the surface color and translucency of the composite resins. Two composite resins (Esthet-X, Dentsply, Milford, USA and Charisma, Kulzer, Domagen, Germany) were used to investigate the surface color. Charisma was used to investigate the translucency. 40 disc samples (diameter: 8 mm, thickness: 5 mm) were made by each product to measure the surface color. Polymerized each sample's one side was treated by Sof-Lex finishing and polishing system (Group C, M, F, SF). 40 disc samples (diameter: 6 mm, thickness: 1 mm) were prepared to measure the opacity. 1 mm samples were ground one side with #600, #1000, #1500 and #2000 sandpapers. CIE $L^{*}a^{*}b^{*}$ values of each 5 mm thickness samples, and XYZ values of 1 mm thickness samples on the white and black background were measured with spectrophotometer (Spectrolino, GretagMacbeth, Regensdorf, Switzerland). Mean surface roughness (Ra) of all samples before and after surface treatment was measured using the Surface Roughness Tester SJ-301 (Mytutoyo, Tokyo, Japan). Regardless of type and shade of the composite resin, $L^{*}$ values measured in group C were higher than others (p < 0.05), and $L^{*}$ value decreased as the Ra value decreased except B3 shade of Esthet-X. But there were no significant difference in $a^{*}$ values among groups. In control group and SF, highest $b^{*}$ values were measured (p < 0.05), except B1 shade of Esthet-X. Contrast ratio decreased as the Ra value decreased (p < 0.05). With the above results, difference of surface roughness has influence on surface color and translucency of dental composite resins.

재래산양에 있어서 핵이식란의 융합조건이 융합 및 체외발달에 미치는 영향

  • 박희성;김태숙;이윤희;정수영;이명열;홍승표;박준규;김충희;정장용
    • Proceedings of the KSAR Conference
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    • 2004.06a
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    • pp.294-294
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    • 2004
  • 오늘날 생명공학기술의 눈부신 발달로 인하여 우리나라 재래산양은 그 모델동물로서 번식ㆍ생리학적으로 매우 중요한 가치를 지니고 있을 뿐만 아니라 고유의 유전자원 보존 측면에서도 산양복제와 같은 다양한 연구를 통하여 개량체계의 확립이 절실히 요구된다. 본 연구는 우량가축의 증식, 인간의 질병치료 및 멸종위기동물의 종 보존 등에 활용할 수 있는 기초자료를 얻고자 재래산양의 핵이식을 실시하여 공여세포의 조건, 전기적 세기 및 융합횟수 등이 융합율과 체외발달율에 미치는 영향을 조사하였다. (중략)

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특집 : 레이저 기반 초정밀 초고속 가공시스템 - PCB pattern 미세화에 따른 UV laser driller의 개발

  • Park, Hong-Jin;Seo, Jong-Hyeon
    • 기계와재료
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    • v.22 no.1
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    • pp.22-29
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    • 2010
  • 최근 휴대폰 등 모방일 전자기기 산업에서 차세대 고부가 PCB(MLB, HDI, FPC, 등) 및 고기능 PCB(COF, MOF, SOF)의 급속한 적용 확대로 직경$20{\mu}m$급의 비아홀(viahole) 및 interconnection 홀 가공을 위한 초정밀/초고속 레이저 드릴링 공정 및 장비기술 개발에 대한 시장의 요구가 급증하고 있다. 이에 반해 기존의 CO2 레이저 드릴링은 기술적 한계에 도달하여 시장의 요구에 대응이 불가하며, 선진업체에서는 최근 UV 레이저 드릴링 장비에 대한 시장 점유율을 높여가고 있다. 특히 국내시장은 미국의 ESI사가 독점하고 있어 기술개발 투자를 통한 국산화가 절실한 상황이다. 이에 당사에서는 초고속/초정밀 UV laser 시스템을 이용한 FPC iva hole drilling을 연구과제로 개발을 진행하고 있으며 국산화를 넘어서 세계시장점유를 목표로 공정장비개발을 진행중이다.

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Comparison on Nano-particle Number Measurement Characteristics for Different Particle Generators between Spray type and Soot Type (Spray type과 Soot type 입자발생기별 나노입자 개수농도분포 측정특성 비교)

  • Kim, M.S.;Kwon, J.W.;Chung, M.C.;Lee, J.W.
    • Journal of ILASS-Korea
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    • v.17 no.4
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    • pp.185-191
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    • 2012
  • Particulate matters (PM) that is generated by most diesel engine is regulated by the mass concentration measured by the conventional method it had been. Recently, Europe PMP (Particle Measurement Program) decided to start the regulation of vehicle's nano-sized particle number (PN) from the year of 2011 because of nano-particle's higher degree of harm to the human body. So firstly, the standard level of PN emission is introduced in the Euro 5/6 emissions regulation with a limit of $6{\times}10^{11}$ per km for light duty vehicle. Also KPMP(Korea Particle Measurement Program) was organized to copy quickly international technical trend. In this paper, it was investigated the nano-sized PN measurement characteristics for different particle generators between spray type and soot type. And the difference ratio between particle generators, the characteristic of PN concentration, counting efficiency and linearity was analyzed. Then, we make conclusions as followed. When particle diameter is increased, counting efficiency of two generators is decreased. Also Secondary calibration method is more higher 3% than Primary calibration method. Finally, SOF which is included in soot particles is not totally removed so it have great influence on test result of counting efficiency and linearity.

Effect of Supplementation of Vitamin E on In Vitro Maturation and Activation of Bovine Oocytes

  • Park, Jong-Im;Jang, Yun-I
    • Journal of Embryo Transfer
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    • v.25 no.3
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    • pp.165-169
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    • 2010
  • This study was carried out to assess the effect of vitamin E against the reactive oxygen species (ROS) on chemical activation of in vitro matured oocytes. Bovine oocytes were aspirated from slaughtered ovaries and transferred to maturation medium with or without vitamin E ($100\;{\mu}M$). After 22 hours of culture, oocytes with polar bodies were selected and submitted to activation treatments with or without vitamin E. After activation, oocytes were cultured in mSOF medium and rate of development was monitored. For ROS ($H_2O_2$) detection, in vitro matured and activated oocytes were selected and stained with DCFDA and observed under fluorescence microscope. The ROS contents were not significant differences in IVM rate, activation process and embryonic development to blastocysts with or without vitamin E. The cell number of blastocyst showed significant difference (p<0.05) in embryos matured and activated with vitamin E. The results of the present study demonstrated that the exposure of vitamin E in IVM and activation process improved the quality of embryos evaluated by the cell number of blastocysts.

Re-Cloning by Somatic Cell Nuclear Transfer from a Cloned Korean Native Goat (복제 산양(진순이)의 체세포 핵이식에 의한 Re-Cloning에 관한 연구)

  • Jung, S.Y.;Park, H.S.
    • Journal of Embryo Transfer
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    • v.22 no.2
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    • pp.89-95
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    • 2007
  • The present study was conducted to examine some factors affecting in vitro development and fecundity of embryos recloned with somatic cell nuclear transfer (SCNT). Fibroblast cells retrieved from the ear of a 3-week-old, cloned Korean goat (Jinsoonny) were used as karyoplast donors and serum-starvation was conducted in tissue culture medium (TCM)-199 supplemented with 0.5% FBS. Recipient oocytes were surgically collected by flushing the oviducts 35 h after hCG injection following FSH priming. The zonae pellucidae of the oocytes were partially perforated with a laser drill and a donor cell was transferred into an enucleated oocyte. The couplets were electrically fused and activated by ionomycin (5 min) and 6-DMAP (4 h). The reconstructed embryos were cultured in mSOF medium containing 0.8% BSA at $39^{\circ}C$ in an atmosphere of 5% $CO_2$, 5% $%O_2$, 90% $N_2$ for 12 to 15 h. Re-cloned embryos (2- to 4-cell stages) were surgically transferred into the oviducts of the recipients and pregnancy was subsequently diagnosed by progesterone assay and ultrasound on Days 21 and 63 of pregnancy. The fusion rate following 1st fusion pulse was higher (p<0.05) in 2nd cloning (65.9%) compared to 1st cloning (51.0%), but it was not different in the other groups. The rate of cleavage after fusion was significantly higher (p<0.05) in 1st (77.7%) than in 2nd cloning (56.0%). A total of 175 re-cloned embryos were transferred into 28 recipients. On day 21 and 60 after transfer, 11 (39.3%) and 4 recipients (17.4%) were pregnancy, respectively. In comparison of pregnancy rate by estrous synchronization, a total of 66 and 109 re-cloned embryos were transferred into 11 recipients in natural estrus and 17 recipients in induced estrus, respectively. Five (45.4%) and 2 recipients (18.2%) in natural estrus were pregnant on days 21 and 63 while 6 (35.3%) and 2 (11.8%) recipients in induced estrus were pregnant, respectively. These results show that recloning of goat can be achieved by SCNT and estrous synchronization between donor and recipient animals may be one of the major factors affecting success rate.

Effects of Heat Stress on the Developmental Competence of Bovine Cumulus-Oocyte Complex During in vitro Maturation (Heat Stress가 소 난자의 체외성숙과 배반포 발달에 미치는 영향)

  • Kim, Min-Su;Kim, Chan-Lan;Seong, Hwan-Hoo;Kim, Namtae;Kim, Sung Woo
    • Journal of Embryo Transfer
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    • v.32 no.3
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    • pp.65-71
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    • 2017
  • The elevated temperature and high humidity has been known as main reason for heat stress on animals and cause detrimental effects on productivity of organisms and physiological conditions of normal bioactivities. The aims of this study were to evaluate the relationship between time of heat shock simulation during in vitro maturation and developmental competence of subsequent embryo after in vitro fertilization. Heat shocked cumulus-oocyte complexes (COCs) of Korean native cattle were subjected to normal conditions for 22, 21, 18 and 12 h respectively and transferred to heat stress inducing condition at $40.5^{\circ}C$ in other incubator for 0 (control), 1 and 4 h. After maturation for 22 h, the oocytes were fertilized and cultured in mSOF media for 8 d and examined the developmental capacity of embryos. There were no differences in maturation and cleavage rates between 0, 1 and 4 h heat socked oocytes, but blastocysts formation were lower in the 4 h heat stressed oocytes. The apoptotic cells of developed blastocysts were also increased in at day 8 with 4 h heat shocked oocytes. These results indicate that heat shock on oocytes during maturation could cause negative effects on the developmental competence of embryos.

Studies on the Developmental Rate of Oocyter Obtained fly Intracytoplasmic Sperm Injection with Epididymal Spermatozoa in Domestic Dogs (개 난자에 부고환 정자로 ICSI후 배양하였을 때 체외발생율에 관한 연구)

  • 김상근;이동수;이만희
    • Korean Journal of Animal Reproduction
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    • v.26 no.2
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    • pp.105-110
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    • 2002
  • The objective of this study was to determine the developmental competence of in vitro matured oocytes after intracytoplasmic sperm injection(ICSI) with epididymal spermatozoa. The ovaries were obtained from slaughtered small species dogs. Oocytes matured in vitro for 24 hrs were fertilized by ICSI with epididymal spermatozoa. After ICSI, one group of oocytes was activated with 2.0 mM dimethylaminopurine or 7% ethanol for 5 min. and second group was not activated. The follicular oocytes were cultured in synthetic oviductal fluid(SOF) and TCM-199 medium containing hormones and 10% FCS for 24~48 hrs in a incubator with 5% $CO_2$ in air at 38.5$^{\circ}C$. 1. Results of IVM showed that the percentage of oocytes reaching MII after 24 h and 48 hrs of incubation were significantly higher(p<0.05) after culture with 48 hrs(9/30, 30.0%) than that after culture with 24hrs(a/30, 26.7%). 2. Results of IVM showed that the percentage of oocytes reaching MII after 48 hrs of incubation were significantly higher(p<0.05) after culture with SOF media(10/30, 30.3%) than TCM-199 media (7/30, 23.3%). 3. The rate of cleavaged embryos to blastocyst obtained by ICSI treated activation oocytes was significantly higher(p<0.05) than that of nonactivation oocytes(5/16, 25.0% vs 1/13, 5.0%). 4. The rates of development of cleavaged embryos to blastocyst obtained by ICSI treated sperm of fresh, epididymal and frozen-thawed epididymal were 8/18(44.43%), 5/16(31.3%), 2/14(14.3%), respectively. and these values of frozen-thawed epididymal sperm injection were lower than fresh sperm injection.