• Title, Summary, Keyword: Microinjection

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Replication of Microstructured Surfaces by Microinjection Molding (초소형사출성형 공정을 이용한 마이크로 구조 표면의 성형)

  • Lee, Bong-Kee;Kim, Young-Bae;Kwon, Tai-Hun
    • Journal of the Korean Society for Precision Engineering
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    • v.26 no.9
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    • pp.135-142
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    • 2009
  • In the present study replication of microstructured surfaces by microinjection molding was carried out. For a fabrication of mold inserts, nickel microstructures having various characteristic dimensions were fabricated by nickel electroforming onto Si mother microstructures. In addition, reverse nickel microstructures based on the electroformed nickel microstructures were successfully realized by electroforming with passivation process. The fabricated nickel microstructures were used as mold inserts for a replication of microstructured surfaces by microinjection molding. Microinjection molding experiment was carried out under three different processing conditions, which revealed effects of a packing stage and mold wall temperature. The microinjection-molded microstructured surfaces were characterized by using an atomic force microscope (AFM). It was found that mold wall temperature could enhance replication quality resulting in the precise microstructured surfaces.

Estimation of the Efficiency of Transgenic Rabbit Production Following GFP Gene Microinjection into Rabbit Zygotes

  • Jin, D.I.;Im, K.S.;Kim, D.K.;Choi, W.S.
    • Asian-Australasian Journal of Animal Sciences
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    • v.13 no.10
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    • pp.1367-1372
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    • 2000
  • The present study was conducted to evaluate the efficiency of transgenic rabbit production by DNA microinjection using EGFP (Enhanced Green Fluorescent Protein) gene. In this experiment EGFP coding sequences fused to CMV promoter were microinjected into rabbit one-cell embryos, and then GFP expression and gene integration were evaluated in preimplantation embryos and fetuses recovered on day 15 of pregnancy to determine efficiency of transgenic rabbit production. Effect of DNA concentration was also tested on development in vitro following microinjection and transgene integration in fetuses. Development of embryos in vitro was decreased by DNA microinjection, but the rates of pregnancy and implantation were not significantly affected by microinjection. As development progressed in vitro percentage of GFP expression in rabbit embryos was decreased, resulting GFP expression detected in 37.5% of blastocysts. The efficiencies for production of transgenic fetuses were 4.0% and 7.6%, respectively, when $10ng/{\mu}l$ and $20ng/{\mu}l$ of DNA concentration were microinjected. Transgenic fetuses were confirmed by GFP expression and PCR analysis of fetus genomic DNA. These results indicated that DNA microinjection itself damaged embryo development and DNA concentration affected the efficiency of transgenic rabbit production.

Functional Expression of a Dipeptide Transporter Obtained from Intestinal HT-29 Cells Using Xenopus Oocytes (장관세포인 HT-29에 존재하는 디펩티드수송체의 Xenopus oocyte에서의 발현)

  • Oh, Doo-Man;Yang, Chae-Ha
    • Journal of Pharmaceutical Investigation
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    • v.25 no.4
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    • pp.299-305
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    • 1995
  • Cloning the gene encoding a dipeptide transporter is necessary for understanding the absorption mechanism of peptides and peptide-like drugs in the gastrointestinal tract. Functional expression of a dipeptide transporter after microinjection into Xenopus laevis oocytes was performed using the mRNA purified from human intestinal HT-29 cells. Fifty nanoliters of purified mRNA (1 mg/mL) were microinjected into healthy oocytes followed by incubation for 4 days in order to express a dipeptide transporter. Functional expression was determined by a uptake assay using 10 Ci/mL $[^3H]-glycylsarcosine$, a dipeptide substate of the transporter. Seasonal variability and batch-to-batch variability were greater in summer. The usage of beveled micropipettes improves viability of oocytes at 4 days after microinjection. Expression of a dipeptide transporter in oocytes after microinjection of mRNA obtained from HT-29 cells was significantly larger than those after microinjection of water or mRNA obtained from the rabbit intestine.

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The Influence of Microinjection of Foreign Gene into the Pronucleus of Fertilized Egg on the Preimplantation Development, Cell Number and Diameter of Rabbit Embryos

  • Makarevich, A.V.;Chrenek, P.;Fl’ak, P.
    • Asian-Australasian Journal of Animal Sciences
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    • v.19 no.2
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    • pp.171-175
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    • 2006
  • The aim of this in vitro study was to test the effect of microinjection (Mi) of foreign gene into the rabbit egg pronucleus and epidermal growth factor (EGF) addition on the blastocyst rate, the cell number and the diameter of embryos, and to determine possible relationships between embryo cell number and embryo diameter. Blastocyst rate was significantly decreased in gene- Mi (G-Mi/E0) group (63.1%) comparing to intact ones (83.5%, $p_1$<0.05). The addition of EGF at 20ng/ml (G-Mi/E20) or 200 ng/ml (GMi/ E200) to gene-Mi embryos did not affect blastocyst rate (65.6 and 55.2% resp.). As a control for Mi, the eggs were microinjected with the same volume of phosphate-buffered solution (PBS-Mi) instead of the gene construct solution. Cell numbers and embryo diameters were measured from embryo images obtained on confocal laser scanning microscope. Bonferroni-modified LSD test showed that the embryo cell number in PBS-Mi group was significantly lower ($p_1$<0.05) and in gene-Mi group was tended to decrease compared with intact embryos. Embryo diameter was not different among experimental groups. No effect of EGF given at any doses both on the cell number and embryo diameter was found. A positive correlation between cell number and embryo diameter was observed in all groups of embryos. Since embryo diameter was not changed under the influence of Mi or EGF addition in this study, this seems to be more conservative characteristics of the embryo morphology. These results suggest that the pronuclear microinjection compromises developmental potential of embryos, decreasing blastocyst rate and embryo cell number, whilst embryo diameter is not affected. No effects of EGF on studied parameters were confirmed. Declined quality of Mi-derived embryos is caused by the microinjection procedure itself, rather than by the gene construct used.

Production of Transgenic Homozygous Diploid in Mud Loach(Misgurnus mizolepis) I. Transfer of Luciferase Gene and Evaluation of Mud Loack Expression Vector

  • Nam Yoon Kwon;Kim Moo-Sang;Lee Hyung-Ho;Kim Dong Soo
    • Journal of Aquaculture
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    • v.9 no.3
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    • pp.293-300
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    • 1996
  • Validities of several gene transfer methods including microinjection, electroporation and lipo-fection with luciferase gene (pRSVL), and effectiveness of mud loach expression vector which contains ARS from mud loach on production of transgenic mud loach were evaluated. Microiniection revealed the $0\~8\%$ of transgene incidence in 2-week-old fish with significant mosaicism. Electroporation and lipofection of mud loach sperm also successfully introduced the transgene into sperm cells, and transferred the foreign DNA into zygote. Gene transfer by electroporation and lipofection showed a range of $0\~28\%$ and $0\~48.1\%$ of transgene incidence, respectively in newly hatched larvae, altough most DNA introduced were gradually degraded with the development of fish. Microinjections of mud loach expression vector caused a significantly reduced survival rate of mud loach embryos with severe teratogenic effects, and ARS/Luc transgene could not be detected in normally developed fish after microinjection.

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Development of Bovine Embryos Reconstructed by Microinjection of Cultured Fetal Fibroblast Cells into In-Vitro Matured Oocytes

  • Kim, Sungmin;Kim, Sangkeun
    • Proceedings of the KSAR Conference
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    • pp.41-41
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    • 2002
  • Bovine cumulus-oocyte complexes were recovered from ovaries at a slaughter and then divided into five groups: control group(unvitrified oocytes), 0 hr. group(composed of oocytes vitrified before the onset of maturation) and 10, 14, and 20 hrs groups(vitrified respectively at 10, 14 and 20 hrs after the onset of maturation). The oocytes remained vitrified for 24 hrs, and then were thawed in 30℃ water bath. Survival and cleavage rates were defined as development rate on in vitro culture and stained with aceto-orcein or FDA test.

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Expression of Human KCNE1 Gene in Zebrafish (Zebrafish에서 인간 KCNE1 유전자 발현에 관한 연구)

  • Park, Hyeon Jeong;Yoo, Min
    • Journal of Life Science
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    • v.27 no.5
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    • pp.524-529
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    • 2017
  • This study was aimed to produce a transgenic zebrafish expressing the human KCNE1 gene. Initially, the entire CDS of the human KCNE1 gene was amplified from a human genomic DNA sample by polymerase chain reaction using a primer set engineered with restriction enzyme sites (EcoRI, BamHI) at the 5' end of each primer. The resultant 402 bp KCNE1 amplicon flanked by EcoR1 and BamH1 was obtained and subsequently cloned into a plasmid vector pPB-CMVp-EF1-GreenPuro. The integrity of the cloned CDS sequence was confirmed by DNA sequencing analysis. Next, the recombinant vector containing the human KCNE1 (pPB-CMVp-hKCNE1-EF1-GreenPuro) was introduced into fertilized eggs of zebrafish by microinjection. Successful expression of the recombinant vector in the eggs was confirmed by the expression of the fluorescence protein encoded in the vector. Finally, in order to assure that the stable expression of the human KCNE1 gene occurred in the transgenic animal, RNAs were extracted from the animal and the presence of KCNE1 transcripts was confirmed by RT-PCT as well as DNA sequencing analysis. The study provides a methodology to construct a useful transgenic animal model applicable to the development of diagnostic technologies for gene therapy of LQTS (Long QT Syndrome) as well as tools for cloning of useful genes in fish.

Replication of Multi-level Microstructures by Microinjection Molding Using Modularized and Sectioned Micromold System (모듈화된 초소형 몰드 시스템(MSMS)을 이용한 다단 마이크로 구조물의 초소형 사출성형 공정)

  • Lee, Bong-Kee;Kwon, Tai-Hun
    • Transactions of the Korean Society of Mechanical Engineers A
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    • v.34 no.7
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    • pp.859-866
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    • 2010
  • In this study, microinjection molding process using the newly developed micromold system, namely modularized and sectioned micromold system (MSMS), has been carried out for a replication of multi-level microstructures. The present MSMS consisted of several micromold modules, each having cross-sectional microstructures on the top surface. The micromold modules were precisely fabricated by deep X-ray lithography and subsequent nickel electroforming. By assembling the micromold modules, an MSMS having multi-level microstructures, which could be used as a mold system in micromolding processes, was obtained. In this manner, polymeric multi-level microstructures, such as the triangular prism microstructures on a stepped surface, were successfully replicated by the microinjection molding process.

A possible role of lipopolysaccharides in the prevention of lysosome0symbiosome fusion as studied by microinjection of an anti-LPS monoclonal antibody (리소솜과 공생낭의 융합저해에서의 Lipopolysaccharide의 역할에 관한 연구)

  • Choi, Eui-Yul
    • Korean Journal of Microbiology
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    • v.32 no.4
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    • pp.280-284
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    • 1994
  • Lack of lysosomal fusion with symbiosomes in symbiont-bearing Amoeba proteus may be due either to the presence of a component in the symbiosome membrane or to the absence of a component needed in the fusion process. Using monoclonal antibody as a probe, lipopolysaccharides were identified as symbiosome-membrane components contributed by symbionts and were found to be exposed on the cytoplasmic side of the membrane. In order to test whether lipopolysaccharides may play a role in the prevention of lysosome-symbiosome fusion, the antilipopolysaccharides antibody was microinjected and processed for double immunostaining in conjuction with anti-lysosome antibody as a lysosome-fusion indicator. Microinjection of the anti-LPS antibody caused symbiosomes to fuse with lysosomes, suggesting that X-bacterial lipopolysaccharides could be 'fusion-preventing' factors.

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Production of Transgenic Pig Harboring Tissue-type Plasminogen Activator Gene with Bovine-$\beta$-Casein Promoter

  • Park, J.K.;Lee, Y.K.;Lee, P.Y.;Kim, S.W.;Jeon, I.S.;Lee, H.G.;Han, J.H.;Park, C.G.;Lee, S.E.;Beak, K.N.;Chang, W.K.
    • Proceedings of the KSAR Conference
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    • pp.190-190
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    • 2004
  • Tissue plasminogen activator (tPA) plays important roles in the brain after excitotoxic injury. This study was conducted to produce transgenic pig harboring human tissue plasminogene activator (htPA) gene. Recombinent htPA(rhtPA) genes containing bovine-β-casein promoter (bBC) were prepared for microinjection and testified the expression level of htPA protein from the Chinese hamster ovary (CHO) cell lines before NDA microinjection into the porcine pronuclei. (omitted)

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