• 한국정밀공학회지
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• 제26권9호
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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.

• Asian-Australasian Journal of Animal Sciences
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• 제13권10호
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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.

• Journal of Pharmaceutical Investigation
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• 제25권4호
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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.

• BMB Reports
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• 제32권2호
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• pp.196-202
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• 1999

The existence of the Ras-independent signal transduction pathway of insulin leading to DNA synthesis was investigated in Rat-1 fibroblasts overexpressing human insulin receptor (HIRc-B) using the single-cell microinjection technique. Microinjection of a dominant-negative mutant $Ras^{N17}$ protein into quiescent HIRc-B cells inhibited the DNA synthesis stimulated by insulin. Microinjection of oncogenic H-$Ras^{V12}$ protein ($H-Ras^{V12}$) (0.1 mg/ml) induced DNA synthesis by 35%, whereas that of control-injected IgG was induced by 20%. When the marginal amount of oncogenic H-$Ras^{V12}$ protein was coinjected with a dominant-negative mutant of the H-Ras protein ($Ras^{N17}$), DNA synthesis was 35% and 74% in the absence and presence of insulin, respectively. This full recovery of DNA synthesis by insulin suggests the existence of the Ras-independent pathway. The same recovery was observed in the cells coinjected with either H-$Ras^{V12}$ plus H-$Ras^{N17}$ plus SH2 domain of the p85 subunit of PI3-kinase ($p85^{SH2-N}$) or H-$Ras^{V12}$ plus H-$Ras^{N17}$ plus interfering anti-Shc antibody. When co-injected with a dominant-negative H-$Ras^{N17}$, the DNA synthesis induced by the Ras-independent pathway was blocked. These results indicate that the Ras-independent pathway of insulin leading to DNA synthesis exists, bypassing the p85 of PI3-kinase and Shc protein, and requires Rac1 protein.

• Asian-Australasian Journal of Animal Sciences
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• 제19권2호
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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.

• 한국응용곤충학회지
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• 제61권1호
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• pp.249-254
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• 2022

오래전부터 산업곤충으로서 이용되어온 누에는 최근 바이오 신소재 생산을 위한 생체공장으로써 주목받고 있다. 소재생산을 위해서는 주로 형질전환 기술을 이용하게 되며, 이는 배아가 있는 알 속으로 목적 유전자를 삽입하는 마이크로인젝션(microinjection) 방식으로 이루어진다. 마이크로인젝션을 위해서는 알을 고정하는 과정이 필수적이며 시간 소모 및 피로도가 높은 작업이다. 따라서 본 연구에서는 시간 소모 및 피로도 개선을 통하여 형질전환 효율을 높이고자 3D 프린팅을 이용하여 알의 정렬 및 고정에 도움을 줄 수 있는 알 배열판(egg liner) 및 접착제 줄눈판(glue drawer)을 3DCADian 프로그램을 이용하여 모델링하고, Fusion 360를 이용하여 3차원 도면을 제작 후 프린팅하여 제작하였다. 제작된 두 도구를 이용하여 슬라이드 글라스에 알을 고정하고, 소요된 시간을 분석한 결과 도구를 사용하지 않았을 때에 비하여 2가지 도구를 이용했을 때 작업시간이 약18.6% 감소하였으며, 연구자의 작업 편의성을 향상시키고 마이크로인젝션을 위한 현미경 및 로봇 팔(manipulator) 조작을 유리하게 하였다. 따라서 알의 배열 수 또는 조작 편의성을 개선할 수 있는 추가적인 연구 및 개량이 이루어진다면, 알 배열판 및 접착제 줄눈판이 누에 형질전환 효율성 개선 및 다른 산업 곤충의 형질전환 연구에도 이용될 수 있을 것으로 판단된다.

• 대한기계학회논문집A
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• 제34권7호
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• pp.859-866
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• 2010

본 연구에서는 다단 마이크로 구조물의 대량성형을 위하여, 모듈화된 초소형몰드 시스템(MSMS)을 이용한 초소형 사출성형 공정을 수행하였다. 본 연구에서 적용된 초소형몰드 시스템은 여러 모듈들로 구성되어 있으며, 각 모듈들은 다양한 단면 마이크로 구조물을 가지고 있다. 초소형몰드 시스템의 모듈들은 X-선 리소그래피 공정 및 니켈 전주도금 공정으로 제작되었으며, 다양한 모듈들을 조합 및 결합함으로써 복잡한 형상을 가지는 초소형몰드 시스템을 효과적으로 구현할 수 있다. 이와 같은 초소형몰드 시스템을 적용함으로써, 본 연구에서는 다단 구조물의 표면에 마이크로 삼각 프리즘이 주기적으로 배열되어 있는 다단 마이크로 구조물의 초소형 사출성형 공정을 성공적으로 수행하였다.

• 생명과학회지
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• 제27권5호
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• pp.524-529
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• 2017

본 연구에서는 zebrafish에 인간의 KCNE1 유전자가 삽입된 형광단백질 vector를 microinjection하고, 그 발현 여부를 확인하고자 하였다. 먼저 양 말단에 제한효소(EcoRΙ, BamHΙ) site를 넣어 제작한 primer들로 genomic DNA에서 KCNE1 유전자를 분리하였다. 그 결과는 약 402 bp 크기의 DNA band였고 이 PCR 산물을 형광단백질 vector인 pPB-CMVp-EF1-GreenPuro 속에 클로닝하여 pPB-CMVp-hKCNE1-EF1-GreenPuro plasmid를 제작하였다. 이렇게 준비된 형광 vector를 zebrafish 수정란에 microinjection하였고, 부화된 치어에서 RT-PCR과 DNA sequencing을 통해 GFP 및 hKCNE1의 발현을 최종 확인하였다. 본 연구는 향후 QT 연장증후군(LQTs)에 대한 동물 모델로써 신경자극 전도, 유전자 치료, 유용 유전자 클로닝을 위한 기술 개발에 응용될 수 있을 것으로 기대된다.

• 한국양식학회지
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• 제9권3호
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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.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2004년도 춘계학술발표대회
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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)