• 농업과학연구
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• 제30권1호
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• pp.76-88
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• 2003

외국의 경우 게놈 연구 및 바이오산업에 DNA 칩을 제작할 수 있는 로봇 시스템을 싼 가격에 사용하고 있으나 우리나라의 경우 자동화 시스템을 비싼 가격에 외국에서 도입하여 사용하기 때문에 바이오산업 및 연구 분야에서의 생산비를 높이게 돼 국내외적으로 생명공학의 경쟁력을 저하시키는 원인이 된다. 따라서, 본 연구에서는 유전체 연구에 필수적인 DNA 칩 제작을 위한 연구용 pin 타입 microarrayer를 개발하였으며, 그 구체적인 연구결과는 다음과 같다. 1. 본 연구에서는 DNA칩 제작을 위한 연구용 pin 타입 microarrayer를 개발하였으며 3축 직교좌표형 로봇 본체, DNA를 묻혀 silylated 슬라이드에 점착하는 DNA 점착 헤드, 칩 및 웰 플레이트 고정부, 핀을 세척 및 건조하는 세척 및 건조장치 등으로 시스템을 구성하였다. 2. DNA 점착 헤드는 DNA 점착시 제도용 펜촉을 사용하도록 설계, 제작하였으며, 슬라이드에 DNA를 점착할 때는 핀이 일정한 힘으로 슬라이드를 누르며 점착할 수 있도록 자석의 반발력을 이용하였다. 3. DNA 점착 헤드 핀의 세척을 위하여 증류수 분사 및 진동 브러쉬를 이용하였으며 세척실험 결과, 핀을 1mm/s로 이동시키며 브러쉬를 통과하도록 하는 방법이 세척효과가 높은 것으로 나타났으며, 핀 건조실험결과는 $8.5kg_f/cm^2$의 압축공기를 30초 동안 핀에 분사하였을 때 핀이 건조되는 것으로 나타났다. 4. 본 로봇 시스템을 이용하여 DNA를 12장의 슬라이드에 모두 점착시키기 위하여 웰 플레이트에서 핀이 DNA를 묻히는 실험을 실시한 결과, 10초 이상 핀에 DNA를 묻혔을 때 슬라이드 12장을 모두 찍는 것으로 나타났으며, 슬라이드에 핀이 1초간 접촉할 때의 DNA 스팟의 크기는 평균$280{\mu}$ 가 되는 것으로 나타났다. 최소 점 간격을 0.32mm로 설정한 후 DNA를 점착해 본 결과 최대 8,100여 점의 DNA 스팟을 한 슬라이드에 점착할 수 있는 것으로 나타났다. 5. 본 로봇 시스템은 12장의 동일 DNA 칩을 생성하기 위해 핀의 세척, 건조, DNA를 묻히는 과정 및 DNA 점착 등의 한 과정을 2분 50초 동안 수행할 수 있는 것으로 나타났다.

• Journal of Plant Biotechnology
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• 제45권3호
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• pp.196-206
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• 2018

고구마는 식량뿐만 아니라 전분을 비롯하여 카로티노이드, 비타민C, 비타민E, 안토시아닌과 같은 저분자 항산화물질을 생산하는 중요한 산업용 뿌리작물로 건조 등 조건 불리지역에 적용이 가능한 최고의 전분작물로 각광받고 있다. 이러한 관점에서 중국, 일본을 비롯한 세계 각국에서 오믹스 기반 유용유전자 발굴 및 활용에 대한 연구가 활발히 진행되고 있다. 또한 2014년부터 한 중 일 고구마연구협의회(TRAS)를 중심으로 Xushu 18(6배체) 고구마 유전체 해독 연구가 진행되고 있으며 거의 완성단계에 이르고 있다. 향후 고구마 유전체 해독이 완성되면 오믹스 기반 연구결과와 더불어 전분대사, 항산화물질 대사, 환경스트레스, 기능성 등의 기작에 관여하는 유용유전자 분리 및 활용 연구의 활성화에 기여할 것이며 6배체 고구마 유전체 해독 연구는 식물 유전체 해독에 있어 가장 문제시되는 다배수체 식물의 유전체 해독 문제해결에 가장 큰 기여를 할 것으로 기대 된다. 본 논문은 현재까지 연구된 고구마 생명공학 연구 현황과 조건 불리지역 분자육종 전망에 대해 기술하였다. 이러한 연구 동향 분석은 고구마를 활용한 글로벌 식량, 에너지, 환경문제 해결을 위한 실용화 연구에 도움이 될 것으로 생각된다.

• Plant Biotechnology Reports
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• 제5권2호
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• pp.147-156
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• 2011

Efficient Agrobacterium-mediated genetic transformation of Scoparia dulcis L. was developed using Agrobacterium tumefaciens strain LBA4404 harboring the binary vector pCAMBIA1301 with ${\beta}$-glucuronidase (GUS) (uidA) and hygromycin phosphotransferase (hpt) genes. Two-day precultured leaf segments of in vitro shoot culture were found to be suitable for cocultivation with the Agrobacterium strain, and acetosyringone was able to promote the transformation process. After selection on shoot organogenesis medium with appropriate concentrations of hygromycin and carbenicillin, adventitious shoots were developed on elongation medium by twice subculturing under the same selection scheme. The elongated hygromycin-resistant shoots were subsequently rooted on the MS medium supplemented with $1mg\;l^{-1}$ indole-3-butyric acid and $15mg\;l^{-1}$ hygromycin. Successful transformation was confirmed by PCR analysis using uidA- and hpt-specific primers and monitored by histochemical assay for ${\beta}$-GUS activity during shoot organogenesis. Integration of hpt gene into the genome of transgenic plants was also verified by Southern blot analysis. High transformation efficiency at a rate of 54.6% with an average of $3.9{\pm}0.39$ transgenic plantlets per explant was achieved in the present transformation system. It took only 2-3 months from seed germination to positive transformants transplanted to soil. Therefore, an efficient and fast genetic transformation system was developed for S. dulcis using an Agrobacterium-mediated approach and plant regeneration via shoot organogenesis, which provides a useful platform for future genetic engineering studies in this medicinally important plant.

• Reproductive and Developmental Biology
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• 제31권2호
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• pp.97-102
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• 2007

The objective of this study was to analyzed pattern of proteins expression abnormally in cloned bovine placenta. TIMP-2 protein whose function is related to extracellular matrix degradation and tissue remodeling processes was one of differentially up-regulated proteins in SCNT placenta. And one of down-regulated protein in SCNT placenta was identified as vimentin protein that is presumed to stabilize the architecture of the cytoplasm. The expression patterns of these proteins were validated by Western blotting. To evaluate how regulatory loci. of TIMP-2 and vimentin genes was programmed reprogramming in cloned placenta. the status of DNA methylation in the promoter region of TIMP-2 and vimentin genes was analyzed by sodium Bisulfite mapping. The DNA methylation results showed that there was not difference in methylation pattern of TIMP-2 and vimentin loci between cloned and normal placenta. Histone H3 acetylation state of the nucleosome was analyzed in the cloned placental and normal placenta by Western blotting. A small portion of the protein lysates were subjected to Western blotting with the antibodies against anti acetyl-Histone H3. Overall histone H3 acetylation state of SCNT placenta was significantly higher than those of normal placenta cells. It is postulated that cloned placenta at the end of gestation seems to be unusual in function and morphology of placenta via improper expression of TIMP-2 and vimentin by abnormal acetylation states of cloned genome.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XII)
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• pp.568-570
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• 2003

The Hyoscyamus niger hyoscyamine $6{\beta}-hydroxylase$ (H6H, EC 1.14.11.11) gene was introduced into the genome of a Scopolia parviflora by the binary vector system using the disarmed Agrobacterium rhizogenes strain KCTC 2703. Expression of H6H enzyme which are involved in alkaloids pathway by western blot analysis using proteins extracted from leaf, stem flower, branch root and main root were examined The enzyme expression was found only in the roots, with no expression in leaf, stem and flower. The alkaloids contents were the most higher in root and then leaf and stem has very small amount of alkaloid contents were analyzed by HPLC. The expression level of H6H in transgenic plants were two or more times than wild type plants. In transgenic plant which constitutively expresses H6H enzyme, high concentration of scopolamine was accumulated.

• Molecules and Cells
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• 제23권1호
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• pp.80-87
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• 2007

Beet curly top virus (BCTV) and Beet severe curly top virus (BSCTV), members of curtoviruses, encode seven open reading frames (ORFs) within a ~3 kb genome. One of these viral ORFs, C1, is known to play an important role in the early stage of viral infection in plants during initiation of viral DNA replication. We used promoter:: reporter (${\beta}$-glucuronidase) gene fusions in transgenic Arabidopsis to identify the putative promoter region of BCTV ORF C1. Unlike other geminiviruses, the intergenic region of BCTV was not sufficient to promote C1 expression in transgenic plants. When sequences extending into the coding region of C1 were tested, strong expression of the reporter protein was observed in vascular tissues of transgenic plants. This expression was not dependent on the presence of the intergenic regions or proximal 5' portions of the C1 coding region. Transgenic plants expressing a reporter gene under control of the putative complete C1 promoter were inoculated with virus to determine if any viral transcript affected C1 expression. Virus inoculated plants did not show any altered pattern or change in of reporter gene expression level. These results suggest that (1) important transcriptional activator elements for C1 expression reside in the 3' portion of C1 coding area itself, (2) C1 protein does not auto-regulate its own expression and (3) C1 expression of two curtoviruses is controlled differently compared to other geminiviruses.

• International Journal of Industrial Entomology and Biomaterials
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• 제2권1호
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• pp.37-53
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• 2001

Two regions of mtDNA genome, cytochrome oxidase subunit I (COI) and 165 ribosomal RNA (165 rRNA) genes, were sequenced for 15 species of the long-horned beetle belonging to four subfamilies and geographic samples of mulberry longicorn beetle, Apriona germari, from two localities in Korea. Ten samples of A. germari collected from Suwon and Busan revealed three COI haplotypes ranging in nucleotide divergence of 0.3% to 0.5%, and the two populations shared one common COI haplotype (80%). The sequence divergence among 15 species of the long-horned beetle was much higher in COI gene (12.3%∼39.4%) than 16S rRNA gene (7.2% to 23.1), and the maximum value in the COI gene is exceptional compared with other relevant studies, including that of Coleoptera. The greatly increased divergence in the COI gene, in facto was stemmed from a peculiar sequence of Prionus insularis belonging to Prioninne, divergence of which ranges from 31.2% to 39.3% from other species. We discussed possible reason of the divergence in this species. Due to the abnormality of COI gene divergence, decrease in phylogenetic signal was severe in COI nucleotide and, subsequently, the converted amino acid sequences, rendering us to put more confidence on the 16S5 rRNA gene data. Although the molecular phylogeny confidently supports the monophyletic origin of Lepturinae, the presence of discrepancy between molecular data and traditional taxonomic views also is a testable hyothesis. One such discrepancy includes taxonomic position of Sophronica obrioides and Theophilea cylindricollis belonging to Lamiinae.

• Asian-Australasian Journal of Animal Sciences
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• 제23권7호
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• pp.855-862
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• 2010

Among the known interferon-induced antiviral mechanisms, the Mx pathway is one of the most powerful pathways. The Mx protein has direct antiviral activity and inhibits a wide range of viruses by blocking an early stage of the viral replication cycle. Cloning, characterization, and expression of Mx in vivo and in vitro have been conducted. The chicken Mx gene spans 21 kb and is made up of 14 exons and 13 introns, of which the promoter region was analyzed. The real-time PCR results showed that Mx expression was increased in chicken embryo fibroblasts (CEF) after 12- and 24-h induction with polyI: C. Induction of Mx expression by poly I: C in vivo revealed tissue-specific patterns among the chicken tissues tested. A trace expression of Mx was detected in healthy chicken liver tissues from adult chickens without inducement; the expression levels in the liver, heart, and gizzard were higher than in the muscle and kidney. This is the first report to demonstrate the expression of a glutathione-S-transferase-tagged-Mx fusion protein of 75 KDa, as well as the biological activity tested by SDS-PAGE and western blotting.

• 식물병연구
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• 제21권4호
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• pp.346-350
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• 2015

국내에서 재배되는 사과 '홍로'에 발생하는 Apple scar skin viroid병의 유전자 계통분석을 하기 위하여 바이로이드 증상이 나타나는 8개 지역(봉화, 청송, 당진, 김천, 무주, 문경, 수원, 영월)의 농가에서 수집한 시료를 RT-PCR 방법을 이용하여 바이로이드 검정을 실시하였다. 검출된 바이로이드의 클로닝과 유전자 염기서열 분석을 통해 8종의 분리주들을 확인하였고 한국, 인도, 중국, 일본 및 그리스에서 보고된 21종의 분리주와 염기서열을 비교하였다. 8개의 분리주들의 핵산 서열은 기존에 보고된 분리주들과 92.2-99.7%의 상동성을 나타냈다. 계통분석을 통해 본 연구에서 보고한 7종의 분리주들은 기존의 것들과 독립된 그룹에 속하는 것으로 나타났다.

• IMMUNE NETWORK
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• 제2권4호
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• pp.233-241
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• 2002

Background: Monoclonal antibodies (mAb) of rodent origin are produced with ease by hybridoma fusion technique, and have been successfully used as therapeutic reagents for humans after humanization by genetic engineering. However, utilization of these antibodies for therapeutic purpose has been limited by the fact that they act as immunogens in human body causing undesired side effects. So far, there have been several attempts to produce human mAbs for effective in vivo diagnostic or therapeutic reagents including the use of humanized xenomouse that is generated by mating knockout mice which lost Ig heavy and light chain genes by homologous recombination and transgenic mice having both human Ig heavy and light gene loci in their genome. Methods: Genomic DNA fragments of mouse Ig heavy and light chain were obtained from a mouse brain ${\lambda}$ genomic library by PCR screening and cloned into a targeting vector with ultimate goal of generating Ig knockout mouse. Results: Through PCR screening of the genomic library, three heavy chain and three light chain Ig gene fragments were identified, and restriction map of one of the heavy chain gene fragments was determined. Then heavy chain Ig gene fragments were subcloned into a targeting vector. The resulting construct was introduced into embryonic stem cells. Antibiotic selection of transfected cells is under the progress. Conclusion: Generation of xenomouse is particularly important in medical biotechnology. However, this goal is not easily achieved due to the technical difficulties as well as huge financial expenses. Although we are in the early stage of a long-term project, our results, at least, partially contribute the successful generation of humanized xenomouse in Korea.