• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2000.04a
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• pp.193-198
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• 2000

PCBs를 분해하는 bphABC유전자를 plasmid vactor pMFB2에 유전자조작한 Pseudomonas putida AC30(pMFB2)를 PCBs에 오염된 연안해역의 해수와 저니로 만든 microcosm에 도입한 결과, 각각 도입 4일과 7일만에 사멸하였다. 그러나, 도입한 P. putida AC30(pMFB2)는 사멸하였지만, 연안해수와 저니 microcosm에서 plasmid pMFB2가 전이한 토착미생물이 검출되었다. 도입한 P. putida AC30(pMFB2)의 생잔실패의 원인을 분석한 결과 공경 0.2$\mu\textrm{m}$의 filter를 통과하는 물질과 생물이 가장 크게 영향을 미치는 것으로 나타났다. 유전자조작 P. putida AC30(pMFB2)의 도입과 bphABC유전자의 토착미생물로의 전이에 따른 토착미생물군집에 미치는 영향을 개체수 변동으로 조사한 결과, 토착미생물 군집에 미치는 영향은 보이지 않았다. P. putida AC30(pMFB2)의 도입에 의한 PCBs의 생분해성을 분석하였다. 그러나, 도입한 유전자조작 균주가 생잔에 실패함으로써 잔류하고 있는 PCBs의 농도변화는 보이지 않았다.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.70-70
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• 2002

형질전환 가축을 생산하기 위하여 최근 체세포 복제 기법을 이용하고 있다. 이러한 체세포를 이용한 형질전환 동물의 생산에는 체세포내에 유전자의 도입 효율이 직접적인 영향을 주게 된다. 따라서 본 연구는 세포내 유전자의 transfection 효율을 높이고자 한우의 체세포를 이용하여 여러 가지 조건에서 유전자 도입을 실시하였다. 세포내 유전자 도입 방법은 electroporation (EP) 방법을 이용하였다. 사용한 세포는 소의 귀세포(KbESF), 태아섬유아세포 (KbFF), 그리고 대조구로서 CHO cell을 이용하여 GFP 유전자를 도입하였다. EP는 0.4 cm cuvette을 사용하였고, voltage는 0.25 kV, 그리고 field strength 는 0.625 kV/cm 조건으로 실시하였으며, pulse times은 각각 1, 2, 또는 3회를 사용하였다. KbFF와 KbESF에서는 각각 pulse times을 증가시킬수록 유전자도입 세포수가 증가하였으나 (KbFF: 81, 634, 1,065 cells/$10^{6}$ cells, KbESF: 1,011, 5,567, 15,408 cells/$10^{6}$ cells), CHO cell에서는 pulse times을 증가시킬 수록 오히려 유전자도입 세포수가 감소하였다 (CHO: 1,591, 687, 297 cells/$10^{6}$ cells). 그리고 2주 동안 neo selection을 실시 한 결과 KbFF, KbESF, CHO에서 각각 93, 35, 184 colony가 선발되었으며, 이 중 65.6%, 8.6%, 4.3% 가 GFP 형광 발현 colony로 나타났다. 한편 CHO cell에서 transfection cell수가 감소된 것은 EP의 자극으로 인해 손상된 세포가 많이 발생한 것으로 나타났다. 또한 neo selection에서 선발된 colony중 GFP가 발현되지 않거나 일부만 발현되는 colony들이 많이 발생하였는데, 이것은 세포내 유전자가 transfection되지 않은 세포도 neo selection에서 선발된다는 것을 제시하고 있다. 따라서 체세포를 이용한 형질전환동물 생산을 위해서는 세포내 유전자 도입과 선발 과정에서 나타난 colony에 대하여 보다 엄격한 screen을 하는 것이 필요한 것으로 생각된다.

• Proceedings of the Korea Society of Poultry Science Conference
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• 1999.11a
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• pp.64-65
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• 1999

• Horticultural Science & Technology
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• v.30 no.2
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• pp.192-200
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• 2012

For the development of genetically modified plants, it is important to verify various factors which potentially affect the risk assessment as well as to establish an experimental program to produce scientific and reliable data. However, it is a time and cost consuming process to develop GM plants as well as to prepare scientific and convincible data for government's approval. Therefore, using the transgenic hot pepper tolerant to a new CMV pathotype, we attempted to suggest few methodological procedures, such as probe saturation for southern blot analysis and RT-PCR and ELISA for expression analysis, for identification and stability evaluation of inserted gene in genetically modified plant which are required for submission for approval. Ten partially overlapped probes covering full length of inserted gene were produced. We could identify that the inserted gene was stacked as a single copy as well as no partial element existed. Also, we could identify the stability of the inserted gene stacked in hot pepper using probe saturation. In the expression analysis with RT-PCR and ELISA, we also could provide the stable expression of transcript and proteins in leaves and placenta and pericarp of fruits of the CMV-resistant hot pepper.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.49 no.3
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• pp.227-231
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• 2004

A total of 219 polymerase chain reaction tests of genetically modified (GM) DNA sequences in soybean seeds and soybean sprouts were conducted during 2000-2001. No CM gene was found in 96 tests of soybean seeds. However, either a functional CP4EPSPS gene or the 355 promoter gene was found three times in 2000 and eight times in 2001, in between 0.01 and 0.17％ of soybean spout products, in 123 tests. Since the amount of GM genes was much less than the threshold limit of 3％, none of the 11 positive soybean-sprout samples needed to be libeled GM crops. Of these, seven sprout samples were from domestic seeds and four were from seeds imported from China. To find the contamination route, the raw materials, seed surface, floor of the storage room, area around the selection machine, surface of the packaging film and corn powder used in the package were tested. The 35S promoter gene was detected in only two samples of the corn powder (0.1％). Although we could not find the cause of the GM contamination, the sprout package film is one possibility. In total,8.9％ of the soybean sprout tests were GM positive, but the amounts were much less than the threshold of 3％. This means that there are frequent false-positives and these would threaten the sprout industry if GMO were decided qualitatively. Food companies should make their safety data available to the public and make an effort to address people's concerns about GM food more openly. In addition, there is a need to establish a quantitative test for GM genes in sampled water and a sampling method for raw materials.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1994.04a
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• pp.266-266
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• 1994

$B^{30}$ 위치에 homoserine이 치환된 사람 insulin 유사체 ($B^{30}$ -homoserine) insulin을 생산하기 위해, insulin의 B 사슬 유전자에 A 사슬 유전자를 직접 연결한 insulin 유전자를 설계하였다. 이 유전자는 10개의 oligonucleotide로 나누어 합성하여 T4 DNA ligase로 결합시킨 후, pUC19 plasmi의 polylinker 영역에 삽입하였다. 이 유전자의 발현을 높이기 위해 이 유전자는 다시 tac promoter의 지배를 받는 lacZ 유전자의 Cia I 또는 Hpa I 제한부위에 도입하여 융합시켰다. 이렇게 구축된 운반체 pTBA나 pKBA를 Escherichia coli JM103 균주에 형질도입시킨 후, 이를 4시간 배양한 후 0.05mM이상의 isopropyl-$\beta$-D-thiogalactopyranoside (IPTG)를 배지에 공급해 주고 2시간 더 배양하였을 때 유전자 발현이 잘 유도되어짐을 알 수 있었다. 이 때 생산된 insulin 전구체들은 세포내 불온성인 inclusion body로 축적되어지는 것을 관찰하였으며, 그 생산량은 세포내 전체 단백질량의 30%에 달하였다.

• Horticultural Science & Technology
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• v.29 no.4
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• pp.345-351
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• 2011

This study was carried out to develop a model system using selection method for disease resistant plant breeding programs using a herbicide bialaphos-resistant patII gene as a gene-based marker. Spraying bialaphos could eliminate the susceptible plants from the segregating populations such as ${F_2}^{\prime}s$ and thereafter. Tomato cv. Momotaro-yoke was transformed with patII gene 60 independent transformants were acquired. Total 42 transformants were analyzed in transgene copy numbers by Southern blotting and the segregation ratios for the bialaphos resistance. Statistical analysis revealed that the transgene copy numbers and the segregation ratios were not always coincided, especially having the tendency of underestimating the real numbers of the transgenes in the multicopy lines. A two-stepwise screening method was applied to select $T_1$ tomato plants which linked the transgenic patII to a disease resistance gene (I2 and Ve). Based on the resistant to susceptible ratios, T-20 plant was finally selected due to the estimated linkage 12-13 cM between the patII gene to the I2 gene on chromosome 11. This newly developed system could be applied to any economical crop in breeding programs.

• Journal of Plant Biotechnology
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• v.40 no.3
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• pp.135-140
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• 2013

A less simple approach developed for generation of marker-free transgenic plants is to select transformants without the use of selective marker genes. Some results about development of marker-free transgenic plants were obtained using a non-selective approach in several crops such as rice, potato and tobacco. However, the study did not provide evidence on detailed characterization of introduced gene on genome, a critical step for confirming the stable integration and transmission of a foreign gene. In this study, we evaluated structure and integration sites of transgene (mCry1Ac) in the transgenic Bt rice plants which were made via conventional Agrobacterium-mediated transformation by non-selective method. Structure and integration sites of transgene in these transgenic plants had similar fashion as those recovered under selection.

• FLOWER RESEARCH JOURNAL
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• v.19 no.4
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• pp.269-273
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• 2011

This study was conducted to investigate the resistance to abiotic stress in the progeny obtained by a cross between NDPK2-transgenic line (NDPK2-7-1) and MnSOD (SOD2) transgenic line (SOD2-2-1-1-35) to develop transgenic petunia highly resistant to environmental stress. At the treatment of 100 and $200{\mu}M$ methyl viologene (MV), the progeny was significantly less damaged than its parental plants (SOD2- or NDPK2-transgenic lines) as well as non-transgenic plants, implying its resistance to oxidative stress enhanced than SOD2- or NDPK2-transgenic plants. In an expression of 11 quantitative traits, the progeny remained similar to control plants, although it infrequently displayed slightly longer or wider than non-transgenic control plants. In the color and shape of flowers, there was no significant difference between the progeny and its parents or non-transgenic control.

• Reproductive and Developmental Biology
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• v.29 no.1
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• pp.49-55
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• 2005

We cloned cDNA of the PGH(porcine growth hormone) gene and constructed retrovirus vector designed to express PGH gene under the regulation of CMV (cytomegalovirus) promoter. To maximize the expression, WPRE(woodchuck hepatitis virus posttranscriptional regulatory element) sequence was placed at the downstream of the PGH gene. After infection with recombinant viruses, approximately 1×10/sup 6/ PFF(porcine fetal fibroblast) cells released PGH protein into the media as much as 1,400 ng. In a subsequent experiment, a modifications of the retrovirus vector was made to express the PGH gene in a teracycline-inducible manner. In PFF cells carrying these viral vector sequences, addition of doxycycline to the media resulted in 2∼6 fold increase in PGH synthesis. In the modified retrovirus vectors, the WPRE sequence also played a role in boosting the effect of the tetracycline induction. This result indicates that our tetracycline-inducible expression system might be a promising candidate in alleviating the complicate physiological problems caused by constitutive expression of the exogenous genes in the transgenic animals.