• Journal of Genetic Medicine
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• 제3권1호
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• pp.33-37
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• 1999

Recombinant adenovirus vector systems with strong promoters have been used to achieve high level production of recombinant protein. However, this overexpression system cause some problems such as disturbance of cell physiology and increment of cellular toxicity. Here, we showed a tetracycline-regulated adenovirus expression vector system. Our results showed that the expression level of transgene(p-53) was high and easily regulated by tetracycline. In addition, the maximal gene expression level of the tetracycline-controlled gene expression system was higher than that of the wild type CMV promoter system. Therefore, tetracycline-regulated adenoviral vector system could be applicable for regulatory high-level expression of toxic gene. Also, this system will be useful for functional studies and gene therapy.

• 한국가축번식학회지
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• 제27권3호
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• pp.225-231
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• 2003

본 연구는 생체 내 세포 및 조직배양기로서의 면역결핍동물을 개발할 목적으로 proximal lck promoter와 DT-A gene를 이용하여 형질전환생쥐을 생산하고 이 형질전환생쥐의 면역세포에서 DT-A gene이 발현되는지를 분석하였다. 형질전환생쥐와 정상생쥐로부터 thymus, spleen 및 liver에서 RNA를 추출하여 RT-PCR수행하였는데 정상생쥐의 조직에서는 어떠한 DT-A gene의 발현양상을 얻을 수 없었으나 형질전환생쥐의 thymus, spleen, liver에 DT gene의 발현을 확인할 수 있었고, Northern blotting을 이용하여 형질전환생쥐의 thymus, spleen 및 liver에서 DT-A gene이 강하게 발현되는 것으로 나타났다. 형질전환생쥐 $F_1$ 및 $F_2$ 산자의 혈액에서 T-cell 발달의 분포도를 확인하기 위해 CD4 및 CD8 antibody를 이용하여 FACS analysis를 실시하였는데 형질전환생쥐의 혈액 내 mature T-cell인 single positive thymocyte의 수가 정상생쥐에 비해 감소하는 경향을 나타냈다. 정상생쥐의 혈액 내 T-cell 중 $CD8^{+}$ T-cell의 경우 약 50%를 나타냈으나 형질전환생쥐의 경우 33%로 감소하였고, $CD4^{+}$ T-cell은 정상생쥐에서 10%를 차지하고 있으나 형질전환생쥐에서는 5.9%로 감소되는 것으로 분석되었다. 그러므로 본 연구의 형질전환생쥐에서 lck promoter에 의해 초기 immature한 상태의 T-cell에서 DT-A gene이 발현되어 발육중인 T-cell이 파괴 되어 mature 상태인 $CD4^{+}CD8^{-}$나 $CD4^{-}CD8^{+}$ cells (single-positive)들이 감소된 것으로 확인되었다.

• Journal of Plant Biotechnology
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• 제42권4호
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• pp.356-363
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• 2015

나무의 유전 공학적 연구를 위해서는 목본 고유의 유전자 및 프로모터 연구가 필수적이다. 우리는 포플러(P. alba ${\times}$ P. glandulosa)의 Pagns-LTP 유전자의 867 bp 프로모터를 분리하였고, ${\beta}$-glucuronidase (GUS) reporter 유전자를 이용한 프로모터의 형질전환 포플러를 제작하여 특성 분석하였다. Pagns-LTP 유전자는 어린뿌리에서 강하게 발현되었고 어린잎에서는 약하게 발현되었으며, 그밖에 다른 조직에서는 발현되지 않았다. 또한, 프로모터의 활성은 뿌리와 어린잎에서 한정되었으며 어린뿌리의 세포 전체에서 강한 활성을 나타내었다. 이에 포플러 ns-LTP 프로모터 내의 cis-element를 조사하고 현사시나무에서 Pagns-LTP 프로모터를 분리한 후 활성을 분석하였다. 프로모터 내의 cis-element를 분석한 결과, 조직 특이적 발현과 호르몬 및 스트레스에 반응하는 다양한 cis-element가 존재함을 확인하였다. 이를 통해 포플러의 ns-LTP는 생장뿐만 아니라, 스트레스에도 관여할 것이라고 추측할 수 있었다. 본 연구는 목본의 유전자 기능 분석 및 다양한 응용 연구를 위해 유용하게 이용될 수 있는 도구로서의 가능성을 제시하였다.

• 생명과학회지
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• 제21권12호
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• pp.1784-1788
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• 2011

전형적인 마이크로 RNA는 주로 해당 마이크로RNA의 호스트 유전자와 동시에 발현하는 형상을 보인다. 마이크로RNA miR-7b와 그 호스트 유전자인 FICT는 유전자 발현 조절부위인 프로모터를 함께 공유할 것으로 추정되며, 이는 이 유전자들의 뇌 특이적인 발현 양상에 기여할 것으로 추정된다. 바이오인포메틱 방법을 이용하여 사람과 마우스의 miR-7혹은 miR-7b의 프로모터 부위가 상호 유사성을 가짐을 확인하였고, 이 부위에 다양한 전자조절 부위가 있는 것을 확인 하였다. 또한 이 가설을 증명하기 위하여 형광발현 리포터 유전자 시스템을 사용하여 형광발현 벡터에 마이크로 RNA miR-7b와 그 호스트 유전자인 FICT의 5' 전부위를 클로링하여 프로모터의 활성정도를 다양한 세포주에서 확인하였다. 이 결과를 통하여 마이크로 RNA와 그 호스트 유전자인 FICT의 프로모터에는 네거티브 유전자 조절인자를 포함하는 것을 확인 할 수 있었다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제5권1호
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• pp.1-6
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• 2000

For the rapid selection of higher recombinant hirudin producing strain in a methylotrophic yeast Hansenula polymorpha, a multiple gene integration and dose-dependent selection vector, based on a telomere-associated ARS and a bacterial aminoglycoside 3-phosphotransferase (aph) gene, was adopted. Two hirudin expression cassettes (HV1 and HV2) were constructed using the MOX promoter of H. polymorpha and the mating factor $\alpha$ secretion signal of S. cerevisiae. Multiple integrants of a transforming vector containing hirudin expression cassettes were easily selected by using an antibiotic, G418. Hirudin expression level and integrated plasmid copy number of the tested transformants increased with increasing the concentration of G418 used for selection. The expression level of HV1 was consistently higher than that of HV2 under the similar conditions, suggesting that the gene context might be quite important for the high-level gene expression in H. polymorpha. The highest hirudin producing strain selected in this study produced over 96 mg/L of biologically active hirudin in a 500-mL flask and 165 mg/L in a 5-L fermentor.

• Journal of Microbiology and Biotechnology
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• 제11권6호
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• pp.915-921
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• 2001

Phytase improves the bioavailability of phytate phosphorus in plant foods to humans and animals, and reduces the phosphorus pollution of animal waste. In order to express a high level of fungal phytase in Saccharomyces cerevisiae, various expression vectors were constructed with different combinations of promoters, translation enhancers, signal peptides, and terminator. Three different promoters fused to the phytase gene (phyA) from Aspergillus niger were tested: a galactokinase (GAL1) promoter, glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter, and yeast hybrid ADH2-GPD promoter consisting of alcohol dehydrogenase II (ADH2) and a GPD promoter. The signal peptides of phytase, glucose oxidase (GO), and rice amylase 1A(RAmy1A) were included. Plus, the translation enhancers of the ${\Omega}$ sequence and UTR70 from the tobacco mosaic virus (TMV) and spinach, respectively, were also tested. Among the recombinant vectors, pGphyA06 containing the GPD promoter, the ${\Omega}$ sequence, RAmy1A, and GAL7 terminator expressed the highest phytase activity in a culture filtrate, which was estimated at 20 IU/ml. An intracellular localization of the expressed phytase activity in a culture filtrate, which was estimated at 20 IU/ml. An intracellular localization of the expressed phytase was also performed by inserting an endoplasmic reticulum (ER) retention signal, KDEL sequence, into the C-terminus of the phytase within the vector pHphyA-6. It appeared that the KDEL sequence directed most of the early expression of phytase into the intracellular compartment yet more than $60\%$ of the total phytase activity was still retained within the cell even after the prolonged (>3 days) incubation of the transformant. However, the intracellular enzyme activity of the transformant without a KDEL sequence was as high as that of the extracellular one, thereby strongly suggesting that the secretion of phytase in S. cerevisiae appeared to be the rate-limiting step for the expression of a large amount of extracellular recombinant phytase, when compared with other yeasts.

• Journal of Microbiology and Biotechnology
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• 제13권4호
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• pp.598-606
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• 2003

Salmonella encodes an enterotoxin (Stn) which possesses biological activity similar to the cholera toxin. Stn contributes significantly to the overall virulence of S. typhimurium in a murine model. The production of Stn is enhanced in a high-osmolarity medium and by contact with epithelial cells. In the present study, the in vitro and in vivo transcriptional regulations of the sin promoter revealed two promoters, P1 and P2. The P1 promoter identified by a primer extension analysis of stn mRNA exhibited a switching mechanism in vivo. Depending on the growth stage, transcription was initiated from different start sites termed $P1_S\;and\;P1_E$. $P1_S$, recognized by RNA polymerase containing ${\sigma}^S(E{\sigma}^S),\;and\;P1_E$, recognized by $E{\sigma}^70$, were activated during the stationary and exponential phases, respectively, while $P1_S\;and\;P1_E$ were both negatively regulated by CRPㆍcAMP and H-NS. Results revealed that $P1_S$ was the responsible promoter activated under a high osmolarity and low pH. The P2 promoter was identified 45 nucleotides downstream from $P1_E$ and negatively controlled by CRPㆍcAMP in vitro. No P2 activity was detected in vivo. The regulation of stn expression monitored using a Pstn::egfp fusion indicated that $E{\sigma}^S$ was required for the induction of stn and various factors were involved in stn regulation inside animal cells.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2007년도 International Meeting of the Microbiological Society of Korea
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• pp.83-85
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• 2007

Deep-sea bacteria are adapted to extreme environments, such as high pressures and cold temperatures. We have isolated many piezophiles which grow well even under high pressures from deep-sea sediment. Shewanella violacea DSS12 and Moritella japonica DSK1 have the ability to grow at up to 70 MPa, and those bacteria have unique mechanisms of gene expression in response to high pressure conditions. The combination of gene expression systems in piezophiles, like the high pressure-dependent promoters and GFP reporter gene, may reveal highly fluorescent cells when exposed to high hydrostatic pressure conditions. It is predicted that a novel bio-sensing system can be made to probe high pressure environments using living bacteria. First, gene transformation into our piezophiles, strains DSS12 and DSK1, were examined. Eschericha coli S17-1 was used for bacterial conjugation with those piezophiles. As a result, the broad host range vector, pKT231, and the shuttle vector, pTH10, were successfully introduced to DSS12 and DSK1, respectively. Next, The pressure regulated promoters from DSS12 and DSK1 were cloned into proper vectors and combined with GFP as a reporter gene downstream of each promoter. The transformants of DSK1 and DSS12 with the recombinant pTH10 and pKT231 plasmid, which has cadA and glnA promoters (each of them is a pressure regulated promoter from DSK1 and DSS12, respectively) and GFP, were grown under high pressure and gene expression of GFP promoted by 50 MPa pressure was confirmed. This is a critical point to create a pressure-sensing bacteria, as the "High Pressure Glow Cells", which will indicate the level of environmental pressure using fluorescence of GFP as a reporter gene.

• Journal of Microbiology and Biotechnology
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• 제7권2호
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• pp.138-143
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• 1997

We have designed and constructed a gene encoding novel high essential amino acid encoding protein(HEAAE). The resultant DNA fragment was tested for in vitro and in vivo expression and then cloned into plant expression vector pBI121, under the control of the cauliflower mosaic virus 35S promoter. Agrobacterium tumefaciens, strain LBA4404, was subsequently transformed with this new construct and Nicotiana tabacum var. Xanthi transgenic plants were obtained. DNA analysis by Southern procedure confirmed the presence of the multi-copy number of genes in the transformed plants. Analysis of RNA and protein synthesized in these transgenic plants demonstrated the stable expression of this gene.

• Molecules and Cells
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• 제25권3호
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• pp.446-451
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• 2008

We assessed heterologous protein expression in 64 strains obtained from the Escherichia coli Reference (ECOR) collection, a collection representing diverse natural E. coli populations. A plasmid generating a glutathione S-transferase and plant carbonic anhydrase fusion protein (GST-CA) under the control of the tac promoter was introduced into the ECOR strains, and the quantity of the fusion protein was determined by SDS-PAGE. The foreign protein was generated at various levels, from very high (40 strains, high producers) to very low (six strains, low producers). Immunoblotting showed that the high producers expressed approximately 250-500 times more GST-CA protein than the low producers. The results of semi-quantitative RT-PCR showed that the low producers generated mRNA levels comparable to those of the high producers, thereby suggesting that, at least in this case, inefficient translation is a major cause of the low production. We introduced a different plasmid, which expressed a maltose binding protein and plant guanylate kinase fusion protein (MBP-GK) into the six low producers. Interestingly, five of these expressed MBP-GK at very high levels. Thus, we conclude that the production of a particular protein from an expression vector can vary considerably, depending on the host strain. Strains in the ECOR collection could function as useful alternative hosts when a desired level of protein expression is not obtained from commonly used strains, such as E. coli K12 or B derivatives.