• Journal of Microbiology and Biotechnology
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• 제19권3호
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• pp.331-337
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• 2009

Interleukin-13 (IL-13) has been proposed as a therapeutic target for bronchial asthma as it plays crucial roles in the pathogenesis of the disease. We developed an in vitro test system measuring transcriptional downregulatory activities on IL-13 as a primary screening method to select drug candidates from natural products. The promoter region of IL-13 (-2,048 to +1) was cloned into the upstream of a luciferase gene in the plasmid pGL4.14 containing the hygromycin resistance gene as a selection marker, generating pGL4.14-IL-13. The EL-4 thymoma and RBL-2H3 mast cells transiently expressing this plasmid highly produced the luciferase activities by responding to PI (PMA and ionomycin) stimulation up to 8-fold and 13-fold compared with the control, respectively, whereas cyclosporin A, a well-known antiasthmatic agent, significantly downregulated the activities. The BF1 clone of RBL-2H3 cells constitutively expressing pGL4.14-IL-13 was established by selecting surviving cells under a constant lethal dose of hygromycin treatment. The feasibility of this system was evaluated by measuring the downregulatory activities of 354 natural products on the IL-13 promoter using the BF1 clone. An extract from Morus bombycis (named TBRC 156) significantly inhibited PI-induced luciferase activities and IL-13 mRNA expression, but not the protein expression. Fisetin (named TBRC 353) inhibited not only PI-induced luciferase activities and mRNA expression, but also the IL-13 protein secretion, whereas myricetin (named TBRC 354) could not suppress the IL-13 expression at all. Our data indicated that this in vitro test system is able to discriminate the effects on IL-13 expression, and furthermore, that it might be suitable as a simple and time-saving primary screening system to select antiasthmatic agents by measuring transcriptional activities of the IL-13 promoter.

• 한국균학회지
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• 제26권4호통권87호
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• pp.450-457
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• 1998

Cryparin은 Cryphonectria parasitica의 세포벽에 풍부한 소수성 단백질에 속한다. cryparin은 비록 하나의 유전자에 의해 발현되지만 액체배양 후 48시간이 지나면 발현된 전체 유전자중에서 22%를 차지할 정도의 높은 발현 양상을 나타낸다. 또한 cryparin은 RNA mycovirus인 Cryphonectria hypovirus 1의 감염에 의해 발현이 현저히 억제되는 유전자로 알려졌다. 이미 지난 실험(Kim et al., 1999)에서 상동염색체간의 재조합을 이용하여 cryparin 유전자를 항생제 hygromycin B 저항성 유전자로 치환한 치환체를 제조하였다. 발현율이 매우 높으면서도 virus에 의해 밀접하게 영향받는 cryparin 유전자의 promoter 분석을 위하여서는 대상이 되는 유전자 치환을 위한 vector만을 포함하며, 분석에 이용될 여러 유전자 운반체들이 어느 한곳에만 삽입되도록 하는 성질을 가진 균주의 개발이 필요하다. 그러나 지난번 실험의 결과 얻어진 cryparin 치환체는 치환용 vector외에도 무작위로 삽입된 vector가 존재하고 나아가 새로운 vector들이 어느 한곳에만 삽입되도록 하는 성질을 갖지 못하였다. 따라서 본 실험에서는 cryparin 유전자 치환체와 영양요구성 돌연변이체인 균주간의 교잡을 이용하여 분석 대상이 되는 유전자의 치환에 이용된 vector만을 포함하며, 분석에 이용될 여러 유전자 운반체들이 genome내의 어느 한곳에만 삽입되도록 하는 성질을 가진 균주를 제조하였다.

• Journal of Plant Biotechnology
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• 제29권2호
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• pp.85-92
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• 2002

Agrobacterium을 이용한 벼의 효율적인 형질전환을 위하여 몇 가지 형질전환 조건을 조사하였다. 그리고 조사된 최적의 형질전환 방법을 이용하여 내한성에 관련된 GPAT 유전자를 식물에 도입하였다. 형질전환 조건은 GUS 발현을 통하여 조사되었다. 본 실험에서는 벼 (Oryza sativa L. cv. Dongjin)의 성숙 종자 유래 캘러스를 3일간 전배양한 후 Agrobacterium을 접종하였다. 접종이 끝난 캘러스는 50mg/L CaCl$_2$, 30mg/L acetosyringone, 2 mg/L 2,4-D, 120 mg/L betaine이 첨가된 공동배양 배지 위에서 암조건으로 10일간 공동배양하여 높은 GUS 발현을 관찰할 수 있었다. 이와 같은 방법으로 GPAT 유전자를 식물에 도입한 결과 54%의 높은 형질전환율을 나타내었다. 형질전환 식물체를 southern 분석한 결과 wild type 식물체에서는 GPAT 유전자가 검출되지 않았으나, 형질전환 식물체에서는 GPAT 유전자가 검출되었다. 또한 GPAT 유전자로 형질전환된 5 계통의 T1 세대에서 hygromycin에 대한 저항성과 감수성의 유전비율이 3 : 1로 분리되었다. 따라서 본 실험의 결과에서 검토된 고빈도의 형질전환 시스템은 단자엽식물의 형질전환에 있어서 모델계로 이용될 수 있으리라 기대된다.

• Journal of Microbiology and Biotechnology
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• 제10권3호
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• pp.287-292
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• 2000

Recombinant Aspergillus niger was constructed to express and secrete a biologically active murine granulaocyte macrophage-colony stimulating factor (mGM-CSF). A 500 bp fragment encoding the signal peptide and terminator of glyceraldehyde-3-phosphate dehydrogenase (gpd). The hygromycin phosphotrasferase gene (hph) was used as a selection marker for the fungal transformants. An expression vector was introduced into A. niger ATCC 9642, and a Northern blot analysis indicated the presence of a considerable amount of transcripts from the introduced mGM-CSF. The biological activity of recombinant mGM-CSF (rmGM-CSF) isolated from the culture filtrate was confirmend by measuring the proliferationof the GM-CSF dependent FDC-P1 cell line. It appeared that rmGM-CSF was amenable to the proteolytic activity produced by A. niger, since biological actibity was only observed when the transformants were grown in a protease-repressing medium, and the activity of rmGM-CSF dramatically decreased with an increase of age of the culture. The yield of rmGM-CSF, as determined by ELISA. was 640 ng/l of culture filtrate. Accordingly, its specific activity is estimated to be approximately two-and-a-half times higher than that of a commercial preparation from E. coli.

• Journal of Plant Biotechnology
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• 제7권4호
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• pp.241-246
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• 2005

Factors affecting regeneration of different Rubus varieties (blackberry, raspberry and their hybrid) were examined and a reliable regeneration system was established. Media for stock plant maintenance were tested; different explants and media were investigated to find the best circumstances for the regeneration. The effect of the commonly used antibiotics was studied to determine the most suitable one for selection of the transformants. We found that both MS and LS media supplemented by $20\;gL^{-1}$ sucrose are suitable for the stock plant maintenance. The optimal hormone content for the stock plants is $0.125\;mgL^{-1}$ 6-benzylaminopurine (BAP) with $0.01\;mgL^{-1}$ indole-3- butyric acid (IBA). The highest regeneration rate was observed on medium containing MS salts with B5 vitamins complemented with glucose, sucrose, maltose, $10\;gL^{-1}$ each, supplemented with benzylaminopurine riboside (BAR) ($2\;mgL^{-1}$) and indole-3-acetic acid (IAA) ($0.1\;mgL^{-1}$). The regenerated shoots appeared directly from the cut edges, without callus phase. Hygromycin and geneticin proved to be good selection agents for the Rubus explants, but due to their severe effect on the tissues we propose to use marker-free constructions for the transformation.

• 한국초지조사료학회지
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• 제20권2호
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• pp.97-102
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• 2000

To increase thennotolerance of forage crops, transgenic rice plants as a model for transformation of monocots were generated. A cDNA encoding the chloroplast-localized small heat shock protein (small HSP) of rice, Oshsp21, was introduced into rice plants via Agrobacterium-mediated gene transfer system. Calli induced from scutella were co-cultivated with a A. tumefaciens strain EHAlOl canying a plasmid, pIGhsp21. A large number of transgenic plants were regenerated on a medium containing hygromycin. Integration of Oshsp2l gene was confirmed by PCR and Southern blot analyses with genomic DNA. Northern blot and immunoblot analyses revealed that the Oshsp21 gene was constitutively expressed and accumulated as mature protein in transgenic plants. Effects of constitutive expression of the OshspZl on thermotolerance were first probed with the chlorophyll fluorescence. Results indicate that inactivation of electron transport reactions in photosystem I1 (PSII), were mitigated by constitutive expression of the Oshsp21. These results suggest that the chloroplast small HSP plays an important role in protecting photosynthetic machinery during heat stress. (Key words : Thermotolerance, Rice, Transgenic, cDNA)

• Journal of Microbiology and Biotechnology
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• 제25권9호
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• pp.1528-1531
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• 2015

Sclerotia of Wolfiporia cocos are of medicinal and culinary value. The genes and molecular mechanisms involved in W. cocos sclerotial formation are poorly investigated because of the lack of a suitable and reproducible transformation system for W. cocos. In this study, a PEG/CaCl₂-mediated genetic transformation system for W. cocos was developed. The promoter P_gpd from Ganoderma lucidum effectively drove expression of the hygromycin B phosphotransferase gene in W. cocos, and approximately 30 transformants were obtained per 10 μg DNA when the protoplast suspension density was 10⁶ protoplasts/ml. However, no transformants were obtained under the regulation of the PtrpC promoter from Aspergillus nidulans.

• 식물조직배양학회지
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• 제27권2호
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• pp.95-100
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• 2000

배추에서 분리된 cytosolic glutathione reductase (BcGR1)유전자를 벼에 효과적으로 도입하기 위하여 몇 가지 실험을 수행하여 얻어진 결과를 요약하면 다음과 같다. 수정 후 3주된 미숙배에서 유도된 캘러스를 이용하여 형질전환 효율의 품종간 차이를 조사한 바 '대립벼'가 42.5%로 가장 높게 나타났다. 공동배양 배지 내에 acetosyringone (AS)이 첨가되지 않았을 경우 형질전환된 개체를 얻을 수 없었으나, 50$\mu$M을 첨가되었을 때 41.0%의 가장 높은 형질 전환효율을 나타내었다. PCR 증폭과 Southern blot 분석을 실시한바 BcGR1 유전자가 형질전환체의 게놈상에 정상적으로 삽입된 것이 확인되었으며, T$_1$세대 28개통 중 17개체의 hygromycin 저항성 유전형질이 3:1로 분리되었다.

• Journal of Microbiology
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• 제40권3호
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• pp.245-247
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• 2002

A genomic DNA library of the fungus Trametes versicolor was constructed in a yeast integration vector which contains the URA3 gene of the budding yeast Saccharomyces cerevisiae and the gene responsible for hygromycin B resistance, and fragments acting as autonomously replicating sequences (ARSes) in the budding yeast were identified from the genomic DNA library. Sixteen recombinant plasmids from the library transformed the budding yeast Saccharomyces cerevisiae to Ura+ at high frequencies. They were maintained stably under selective conditions, but were gradually lost from yeast cells at different rates under nonselective conditions, indicating that they contain eukaryotic origins of DNA replication and exist as extrachromosomal plasmids. Base sequences of four ARS DNAs among the 16 cloned fragments revealed that all or the four contain at least one 11 bp ［(A/T)TTTA(T/C)(A/G)TTT(A/T)］consensus sequence of the budding yeast ARS.

• Journal of Plant Biotechnology
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• 제6권3호
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• pp.145-150
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• 2004

Cabbage plants were transformed with the potato proteinase inhibitor II (PINII) gene, bar gene, and hpt gene using Agrobacterium. The expression of the PINII gene was driven by its own promoter which was wound-inducible. Ten transgenic plants were obtained from medium containing hygromycin as a selection antibiotic. The integration and expression of PINII and bar genes were confirmed by Southern and Northern hybridization. Growth and development of diamondback moths (Plutella xylostella) and tobacco cutworm (Spodoptera litura) larvae were examined on $T_1$ plants. The weight of the larvae and pupae of these two insects grown on transgenic plants was not different compared to those grown on wild type plants. However, the pupation and emergence rate of diamondback moths and tobacco cutworms fed on some transgenic plants was lower than on wild type plants. These results suggest that the PINII transgene under the control of a wound-induced promoter may be used for control of insects in transgenic cabbage through reduction of insect progeny number.