• Biotechnology and Bioprocess Engineering:BBE
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• 제9권3호
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• pp.217-222
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• 2004

As Agrobacterium tumefaciens, which has long been used to transform plants, is known to transfer T-DNA to budding yeast, Saccharomyces cerevisiae, a variety of fungi were subjected to the A. tumefaciens-mediated transformation to improve their transformation frequency and feasibility. The A. tumefaciens-mediated transformation of chestnut blight fungus, Cryphonectria parasitica, is performed in this study as the first example of transformation of a hardwood fungal pathogen. The transfer of the binary vector pBIN9-Hg, containing the bacterial hygromycin B phosphotransferase gene under the control of the Aspergillus nidulans trpC promoter and terminator, as a selectable marker, led to the selection of more than 1,000 stable, hygromycin B-resistant transformants per 1${\times}$10$\^$6/ conidia of C. parasitica. The putative transformants appeared to be mitotically stable. The transformation efficiency appears to depend on the bacterial strain, age of the bacteria cell culture and ratio of fungal spores to bacterial cells. PCR and Southern blot analysis indicated that the marker gene was inserted at different chromosomal sites. Moreover, three transformants out of ten showed more than two hybridizing bands, suggesting more than two copies of the inserted marker gene are not uncommon.

• Journal of Periodontal and Implant Science
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• 제32권3호
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• pp.603-614
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• 2002

Human gingival fibroblasts have proven to useful as a species specific cell culture system in various system on periodontal disease and regeneration. However, their use is limited, since they are hard to obtain and lifespan is short due to replicative senescence. To overcome these disadvantages, we transfected primary human gingival fibroblasts by the E6 and E7 genes of the Human papilloma virus(HPV) 16. The full length of HPV 16 E6 and E7 was cloned from the pBR322 into BamHl and Sal I of a pBabe vector including hygromycin B resistance. Before pBabeE6/E7 plasmid transfection, peak 8 GFP including G418 resistance was transfected into primary GF to check the transfection efficency. PBabe E6/E7 plasmid was transfected using Lipofectamine plus following manufacter's instruction into primary normal human gingival fibroblasts in 60mm dishes with FBS free DMEM. After 2 days of transfection, the cells were treated with hygromycin for 2 weeks until the transfected control cells died. The resulting hygromycin resistant colonies were pooled, and clonned, and sucessful　transfection was established for immortalized gingival fibroblast cell lines. Immoralized GF cells showed stellate shape, that is similar to that of orange grains, and more rapid growth and higher proliferation than that of primary gingival fibroblasts. This cell lines overcame crisis and could be cultured over 30 subcultured, could be use for three dimentional culture, epithelial-mesenchymal interaction study.

• 한국잠사학회:학술대회논문집
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• 한국잠사학회 2003년도 제46회 춘계 학술연구 발표회
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• pp.40-40
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• 2003

Recombinant plasmids harboring heterologous genes coding enhanced green fluorescent protein (EGFP) and erythropoietin (EPO) were transfected and expressed in Drosophila melanogaster S2 cells. Stably transformed cell populations expressing EGFP or monkey EPO were isolated after 4 weeks of selection with hygromycin B. (omitted)

• Journal of Animal Science and Technology
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• 제46권2호
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• pp.235-242
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• 2004

유용유전자 도입을 통한 신품종 이탈리안 라이그래스를 개발할 목적으로 Agrobacterium을 이용한 효율적인 형질전환 체계를 확립하였다. 이탈리안 라이그래스 성숙종자 유래의 캘러스를 standard binary vector인 pIG121Hm을 가지는 Agrobacterium EHA101을 이용하여 감염시킨 후 공동배양하여 형질전환시켰다. Agrobacterium을 이용한 형질전환에 있어서 중요한 인자로 작용하는 몇 가지 요인에 대한 이탈리안 라이그래스 캘러스의 형질전환 효율을 GUS 유전자의 발현정도로 조사하였다. Agrobacterium 감염시에 접종배지와 공동배양배지에 200${\mu}M$의 acetosyringone(AS)을 첨가해 주었을 때 형질전환 효율이 현저히 증가되었다. 또한 Agrobacterium의 농도를 $OD_{600}$=10. 이상의 높은 농도로 1시간 감염시켜Tdfm 때 형질전환 효율이 증가되었다. 접종배지에 200${\mu}M$의 AS와 0.1%의 Tween20을 첨가해 주었을 때 가장 높은 형질전환 효율을 나타내었다. 50mg/L의 hygromycin이 첨가된 선발배지에서 살아남은 캘러스로부터 정상적인 식물체가 재분화되었으며, 이들 형질전환체의 잎으로부터 GUS 활성 염색과 PCR 분석을 통하여 발현벡터의 T-DNA 영역이 형질전환 식물체의 genome으로 도입되었음을 확인할 수 있었다. 본 연구를 통하여 확립된 효율적인 형질전환 시스템은 분자육종을 통한 신품종 이탈리안 라이그래스의 개발에 유용하게 이용될 수 있을 것이다.

• Journal of Plant Biotechnology
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• 제5권3호
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• pp.163-168
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• 2003

A routine system based on particle bombardment of embryogenic callus for recovery of fertile transgenic rice (Oryza sativa L.) plants was developed. Embryogenic callus was established within 2-3 months from calli derived from mature seeds of Korean rice cultivar, Nagdongbyeo. The callus was bombarded with the plasmid pRQ6 containing the $\beta$-glucuronidase gene (gusA) and hygromycin phosphotransferase gene (hph, conferring resistance to hygromycin B), both driven by CaMV 35S promoter. Placement of cells on an osmoticum-containing medium (0.2 M sorbitol and 0.2 M mannitol) 4 hrs prior to and 16 hrs after bombardment resulted in a statistically significant increase with 3.2-fold in transient expression frequency gusA. In five independent experiments, the average frequency of transformation showing GUS activities was $8.86\%$. A large number of morphologically normal, fertile transgenic rice plants were obtained. Integration of foreign gene into the genome of $R_0$ transgenic plants was confirmed by Southern blot analysis. GUS and HPT were detected in $R_1$ progeny and Mendelian segregation of these genes was observed in $R_1$ progeny.

• Journal of Microbiology
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• 제43권5호
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• pp.417-423
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• 2005

In this study, the nematode-trapping fungus, Monacrosporium sphaeroides, was transformed with a plasmid harboring the hygromycin B phosphotransferase gene, via restriction enzyme-mediated integration (REMI). Frequencies of up to 94 transformants ${\mu}g^{-1}$ per linearized plasmid DNA were obtained by optimizing the PEG concentration, as well as the category and quantity of the added restriction enzyme. $90\%$ of the transformants were determined to be stable for drug resistance when 20 randomly selected transformants were tested. Southern analyses revealed that the transforming DNA was integrated into the M. sphaeroides genome either with or without rearrangement. Five mitotic stable mutant strains were obtained using this approach, all of which had been altered with regard to sporulation capacity and pathogenicity toward nematodes. Southern blot analyses of the five mutants revealed that foreign plasmid DNA had integrated into the genome. Three of the mutants, Tms2316, Tms3583 and Tms1536, exhibited integration at a single location, whereas the remaining two, Tms32 and Tms1913, manifested integration at double or multiple locations. Our results suggest that the transformation of M. sphaeroides via REMI will facilitate insertional mutagenesis, the functional analysis of a variety of genes, and the tagging or cloning of genes of interest.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
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• pp.74-74
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• 2003

Embryonic stem cells have several characteristics suitable for cell replacement therapy. To investigate a possibility of using human embryonic stem cell (hESC) as a carrier of therapeutic gene(s), hESC (MB03) was co-transfected with cDNAS coding for tyrosine hydroxylase (TH) and GTP cyclohydrolase Ⅰ (GTPCH Ⅰ) and bulk-selected using neomycin and hygromycin-B. Successful transfection was confirmed by western immunoblotting and RT-PCR. The genetically modified hESC (bk-THGC) relieved apomorphine-induced asymmetric motor behavior by approximately 54% when grafted into striatum of 6-OHDA-denervated rat brain. The number of rotation, however, increased up to 176+18% in 6 weeks when sham-grafted compared with number of rotation before graft. Immunohistochemical staining revealed that the grafted hESC survived and expressed TH for at least 6 weeks while the experiment was continued.

• Journal of Microbiology and Biotechnology
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• 제10권1호
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• pp.115-118
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• 2000

Recombinant plasmids harboring a heterologous gene coding for the enhanced green fluorescent protein (EGFP) were transfected and expressed in Drosophila melanogaster S2 cells. A stable transformation of polyclonal cell populations expressing EGFP were isolated after 4 weeks of selection with hygromycin B. The recombinant EFGP expressed in transformed S2 cells consisted of a molecular weight of 27 kDa. EGFP expression was also confirmed by fluorometric measurement. The maximum EGFP concentration was about 9.3 mg/I. The present findings demonstrate not only the successful stable expression of EGFP in Drosophuila was about 9.3 mgI. The present findings demonstrate not only the successful stable expression of EGFP in Drosophila S2 cells, but also the use of EGFP as a reporter to analyze gene expression, with its potential of a Drosophila cell expression system for recombinant protein production being an alternative to a baculovirus-insect cell expression system.

• Archives of Pharmacal Research
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• 제15권1호
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• pp.35-40
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• 1992

S. fradiae showed the highest acetanilide p-hydroxylation activity in the tested strains. And S. fradiae was well characterized genetically, especially with respect to tylosin production. Two mutants, which lost hydroxylation, were isolated in 140 regenerated colonies from protoplasts. In restriction enzyme digesion of total DNAs, isolation of giant linear plasmid DNA and determination of antibiotic resistances to chloramphenicol, tylosin, hygromycin B and mitomycin C, any differences among mutants and a wild type strain were not detected. These facts suggest that lesion on 6, 000 Kb chromosomal DNA was responsible for the lack of p-hydroxylation activity induced by protoplast formation and regeneration.

• 한국잠사학회:학술대회논문집
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• 한국잠사학회 2003년도 제46회 춘계 학술연구 발표회
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• pp.41-41
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• 2003

To elucidate the apoptosis mechanism of Trichoplusia ni cell, fundamental studies for apoptosis induction and suppression were performed. Hygromycin B, a known inducer of apoptosis, started the inhibition of T ni cell growth at 200 ug/$m\ell$ concentration. Furthermore, at 400 $\mu\textrm{g}$/$m\ell$ concentration, DNA fragmentation was detected on day 2 of incubation. (omitted)