• 생명과학회지
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• 제17권2호통권82호
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• pp.293-297
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• 2007

형광단백질 (green fluorescent protein, GFP)은 생물공정을 살피데 지표 단백질로 유용하게 사용이 된다. 본 연구에서는 쌀세포에서 외래 단백질의 발현양상을 관찰하기 위해서, 표지 단백질로 GFP를 형질전환 후 이것에서 유도된 현탁세포에서 GFP의 발현 양상을 관찰하였다. 형질전환시 GFP의 발현을 위한 프로모터로 RAmysE를 사용하였으며 이것은 배양액 중에서 당이 고갈되었을 때 강력히 작동된다. 그래서 본 연구에서는 배양액 중에 다양한 슈크로오스 농도로 쌀세포를 배양하여 세포의 성장양태 및 GFP의 발현양에 미치는 영향을 관찰한 결과 세포의 성장은 12%의 당농도에서 7.06g/L로 최적이였으며 GFP는 당을 가장 적게 사용한 3%에서 최적임을 알 수가 있었다. 이것은 세포의 성장과 GFP의 생산에 사용된 당이 반대로 영향을 미침을 알 수가 있었으며 향후 최적의 대량배양을 위해서는 세포의 성장과 산물의 생산시기를 분리한 이단계 배양법이 필요함을 암시한다.

• 미생물학회지
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• 제47권2호
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• pp.174-178
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• 2011

본 연구에서는 외부의 푸마르산을 인식하는 DcuS/R TCS에 의하여 발현이 조절되는 dctA 유전자의 발현 특성을 GFP를 이용하여 관찰하였으며, 1 mM의 푸마르산을 감지하여 GFP를 발현 시킬 수 있음을 확인하였다. 결론적으로, 개량된 E. coli는 간단한 dctA 프로모터와 GFP의 융합을 이용하여 푸마르산을 모니터 할 수 있으며, 이것은 상승된 푸마르산 농도 조건하에서 원활히 작동함을 확인할 수 있었다.

• 한국수정란이식학회지
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• 제15권2호
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• pp.167-173
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• 2000

The principal objective of this study was to clone transgenic embryos in order to improve the efficiency of transgenic animal production by the combination of microinjection and nuclear transplantation techniques. Mature female New Zealand White rabbits were superovulated by eCG and hCG treatments, fllowed by natural mating. Zygotes were collected from the oviducts at 18∼22 h after hCG injection by flushing with D-PBS containing 5% fetal calf serum(FCS). Two to three picoliters of green fluorescent protein(GFP) gene wa microinjected into male pronucleus. The foreign gene-injected zygotes were cultured in TCM-199 or RD medium containing 10% FCS with a monolayer of rabbit oviductal epithelial cells in a 5% CO2 incubator. The morulae expressing GFP gene were selected and their blastomeres were separated for the use of nuclear donor. Following nuclear transplantation of fluorescence-positive morula stage blastomeres, 13 (21.3%) out of 61 fused oocytes developed to blastocyst stage and all of the cloned blastocysts expressed GFP. The results indicate that the screening of transgene in rabbit embryos by GFP detection could be a promisible method for the preselection of transgenic embryos. Also the cloning of preselected transgenic embryos by nuclear transplantatin could be efficiently applied to the multiple production of transgenic animals.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
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• pp.42-42
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• 2003

복제기술은 기존의 형질전환 동물 생산의 효율을 향상시킬 수 있는 기술로서 인정하고 있으며 또한 이를 이용하여 형질전환 동물의 생산이 이루어지고 있다. 따라서 본 연구는 표지유전자 (GFP)와 유용유전자 (hFSH)를 이용하여 임신 45일령에 채취한 태아섬유아세포에 transfection 하고, transfection 된 세포의 효율적인 선발과 이를 이용한 형질전환 복제 수정란을 생산하고자 실시하였다. 대조구 (KbFF), GFP (79KbFF-GFP c-3) 및 hFSH (79KbFF-hFSH n-1)에 공시한 세포는 모두 동일한 태아유래의 세포 (모 79, 부 KPN178,♂)를 이용하였다. pAB-eGFP와 hFSH 유전자는 각파 electroporation 방법을 이용하여 transfection 하고, 이를 2주 동안 G418로 배양하며 selection 하였다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2002년도 생물공학의 동향 (X)
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• pp.232-234
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• 2002

공업적으로 중요성이 날로 더해가는 phospholipase C (PLC) 를 Bacillus cereus를 이용하여 생산하였다. 또한, plc::gfp fusion protein 을 생산하는 재조합 E. coli 를 제조하고 배양하였으며 특히 형광센서를 이용하여 PLC 의 생산 특성을 모니터링하였다.

• Asian-Australasian Journal of Animal Sciences
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• 제30권6호
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• pp.886-892
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• 2017

Objective: Transgenic technology is widely used for industrial applications and basic research. Systems that allow for genetic modification play a crucial role in biotechnology for a number of purposes, including the functional analysis of specific genes and the production of exogenous proteins. In this study, we examined and verified the cumate-inducible transgene expression system in chicken DF1 and quail QM7 cells, as well as loxP element-mediated transgene recombination using Cre recombinase in DF1 cells. Methods: After stable transfer of the transgene with piggyBac transposon and transposase, transgene expression was induced by an appropriate concentration of cumate. Additionally, we showed that the transgene can be replaced with additional transgenes by co-transfection with the Cre recombinase expression vector. Results: In the cumate-GFP DF1 and QM7 cells, green fluorescent protein (GFP) expression was repressed in the off state in the absence of cumate, and the GFP transgene expression was successfully induced in the presence of cumate. In the cumate-MyoD DF1 cells, MyoD transgene expression was induced by cumate, and the genes controlled by MyoD were upregulated according to the number of days in culture. Additionally, for the translocation experiments, a stable enhanced green fluorescent protein (eGFP)-expressing DF1 cell line transfected with the loxP66-eGFP-loxP71 vector was established, and DsRed-positive and eGFP-negative cells were observed after 14 days of co-transfection with the DsRed transgene and Cre recombinase indicating that the eGFP transgene was excised, and the DsRed transgene was replaced by Cre recombination. Conclusion: Transgene induction or replacement cassette systems in avian cells can be applied in functional genomics studies of specific genes and adapted further for efficient generation of transgenic poultry to modulate target gene expression.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2001년도 Proceedings of 2001 International Symposium
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• pp.77-80
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• 2001

A variety of different methods to generate diverse proteins, including random mutagenesis and recombination, are currently available, and most of them accumulate the mutations on the target gene of a protein, whose sequence space remains unchanged. On the other hand, a pool of diverse genes, which is generated by random insertions, deletions, and exchange of the homologous domains with different lengths in the target gene, would present the protein lineages resulting in new fitness landscapes. Here we report a method to generate a pool of protein variants with different sequence spaces by employing green fluorescent protein (GFP) as a model protein. This process, designated functional salvage screen (FSS), comprises the following procedures： a defective GFP template expressing no fluorescence is firstly constructed by genetically disrupting a predetermined region(s) of the protein, and a library of GFP variants is generated from the defective template by incorporating the randomly fragmented genomic DNA from E. coli into the defined region(s) of the target gene, followed by screening of the functionally salvaged, fluorescence-emitting GFPs. Two approaches, sequence-directed and PCR-coupled methods, were attempted to generate the library of GFP variants with new sequences derived from the genomic segments of E. coli. The functionally salvaged GFPs were selected and analyzed in terms of the sequence space and functional property. The results demonstrate that the functional salvage process not only can be a simple and effective method to create protein lineages with new sequence spaces, but also can be useful in elucidating the involvement of a specific region(s) or domain(s) in the structure and function of protein.

• Journal of Plant Biotechnology
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• 제36권3호
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• pp.255-260
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• 2009

The translation elongation factor 1A, eEF1A, catalyzes the binding of aminoacyl-tRNA to the A-site of the ribosome by a GTP-dependent mechanism. By subtractive suppression hybridization technique, we have isolated a soybean low-temperature inducible gene, SLTI100 encoding translation elongation factor 1A. Multiple sequence alignments and phylogenic analysis showed that SLTI100 and other eEF1As originated from diverse organisms are highly conserved. RNA expression of SLTI100 was specifically induced by low temperature, high salt, ABA, or drought stress. Based on the subcellular localization of the corresponding gene product fused to GFP, we were able to confirm that SLTI100-GFP was restricted to the nucleus and cytoplasm. We propose that soybean eEF1A may play an important role in translational regulation during abiotic stress responses in plants.

• Journal of Microbiology and Biotechnology
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• 제29권11호
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• pp.1790-1798
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• 2019

Flock House virus (FHV), an insect RNA virus, has a bipartite genome. FHV RNA1 can be packaged in turnip yellow mosaic virus (TYMV) as long as the FHV RNA has a TYMV sequence at the 3'-end. The encapsidated FHV RNA1 has four additional nucleotides at the 5'-end. We investigated whether the recombinant FHV RNA1 could replicate in mammalian cells. To address this issue, we prepared in vitro transcribed FHV RNAs that mimicked the recombinant FHV RNA1, and introduced them into baby hamster kidney (BHK) cells. The result showed that the recombinant FHV RNA1 was capable of replication. An eGFP gene inserted into the frame with B2 gene of the FHV RNA1 was also successfully expressed. We also observed that eGFP expression at the protein level was strong at 28℃ but weak at 30℃. Sequence analysis showed that the 3'-ends of the RNA1 and RNA3 replication products were identical to those of the authentic FHV RNAs. This indicates that FHV replicase correctly recognized an internally-located replication signal. In contrast, the 5'-ends of recombinant FHV RNA1 frequently had deletions, indicating random initiation of (+)-strand synthesis.

• Journal of Veterinary Science
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• 제22권4호
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• pp.56.1-56.10
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• 2021

Background: Fluorescent antibody virus neutralization (FAVN) test is a standard assay for quantifying rabies virus-neutralizing antibody (VNA) in serum. However, a safer rabies virus (RABV) should be used in the FAVN assay. There is a need for a new method that is economical and time-saving by eliminating the immunostaining step. Objectives: We aimed to improve the traditional FAVN method by rescuing and characterizing a new recombinant RABV expressing green fluorescent protein (GFP). Methods: A new recombinant RABV expressing GFP designated as ERAGS-GFP was rescued using a reverse genetic system. Immuno-fluorescence assay, peroxidase-linked assay, electron microscopy and reverse transcription polymerase chain reaction were performed to confirm the recombinant ERAGS-GFP virus as a RABV expressing the GFP gene. The safety of ERAGS-GFP was evaluated in 4-week-old mice. The rabies VNA titers were measured and compared with conventional FAVN and FAVN-GFP tests using VERO cells. Results: The virus propagated in VERO cells was confirmed as RABV expressing GFP. The ERAGS-GFP showed the highest titer (10^8.0 TCID₅₀/mL) in VERO cells at 5 days post-inoculation, and GFP expression persisted until passage 30. The body weight of 4-week-old mice inoculated intracranially with ERAGS-GFP continued to increase and the survival rate was 100%. In 62 dog sera, the FAVN-GFP result was significantly correlated with that of conventional FAVN (r = 0.95). Conclusions: We constructed ERAGS-GFP, which could replace the challenge virus standard-11 strain used in FAVN test.