• 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.

• 한국해양바이오학회지
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• 제5권4호
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• pp.26-32
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• 2011

Green fluorescent protein (GFP), a very important biological agent that involves shifting the color of bioluminescence from blue to green in luminous coelenterates and to increase the quantum yield of light emission. GFP discovered in medusa, Aequorea victoria is a key factor of various biotechnological and cell biological applications. Beside these applications, GFP of A. victoria is generally stable, which does not require co-factors for activity and can be functionally expressed in different bacterial species. This property of GFPs from A. victoria permits them to be a unique tool to monitor gene expression and protein localization in different organisms. The present review brings out the past milestones and future perspectives on GFPs, with an elaborative reviewing on its applications.

• KSBB Journal
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• 제14권3호
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• pp.377-379
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• 1999

CHO/dhfr- 세포에 대해 LipofectAmine$^{TM}$을 이용한 유전자 이입 효율을 증가시키기 위하여 지질과 DNA의 최적 농도를 구하였다. Reporter 유전자로서 GFP 유전자를 이용하였으며, 여러 농도의 지질 DNA로 유전자 이입된 각 세포군에서 나타나는 green fluorescence intensity를 FACS 분석함으로써 유전자 이입 효율을 정량화 할 수 있었다. 그 결과 24-well plate에서 $2.0{\mu}L$LipofectAmine$^{TM}$과 $0.4{\mu}g$ DNA를 조합하여 사용했을 때 최적의 유전자 이입 효율이 나타남을 알 수 있었다. 또한, GFP는 유전자 이입 최적화를 수행하는 데에 여러가지 면에서 유용한 수단이 될 수 있음을 확인할 수 있었다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVI)
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• pp.408-411
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• 2005

대장균을 이용하여 형광단백질을 형질전환하고 배양하면서 그 특성을 관찰하고 형광세기 및 형광 단백질의 크기를 조사하였다.

• 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.

• 한국수정란이식학회지
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• 제14권1호
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• pp.1-8
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• 1999

The efficiency of transgenic livestock animal production may be improved by early selection of transgenci preimplantation embryos. To examine the possibility of GFP gene as a non-invasive marker for the early screening of transgenic embryo, the GFP gene was microinjected into rabbit zygotes and the later stages of preimplantation embryos were examined for the expression of GFP. The presence of injected DNA was detected by PCR analysis and the expression of GFP was detected by observing green fluorescence in embryos under a fluorescent microscope. Out of 108 GFP gene-injected rabbit zygotes, seventy three(67.6%) were fluorescence-positive. When 11 fluroresecence-positive blastocysts were analyzed for the presence of GFP gene by PCR, 6(54.5%) were positive, and all of the 8 flrouescence-negative blastocysts were also negative by PCR. The results indicate that the screening of transgene in rabbit embryos by PCR analysis and GFP detection could be a promising method for the preselection of transgenic embryos.

• Animal Bioscience
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• 제36권6호
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• pp.973-979
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• 2023

Objective: The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system, which is the most efficient and reliable tool for precisely targeted modification of the genome of living cells, has generated considerable excitement for industrial applications as well as scientific research. In this study, we developed a gene-editing and detection system for chick embryo sexing during the embryonic stage. Methods: By combining the CRISPR/Cas9 technical platform and germ cell-mediated germline transmission, we not only generated Z chromosome-targeted knockin chickens but also developed a detection system for fluorescence-positive male chicks in the embryonic stage. Results: We targeted a green fluorescent protein (GFP) transgene into a specific locus on the Z chromosome of chicken primordial germ cells (PGCs), resulting in the production of Z^GFP-knockin chickens. By mating Z^GFP-knockin females (Z^GFP/W) with wild males (Z/Z) and using a GFP detection system, we could identify chick sex, as the GFP transgene was expressed on the Z chromosome only in male offspring (Z^GFP/Z) even before hatching. Conclusion: Our results demonstrate that the CRISPR/Cas9 technical platform with chicken PGCs facilitates the production of specific genome-edited chickens for basic research as well as practical applications.

• Applied Biological Chemistry
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• 제44권4호
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• pp.215-218
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• 2001

쪽(Polygonum tinctortium) 세포에서 외래 단백질 발현을 검토하기 위하여 green fluorescent protein(GFP)가 내재하는 pCAMBIA1302를 형질 전환시켰으며 Western blot 분석에 의해 GFP의 발현을 확인하였다. Sodium butyrate가 GFP생성에 미치는 효과를 검토한 결과, 10 mM에서 세포성장이 지연되었으며, 15 mM 이상에서는 정지되었다. 세포 내 GFP 생성량은 세포 접종 후 3일째 5 mM sodium butyrate를 첨가하였을 때가 0일째 처리에 비해 120% 증가하였다. 또한 접종후 3일 후 5 mM의 sodium butyrate를 처리한 경우가 10 mM의 경우보다 GFP의 수율이 50% 증가하였다. 본 실험을 통하여 세포 접종 후 3일째, 5 mM의 농도로 처리한 sodium butyrate가 외래 단백질의 발현을 효과적으로 증가시키는 결과를 확인하였다.

• Journal of Microbiology and Biotechnology
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• 제17권10호
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• pp.1727-1732
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• 2007

Phenylacetic acid (PAA) is produced by many bacteria as an antifungal agent and also appears to be an environmentally toxic chemical. The object of this study was to detect PAA using Pseudomonas putida harboring a reporter plasmid that has a PAA-inducible promoter fused to a green fluorescent protein (GFP) gene. Pseudomonas putida KT2440 was used to construct a green fluorescent protein-based reporter fusion using the paaA promoter region to detect the presence of PAA. The reporter strain exhibited a high level of gfp expression in minimal medium containing PAA; however, the level of GFP expression diminished when glucose was added to the medium, whereas other carbon sources, such as succinate and pyruvate, showed no catabolic repression. Interestingly, overexpression of a paaF gene encoding PAA-CoA ligase minimized catabolic repression. The reporter strain could also successfully detect PAA produced by other PAA-producing bacteria. This GFP-based bioreporter provides a useful tool for detecting bacteria producing PAA.

• Parasites, Hosts and Diseases
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• 제49권4호
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• pp.357-364
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• 2011

Various Leishmania species were engineered with green fluorescent protein (GFP) using episomal vectors that encoded an antibiotic resistance gene, such as aminoglycoside geneticin sulphate (G418). Most reports of GFP-Leishmania have used the flagellated extracellular promastigote, the stage of parasite detected in the midgut of the sandfly vector; fewer studies have been performed with amastigotes, the stage of parasite detected in mammals. In this study, comparisons were made regarding the efficiency for in vitro G418 selection of GFP-Leishmania amazonensis promastigotes and amastigotes and the use of in vivo G418 selection. The GFP-promastigotes retained episomal plasmid for a prolonged period and G418 treatment was necessary and efficient for in vitro selection. In contrast, GFP-amastigotes showed low retention of the episomal plasmid in the absence of G418 selection and low sensitivity to antibiotics in vitro. The use of protocols for G418 selection using infected BALB/c mice also indicated low sensitivity to antibiotics against amastigotes in cutaneous lesions.