Journal articles: 'EGFP (enhanced green fluorescent protein)'

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Baumann, Chris T., Carol S. Lim, and Gordon L. Hager. "Simultaneous Visualization of the Yellow and Green Forms of the Green Fluorescent Protein in Living Cells." Journal of Histochemistry & Cytochemistry 46, no. 9 (September 1998): 1073–76. http://dx.doi.org/10.1177/002215549804600911.

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In this study we sought to develop a method for the co-localization of proteins in living cells utilizing the enhanced green fluorescent protein (EGFP) and a redshifted EGFP variant, EYFP (enhanced yellow fluorescent protein). EYFP was expressed as an unsubstituted molecule while EGFP was fused to NF1 (EGFP-NF1), a transcription factor found exclusively in the nucleus. The Leica TCS SP laser scanning confocal microscope was used. This microscope allows the user to monitor the emitted light at defined wavelengths owing to the presence of a monochrometer in the emission light path. pEGFP-NF1 and pEYFP were co-expressed in the same cell and excited with the 476–nm and 488–nm argon laser lines. To separate the EYFP and EGFP fluorescence, EGFP-NF1 emission was recorded between 496 and 505 nm. These wavelengths are on the left shoulder of the EGFP emission peak and exclude most of the EYFP fluorescence. The EYFP emission was followed between 670 and 754 nm, utilizing the tail of EYFP emission that extends well beyond that for EGFP. Under these conditions we obtained excellent discrimination between EYFP fluorescence and EGFP-NF1 emission. These observations demonstrate that EYFP- and EGFP-substituted chimeras can be used for simultaneous detection in living cells.

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Takenaka-Uema, Akiko, Yousuke Murata, Fumihiro Gen, Yukari Ishihara-Saeki, Ken-ichi Watanabe, Kazuyuki Uchida, Kentaro Kato, et al. "Generation of a Recombinant Akabane Virus Expressing Enhanced Green Fluorescent Protein." Journal of Virology 89, no. 18 (July 8, 2015): 9477–84. http://dx.doi.org/10.1128/jvi.00681-15.

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ABSTRACTWe generated a recombinant Akabane virus (AKAV) expressing enhanced green fluorescence protein (eGFP-AKAV) by using reverse genetics. We artificially constructed an ambisense AKAV S genome encoding N/NSs on the negative-sense strand, and eGFP on the positive-sense strand with an intergenic region (IGR) derived from the Rift Valley fever virus (RVFV) S genome. The recombinant virus exhibited eGFP fluorescence and had a cytopathic effect in cell cultures, even after several passages. These results indicate that the gene encoding eGFP in the ambisense RNA could be stably maintained. Transcription of N/NSs and eGFP mRNAs of eGFP-AKAV was terminated within the IGR. The mechanism responsible for this appears to be different from that in RVFV, where the termination sites for N and NSs are determined by a defined signal sequence. We inoculated suckling mice intraperitoneally with eGFP-AKAV, which resulted in neurological signs and lethality equivalent to those seen for the parent AKAV. Fluorescence from eGFP in frozen brain slices from the eGFP-AKAV-infected mice was localized to the cerebellum, pons, and medulla oblongata. Our approach to producing a fluorescent virus, using an ambisense genome, helped obtain eGFP-AKAV, a fluorescent bunyavirus whose viral genes are intact and which can be easily visualized.IMPORTANCEAKAV is the etiological agent of arthrogryposis-hydranencephaly syndrome in ruminants, which causes considerable economic loss to the livestock industry. We successfully generated a recombinant enhanced green fluorescent protein-tagged AKAV containing an artificial ambisense S genome. This virus could become a useful tool for analyzing AKAV pathogenesis in host animals. In addition, our approach of using an ambisense genome to generate an orthobunyavirus stably expressing a foreign gene could contribute to establishing alternative vaccine strategies, such as bivalent vaccine virus constructs, for veterinary use against infectious diseases.

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Tandio Saputro, Shania Safera, Khayu Wahyunita, Astutiati Nurhasanah, Yudhi Nugraha, Irvan Faizal, Sabar Pambudi, and Andri Pramesyanti Pramono. "Expression of modified enhanced green fluorescent polyarginine protein in Saccharomyces cerevisiae INVSc1." F1000Research 12 (January 3, 2023): 1. http://dx.doi.org/10.12688/f1000research.123181.1.

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Background: The enhanced green fluorescent protein (EGFP) gene is a reporter gene that can be used to optimize protein isolation procedures and the functional working of a transduction protein. EGFP, with the addition of eleven arginine residues, has been engineered to functionally improve the protein transduction process, which can later be used for cell reprogramming like induced pluripotent stem cells. The addition of six histidine amino acid residues at its C-terminal is intended for the protein isolation process using the His-tag antibody. Methods: The study aimed to investigate the optimization of the EGFP polyarginine protein expression in Saccharomyces cerevisiae in sufficient quantities for the protein isolation stage. This study also analyzed EGFP expression without polyarginine to analyze the polyarginine addition effect on expression processes. Protein expression was qualitatively measured by looking at expression fluorescence and protein levels of EGFP and EGFP - PolyR proteins. Results: Bands on Western Blots with 6×His-tag monoclonal antibody (primary antibody) and Goat anti-mouse IgG HRP (secondary antibody) showed the EGFP polyarginine and EGFP proteins were expressed in Saccharomyces cerevisiae INVSc1 at relatively low levels. The lyticase incubation time modification and administration of 3-5 kDa microfilter to concentrate increased the yield of isolated protein. Conclusions: The sufficient amount of protein isolation in S. cerevisiae can be achieved by using lyticase and sonicators combination for the lysis process.

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Mi, H. W., M. C. Lee, Y. C. Chiang, L. P. Chow, and C. P. Lin. "Single-Molecule Imaging of Bmp4 Dimerization on Human Periodontal Ligament Cells." Journal of Dental Research 90, no. 11 (August 12, 2011): 1318–24. http://dx.doi.org/10.1177/0022034511418340.

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We expressed bone morphogenetic protein 4 (BMP4) fused with enhanced green fluorescent protein (BMP4-EGFP) in the secretory pathways of producer cells. Fluorescent EGFP was acquired only after we interrupted the transport of BMP4-EGFP by culturing cells at a lower temperature (20°C), and the dynamics of BMP4-EGFP could be monitored by single-molecule microscopy. Western blotting analysis confirmed that exposure to low temperature helped the integrated formation of BMP4-EGFP fusion proteins. In this study, for the first time, we could image the fluorescently labeled BMP4 molecules localized on the plasma membrane of living hPDL cells. The one-step photobleaching with EGFP and the “blinking” behavior of quantum dots suggest that the fluorescent spots represent the events of single BMP4 molecules. Single-molecule tracking showed that the BMP receptors (BMPR) dimerize after BMP4 stimulation, or that a complex of one BMP4 molecule and a pre-formed BMPR dimer develops first, followed by the binding of the second BMP4 molecule. Furthermore, BMP4-EGFP enhanced the osteogenic differentiation of hPDL cells via signal transduction involving BMP receptors. This single-molecule imaging technique might be a valuable tool for the future development of BMP4 gene therapy and regenerative medicine mediated by hPDLs. Abbreviations: BMP4, bone morphogenetic protein 4; BMPR, BMP receptor; EGFP, enhanced green fluorescent protein; hPDL cells, human periodontal ligament cells; QDs, quantum dots; TIRFM, total internal reflection fluorescence microscopy; 293 cells, human embryonic kidney cells; oDM, osteogenic differentiation medium; HcoI, type I collagen; ALP, alkaline phosphatase; BSP, bone sialoprotein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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Mizozoe, Otaki, and Aikawa. "The Mechanism of Chlorine Damage Using Enhanced Green Fluorescent Protein-Expressing Escherichia coli." Water 11, no. 10 (October 16, 2019): 2156. http://dx.doi.org/10.3390/w11102156.

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This study investigated how chlorine inactivates and damages Escherichia coli cells. E. coli that had transformed to express enhanced green fluorescent protein (EGFP) at the cytoplasm was treated with chlorine. Damage to the cell membrane and cell wall was analyzed by measuring the fluorescence intensity of the leaked EGFP, then accounting for the fluorescence deterioration. At pH 7, E. coli was lethally damaged after treatment with chlorine, but significant leakage of EGFP was not observed. In contrast, significant leakage of EGFP was observed at pH 9, even though E. coli was not as inactivated as it was at pH 7. Flow cytometry was used to confirm the fluorescence intensity of the remaining EGFP inside the cells. No significant fluorescence loss was observed in the cells at pH 7. However, at pH 9, the fluorescence intensity in the cells decreased, indicating leakage of EGFP. These results suggest that hypochlorous acid inactivates E. coli without damaging its cell membrane and cell wall, whereas the hypochlorite ion inactivates E. coli by damaging its cell membrane and cell wall. It was possible to confirm the chlorine damage mechanism on E. coli by measuring the fluorescence intensity of the leaked EGFP.

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dos Santos, Nathalia Vieira, Carolina Falaschi Saponi, Tamar Louise Greaves, and Jorge Fernando Brandão Pereira. "Revealing a new fluorescence peak of the enhanced green fluorescent protein using three-dimensional fluorescence spectroscopy." RSC Advances 9, no. 40 (2019): 22853–58. http://dx.doi.org/10.1039/c9ra02567g.

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Krasowska, Joanna, Monika Olasek, Agnieszka Bzowska, Patricia L. Clark, and Beata Wielgus-Kutrowska. "The comparison of aggregation and folding of enhanced green fluorescent protein (EGFP) by spectroscopic studies." Spectroscopy 24, no. 3-4 (2010): 343–48. http://dx.doi.org/10.1155/2010/186903.

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GFP (Green Fluorescent Protein) is well known for its unique chromophore which is formed by autocatalytic cyclization of a polypeptide backbone of Ser65, Tyr66 and Gly67, and is able to emit green visible light. Due to unusual chromophore responsible for the fluorescence GFP and its mutants (e.g., EGFP) have become widely used reporter proteins in molecular biology and biotechnology. GFP can easily be fused to any protein of interest and co-expressed in cells; the GFP fluorescence is then used to visualize the distribution, transport and aggregation of the protein in the cell. However, GFP has a tendency to aggregate itself, and also formation of its chromophore critically depends on the presence of reducing agents. Therefore we have undertaken spectroscopic kinetic studies of EGFP folding and aggregation as a function of pH, and in the presence of various reducing agents, to study the competition between these two processes. The best conditions for folding of EGFP provides BME as a reducing agent. Aggregation of EGFP depends strongly on pH, and on the concentration of the protein. The careful control experiments must therefore be performed during investigations of proteins fused with EGFP, especially at pH lower than 7.

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Hu, Yu, Ziying Li, Wei Shi, Yanxue Yin, Heng Mei, Huafang Wang, Tao Guo, Jun Deng, Han Yan, and Xuan Lu. "Early diagnosis of cerebral thrombosis by EGFP–EGF1 protein conjugated ferroferric oxide magnetic nanoparticles." Journal of Biomaterials Applications 33, no. 9 (January 15, 2019): 1195–201. http://dx.doi.org/10.1177/0885328218823475.

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Cerebral thrombosis disease is a worldwide problem, with high rates of morbidity, disability, and mortality. Magnetic resonance imaging diffusion-weighted imaging was used as an important early diagnostic method for cerebral thrombotic diseases; however, its diagnosis time is 2 h after onset. In this study, we designed EGFP–EGF1–NP–Fe3O4 for earlier diagnosis of cerebral thrombosis by taking advantage of EGFP–EGF1 fusion protein, in which EGF1 can bind with tissue factor and enhanced green fluorescent protein has previously been widely used as a fluorescent protein marker. EGFP–EGF1–NP–Fe3O4 or NP–Fe3O4 reaches the highest concentration in the infarction areas in 1 h. To evaluate the targeting ability of EGFP–EGF1–NP–Fe3O4, a fluorochrome dye, Dir, was loaded into the nanoparticle. As shown by the in vivo organ multispectral fluorescence imaging, Dir-loaded EGFP–EGF1–NP–Fe3O4 exhibited higher fluorescence than those of model rats treated with Dir-loaded NP–Fe3O4. Coronal frozen sections and transmission electron microscope further showed that EGFP–EGF1–NP–Fe3O4 was mainly accumulated in the tissue factor exposure region of brain. The data indicated that the EGFP–EGF1–NP–Fe3O4 targeted cerebral thrombosis and might be applied in the early diagnosis of intracranial thrombosis.

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BOLSOVER, Stephen, Ozbek IBRAHIM, Niamh O'LUANAIGH, Helen WILLIAMS, and Shamshad COCKCROFT. "Use of fluorescent Ca2+ dyes with green fluorescent protein and its variants: problems and solutions." Biochemical Journal 356, no. 2 (May 24, 2001): 345–52. http://dx.doi.org/10.1042/bj3560345.

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We have studied the degree to which fluorescent Ca2+ indicator dyes, and green fluorescent protein and its variants, can be used together. We find that the most commonly used fluorescent protein, enhanced green fluorescent protein (EGFP), seriously contaminates fura 2 signals. We suggest two alternative combinations for which there is no detectable contamination of the Ca2+ indicator signal by the fluorescent protein. Blue fluorescent protein can be used with the Ca2+ indicator Fura Red; EGFP can be used with the Ca2+ indicator X-Rhod 1. The use of these combinations will permit the accurate measurement of Ca2+ signals in cells transfected with fluorescent proteins.

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Spitzer, Dirk, Kurt E. J. Dittmar, Manfred Rohde, Hansjörg Hauser, and Dagmar Wirth. "Green Fluorescent Protein-Tagged Retroviral Envelope Protein for Analysis of Virus-Cell Interactions." Journal of Virology 77, no. 10 (May 15, 2003): 6070–75. http://dx.doi.org/10.1128/jvi.77.10.6070-6075.2003.

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ABSTRACT Fluorescent retroviral envelope (Env) proteins were developed for direct visualization of viral particles. By fusing the enhanced green fluorescent protein (eGFP) to the N terminus of the amphotropic 4070A envelope protein, extracellular presentation of eGFP was achieved. Viruses incorporated the modified Env protein and efficiently infected cells. We used the GFP-tagged viruses for staining retrovirus receptor-positive cells, thereby circumventing indirect labeling techniques. By generating cells which conditionally expressed the GFP-tagged Env protein, we could confirm an inverse correlation between retroviral Env expression and infectivity (superinfection). eGFP-tagged virus particles are suitable for monitoring the dynamics of virus-cell interactions.

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Goh, Yan Y., Bow Ho, and Jeak L. Ding. "A Novel Fluorescent Protein-Based Biosensor for Gram-Negative Bacteria." Applied and Environmental Microbiology 68, no. 12 (December 2002): 6343–52. http://dx.doi.org/10.1128/aem.68.12.6343-6352.2002.

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ABSTRACT Site-directed mutagenesis of enhanced green fluorescent protein (EGFP) based on rational computational design was performed to create a fluorescence-based biosensor for endotoxin and gram-negative bacteria. EGFP mutants (EGFPi) bearing one (G10) or two (G12) strands of endotoxin binding motifs were constructed and expressed in an Escherichia coli host. The EGFPi proteins were purified and tested for their efficacy as a novel fluorescent biosensor. After efficient removal of lipopolysaccharide from the E. coli lysates, the binding affinities of the EGFPi G10 and G12 to lipid A were established. The KD values of 7.16 × 10−7 M for G10 and 8.15 × 10−8 M for G12 were achieved. With high affinity being maintained over a wide range of pH and ionic strength, the binding of lipid A/lipopolysaccharide to the EGFPi biosensors could be measured as a concentration-dependent fluorescence quenching of the EGFP mutants. The EGFPi specifically tagged gram-negative bacteria like E. coli and Pseudomonas aeruginosa, as well as other gram-negative bacteria in contaminated water sampled from the environment. This dual function of the EGFPi in detecting both free endotoxin and live gram-negative bacteria forms the basis of the development of a novel fluorescent biosensor.

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Le, Long P., Jing Li, Vladimir V. Ternovoi, Gene P. Siegal, and David T. Curiel. "Fluorescently tagged canine adenovirus via modification with protein IX–enhanced green fluorescent protein." Journal of General Virology 86, no. 12 (December 1, 2005): 3201–8. http://dx.doi.org/10.1099/vir.0.80968-0.

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Canine adenovirus type 2 (CAV2) has become an attractive vector for gene therapy because of its non-pathogenicity and the lack of pre-existing neutralizing antibodies against this virus in the human population. Additionally, this vector has been proposed as a conditionally replicative adenovirus agent under the control of an osteocalcin promoter for evaluation in a syngeneic, immunocompetent canine model with spontaneous osteosarcoma. In this study, a CAV2 vector labelled with the fluorescent capsid fusion protein IX–enhanced green fluorescent protein (pIX–EGFP) was developed. Expression of the fluorescent fusion-protein label in infected cells with proper nuclear localization, and incorporation into virions, could be detected. The labelled virions could be visualized by fluorescence microscopy; this was applicable to the tracking of CAV2 infection, as well as localizing the distribution of the vector in tissues. Expression of pIX–EGFP could be exploited to detect the replication and spread of CAV2. These results indicate that pIX can serve as a platform for incorporation of heterologous proteins in the context of a canine adenovirus xenotype. It is believed that capsid-labelled CAV2 has utility for vector-development studies and for monitoring CAV2-based oncolytic adenovirus replication.

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Lee, Woonwoo, Hyojin Kim, Yerin Kang, Youngshim Lee, and Youngdae Yoon. "A Biosensor Platform for Metal Detection Based on Enhanced Green Fluorescent Protein." Sensors 19, no. 8 (April 18, 2019): 1846. http://dx.doi.org/10.3390/s19081846.

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Microbial cell-based biosensors, which mostly rely on stress-responsive operons, have been widely developed to monitor environmental pollutants. Biosensors are usually more convenient and inexpensive than traditional instrumental analyses of environmental pollutants. However, the targets of biosensors are restricted by the limited number of genetic operon systems available. In this study, we demonstrated a novel strategy to overcome this limitation by engineering an enhanced green fluorescent protein (eGFP). It has been reported that combining two fragments of split-eGFP can form a native structure. Thus, we engineered new biosensors by inserting metal-binding loops (MBLs) between β-strands 9 and 10 of the eGFP, which then undergoes conformational changes upon interaction between the MBLs and targets, thereby emitting fluorescence. The two designed MLBs based on our previous study were employed as linkers between two fragments of eGFP. As a result, an Escherichia coli biosensor exhibited a fluorescent signal only when interacting with cadmium ions, revealing the prospect of a new biosensor for cadmium detection. Although this study is a starting stage for further developing biosensors, we believe that the proposed strategy can serve as basis to develop new biosensors to target various environmental pollutants.

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Mamontova, Anastasia V., Aleksander M. Shakhov, Konstantin A. Lukyanov, and Alexey M. Bogdanov. "Deciphering the Role of Positions 145 and 165 in Fluorescence Lifetime Shortening in the EGFP Variants." Biomolecules 10, no. 11 (November 13, 2020): 1547. http://dx.doi.org/10.3390/biom10111547.

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The bright ultimately short lifetime enhanced emitter (BrUSLEE) green fluorescent protein, which differs from the enhanced green fluorescent protein (EGFP) in three mutations, exhibits an extremely short fluorescence lifetime at a relatively high brightness. An important contribution to shortening the BrUSLEE fluorescence lifetime compared to EGFP is provided by the T65G substitution of chromophore-forming residue and the Y145M mutation touching the chromophore environment. Although the influence of the T65G mutation was studied previously, the role of the 145th position in determining the GFPs physicochemical characteristics remains unclear. In this work, we show that the Y145M substitution, both alone and in combination with the F165Y mutation, does not shorten the fluorescence lifetime of EGFP-derived mutants. Thus, the unlocking of Y145M as an important determinant of lifetime tuning is possible only cooperatively with mutations at position 65. We also show here that the introduction of a T65G substitution into EGFP causes complex photobehavior of the respective mutants in the lifetime domain, namely, the appearance of two fluorescent states with different lifetimes, preserved in any combination with the Y145M and F165Y substitutions.

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Sen, Tirthendu, Anastasia Mamontova, Anastasia Titelmayer, Aleksander Shakhov, Artyom Astafiev, Atanu Acharya, Konstantin Lukyanov, Anna Krylov, and Alexey Bogdanov. "Influence of the First Chromophore-Forming Residue on Photobleaching and Oxidative Photoconversion of EGFP and EYFP." International Journal of Molecular Sciences 20, no. 20 (October 22, 2019): 5229. http://dx.doi.org/10.3390/ijms20205229.

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Enhanced green fluorescent protein (EGFP)—one of the most widely applied genetically encoded fluorescent probes—carries the threonine-tyrosine-glycine (TYG) chromophore. EGFP efficiently undergoes green-to-red oxidative photoconversion (“redding”) with electron acceptors. Enhanced yellow fluorescent protein (EYFP), a close EGFP homologue (five amino acid substitutions), has a glycine-tyrosine-glycine (GYG) chromophore and is much less susceptible to redding, requiring halide ions in addition to the oxidants. In this contribution we aim to clarify the role of the first chromophore-forming amino acid in photoinduced behavior of these fluorescent proteins. To that end, we compared photobleaching and redding kinetics of EGFP, EYFP, and their mutants with reciprocally substituted chromophore residues, EGFP-T65G and EYFP-G65T. Measurements showed that T65G mutation significantly increases EGFP photostability and inhibits its excited-state oxidation efficiency. Remarkably, while EYFP-G65T demonstrated highly increased spectral sensitivity to chloride, it is also able to undergo redding chloride-independently. Atomistic calculations reveal that the GYG chromophore has an increased flexibility, which facilitates radiationless relaxation leading to the reduced fluorescence quantum yield in the T65G mutant. The GYG chromophore also has larger oscillator strength as compared to TYG, which leads to a shorter radiative lifetime (i.e., a faster rate of fluorescence). The faster fluorescence rate partially compensates for the loss of quantum efficiency due to radiationless relaxation. The shorter excited-state lifetime of the GYG chromophore is responsible for its increased photostability and resistance to redding. In EYFP and EYFP-G65T, the chromophore is stabilized by π-stacking with Tyr203, which suppresses its twisting motions relative to EGFP.

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Nha Trang, Nguyen Thi, Huynh Thi Thu Ha, Nguyen Phuong Thao, Duong Thi Anh Tho, Cao Thi Trang, Le Thi Ha Thanh, Nguyen Hoang Tue, Nguyen Hoang Loc, and Nguyen Ngoc Luong. "Expression of a synthetic gene encoding the enhanced green fluorescent protein in various Escherichia coli strains." Vietnam Journal of Biotechnology 20, no. 2 (June 30, 2022): 359–68. http://dx.doi.org/10.15625/1811-4989/16344.

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Enhanced Green Fluorescent Protein (eGFP) shows much stronger fluorescence than its ancestor, Green Fluorescent Protein (GFP), thus has been widely applied as a reporter for biomedical research. In this study, we reported the expression of a synthetic codon optimized gene encoding eGFP in Escherichia coli (E. coli). The gene was cloned into two expression vectors, pQE30 and pColdII and the resulting recombinant vectors were transformed into E. coli M15 and BL21 De3 RIL codon plus strains, respectively. The expression levels of functional eGFP showed a temperature dependent pattern, in which lowering the induction temperature increased the amount of functional eGFP. Surprisingly, eGFP showed a phenomenon called auto-induction when E. coli TOP10 cells carrying recombinant pQE30 and pColdII were grown on Luria Broth plates. The recombinant eGFP showed robust stability even at room temperature, thus greatly facilitated its purification and handling. Mouse polyclonal antibodies were conveniently generated against the protein. Besides its potential application as a reporter gene in E. coli, the gene and its expression systems reported here are extremely useful as models for teaching recombinant DNA technology at undergraduate level.

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Jin, Xin, Xin Liu, Xiaohua Zhu, Hao Li, Wang Li, Yan Huang, and Shouzhuo Yao. "A label-free fluorescence assay for thrombin activity analysis based on fluorescent protein and gold nanoparticles." Analytical Methods 8, no. 18 (2016): 3691–97. http://dx.doi.org/10.1039/c6ay00290k.

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A label-free and sensitive fluorescence assay has been developed for probing thrombin activity based on an engineered enhanced green fluorescent protein (EGFP) probe and unmodified gold nanoparticles (AuNPs).

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Guan, Xingang, Chun Li, Dan Wang, Weiqi Sun, and Xiaodong Gai. "A tumor-targeting protein nanoparticle based on Tat peptide and enhanced green fluorescent protein." RSC Advances 6, no. 12 (2016): 9461–64. http://dx.doi.org/10.1039/c5ra27411g.

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Faust, Nicole, Florencio Varas, Louise M. Kelly, Susanne Heck, and Thomas Graf. "Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages." Blood 96, no. 2 (July 15, 2000): 719–26. http://dx.doi.org/10.1182/blood.v96.2.719.

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Abstract Pluripotent hematopoietic stem cells have been studied extensively, but the events that occur during their differentiation remain largely uncharted. To develop a system that allows the differentiation of cultured multipotent progenitors by time-lapse fluorescence microscopy, myelomonocytic cells were labeled with green fluorescent protein (GFP) in vivo. This was achieved by knocking the enhanced GFP (EGFP) gene into the murine lysozyme M (lys) locus and using a targeting vector, which contains a neomycin resistant (neo) gene flanked by LoxP sites and “splinked” ends, to increase the frequency of homologous recombination. Analysis of the blood and bone marrow of thelys-EGFP mice revealed that most myelomonocytic cells, especially mature neutrophil granulocytes, were fluorescence-positive, while cells from other lineages were not. Removal of the neogene through breeding of the mice with the Cre-deleter strain led to an increased fluorescence intensity. Mice with an inactivation of both copies of the lys gene developed normally and were fertile.

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Faust, Nicole, Florencio Varas, Louise M. Kelly, Susanne Heck, and Thomas Graf. "Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages." Blood 96, no. 2 (July 15, 2000): 719–26. http://dx.doi.org/10.1182/blood.v96.2.719.014k29_719_726.

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Pluripotent hematopoietic stem cells have been studied extensively, but the events that occur during their differentiation remain largely uncharted. To develop a system that allows the differentiation of cultured multipotent progenitors by time-lapse fluorescence microscopy, myelomonocytic cells were labeled with green fluorescent protein (GFP) in vivo. This was achieved by knocking the enhanced GFP (EGFP) gene into the murine lysozyme M (lys) locus and using a targeting vector, which contains a neomycin resistant (neo) gene flanked by LoxP sites and “splinked” ends, to increase the frequency of homologous recombination. Analysis of the blood and bone marrow of thelys-EGFP mice revealed that most myelomonocytic cells, especially mature neutrophil granulocytes, were fluorescence-positive, while cells from other lineages were not. Removal of the neogene through breeding of the mice with the Cre-deleter strain led to an increased fluorescence intensity. Mice with an inactivation of both copies of the lys gene developed normally and were fertile.

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Krasowska, Joanna, Katarzyna Pierzchała, Agnieszka Bzowska, László Forró, Andrzej Sienkiewicz, and Beata Wielgus-Kutrowska. "Chromophore of an Enhanced Green Fluorescent Protein Can Play a Photoprotective Role Due to Photobleaching." International Journal of Molecular Sciences 22, no. 16 (August 9, 2021): 8565. http://dx.doi.org/10.3390/ijms22168565.

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Under stress conditions, elevated levels of cellular reactive oxygen species (ROS) may impair crucial cellular structures. To counteract the resulting oxidative damage, living cells are equipped with several defense mechanisms, including photoprotective functions of specific proteins. Here, we discuss the plausible ROS scavenging mechanisms by the enhanced green fluorescent protein, EGFP. To check if this protein could fulfill a photoprotective function, we employed electron spin resonance (ESR) in combination with spin-trapping. Two organic photosensitizers, rose bengal and methylene blue, as well as an inorganic photocatalyst, nano-TiO2, were used to photogenerate ROS. Spin-traps, TMP-OH and DMPO, and a nitroxide radical, TEMPOL, served as molecular targets for ROS. Our results show that EGFP quenches various forms of ROS, including superoxide radicals and singlet oxygen. Compared to the three proteins PNP, papain, and BSA, EGFP revealed high ROS quenching ability, which suggests its photoprotective role in living systems. Damage to the EGFP chromophore was also observed under strong photo-oxidative conditions. This study contributes to the discussion on the protective function of fluorescent proteins homologous to the green fluorescent protein (GFP). It also draws attention to the possible interactions of GFP-like proteins with ROS in systems where such proteins are used as biological markers.

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ILK, Nicola, Seta KÜPCÜ, Gerald MONCAYO, Sigrid KLIMT, Rupert C. ECKER, Renate HOFER-WARBINEK, Eva M. EGELSEER, Uwe B. SLEYTR, and Margit SÁRA. "A functional chimaeric S-layer-enhanced green fluorescent protein to follow the uptake of S-layer-coated liposomes into eukaryotic cells." Biochemical Journal 379, no. 2 (April 15, 2004): 441–48. http://dx.doi.org/10.1042/bj20031900.

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The chimaeric gene encoding a C-terminally truncated form of the S-layer protein SbpA of Bacillus sphaericus CCM 2177 and the EGFP (enhanced green fluorescent protein) was ligated into plasmid pET28a and cloned and expressed in Escherichia coli. Just 1 h after induction of expression an intense EGFP fluorescence was detected in the cytoplasm of the host cells. Expression at 28 °C instead of 37 °C resulted in clearly increased fluorescence intensity, indicating that the folding process of the EGFP moiety was temperature sensitive. To maintain the EGFP fluorescence, isolation of the fusion protein from the host cells had to be performed in the presence of reducing agents. SDS/PAGE analysis, immunoblotting and N-terminal sequencing of the isolated and purified fusion protein confirmed the presence of both the S-layer protein and the EGFP moiety. The fusion protein had maintained the ability to self-assemble in suspension and to recrystallize on peptidoglycan-containing sacculi or on positively charged liposomes, as well as to fluoresce. Comparison of fluorescence excitation and emission spectra of recombinant EGFP and rSbpA31-1068/EGFP revealed identical maxima at 488 and 507 nm respectively. The uptake of liposomes coated with a fluorescent monomolecular protein lattice of rSbpA31-1068/EGFP into HeLa cells was studied by confocal laser-scanning microscopy. The major part of the liposomes was internalized within 2 h of incubation and entered the HeLa cells by endocytosis.

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Heck, Susanne, Olga Ermakova, Hiromi Iwasaki, Koichi Akashi, Chiao-Wang Sun, Thomas M. Ryan, Tim Townes, and Thomas Graf. "Distinguishable live erythroid and myeloid cells in β-globin ECFP x lysozyme EGFP mice." Blood 101, no. 3 (February 1, 2003): 903–6. http://dx.doi.org/10.1182/blood-2002-06-1861.

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Abstract We previously described a mouse line that contains green myelomonocytic cells due to the knock-in of enhanced green fluorescence protein (EGFP) into the lysozyme M gene.1 We have now created a transgenic line with fluorescent erythroid cells using a β-globin locus control region driving the enhanced cyan fluorescence protein (ECFP) gene. These mice exhibit cyan fluorescent cells specifically in the erythroid compartment and in megakaryocyte-erythroid progenitors. Crossing the animals with lysozyme EGFP mice yielded a line in which live erythroid and myeloid cells can readily be distinguished by fluorescence microscopy and by fluorescence-activated cell-sorter scanner. This cross allowed unambiguous identification of unstained mixed erythroid-myeloid colonies for the first time. The new mouse lines should become useful tools to dissect the branching between erythroid and myelomonocytic cells during in vitro differentiation of definitive multipotent progenitors.

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Rodríguez-Mejía, José-Luis, Abigail Roldán-Salgado, Joel Osuna, Enrique Merino, and Paul Gaytán. "A Codon Deletion at the Beginning of Green Fluorescent Protein Genes Enhances Protein Expression." Journal of Molecular Microbiology and Biotechnology 27, no. 1 (November 8, 2016): 1–10. http://dx.doi.org/10.1159/000448786.

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Recombinant protein expression is one of the key issues in protein engineering and biotechnology. Among the different models for assessing protein production and structure-function studies, green fluorescent protein (GFP) is one of the preferred models because of its importance as a reporter in cellular and molecular studies. In this research we analyze the effect of codon deletions near the amino terminus of different GFP proteins on fluorescence. Our study includes Gly4 deletions in the enhanced GFP (EGFP), the red-shifted GFP and the red-shifted EGFP. The Gly4 deletion mutants and their corresponding wild-type counterparts were transcribed under the control of the T7 or Trc promoters and their expression patterns were analyzed. Different fluorescent outcomes were observed depending on the type of fluorescent gene versions. In silico analysis of the RNA secondary structures near the ribosome binding site revealed a direct relationship between their minimum free energy and GFP production. Integrative analysis of these results, including SDS-PAGE analysis, led us to conclude that the fluorescence improvement of cells expressing different versions of GFPs with Gly4 deleted is due to an enhancement of the accessibility of the ribosome binding site by reducing the stability of the RNA secondary structures at their mRNA leader regions.

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Kredel, Simone, Michael Wolff, Jörg Wiedenmann, Barbara Moepps, G. Ulrich Nienhaus, Peter Gierschik, Barbara Kistler, and Ralf Heilker. "CXCR2 Inverse Agonism Detected by Arrestin Redistribution." Journal of Biomolecular Screening 14, no. 9 (September 22, 2009): 1076–91. http://dx.doi.org/10.1177/1087057109344616.

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To study CXCR2 modulated arrestin redistribution, the authors employed arrestin as a fusion protein containing either the Aequorea victoria—derived enhanced green fluorescent protein (EGFP) or a recently developed mutant of eqFP611, a red fluorescent protein derived from Entacmaea quadricolor. This mutant, referred to as RFP611, had earlier been found to assume a dimeric quarternary structure. It was therefore employed in this work as a “tandem” (td) construct for pseudo monomeric fusion protein labeling. Both arrestin fusion proteins, containing either td RFP611 (Arr td RFP611) or enhanced green fluorescent protein (EGFP; Arr EGFP), were found to colocalize with internalized fluorescently labeled Gro α a few minutes after Gro α addition. Intriguingly, however, Arr td RFP611 and Arr EGFP displayed distinct cellular distribution patterns in the absence of any CXCR2 activating ligand. Under these conditions, Arr td RFP611 showed a largely homoge neous cytosolic distribution, whereas Arr EGFP segregated, to a large degree, into granular spots. These observations indi cate a higher sensitivity of Arr EGFP to the constitutive activity of CXCR2 and, accordingly, an increased arrestin redistribution to coated pits and endocytic vesicles. In support of this interpretation, the authors found the known CXCR2 antagonist Sch527123 to act as an inverse agonist with respect to Arr EGFP redistribution. The inverse agonistic properties of Sch527123 were confirmed in vitro in a guanine nucleotide binding assay, revealing an IC50 value similar to that observed for Arr EGFP redistribution. Thus, the redistribution assay, when based on Arr EGFP, enables the profiling of antagonistic test compounds with respect to inverse agonism. When based on Arr td RFP611, the assay may be employed to study CXCR2 agonism or neutral antagonism. ( Journal of Biomolecular Screening 2009:1076 1091)

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Doi, Kentaro, Jian Kong, Janos Hargitai, Stephen P. Goff, and Peter Gouras. "Transient Immunosuppression Stops Rejection of Virus-Transduced Enhanced Green Fluorescent Protein in Rabbit Retina." Journal of Virology 78, no. 20 (October 15, 2004): 11327–33. http://dx.doi.org/10.1128/jvi.78.20.11327-11333.2004.

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ABSTRACT The expression of lentivirus-transduced enhanced green fluorescent protein (EGFP) was detectable in rabbit retinal pigment epithelium (RPE) within 3 to 5 days after subretinal injection of the vector. Within 2 to 3 weeks, EGFP-expressing cells were eliminated by rejection. In the current experiments, we monitor serum antibody titers for EGFP before and after transduction and determine whether systemic immunosuppression prevents recognition of EGFP by the immune system. While all control rabbits developed antibodies against EFGP and showed signs of rejection, no such evidence was observed with animals which received immunosuppression. One month of systemic immunosuppression permanently prevented rejection of RPE with EGFP expression. Fluorescence has been maintained for more than a year. If a control eye was injected with the same virus after terminating immunosuppression, both eyes showed signs of rejection. The lack of rejection is not due to tolerance but to a failure of the animals to detect the foreign protein. Detection must depend upon a brief window of time after surgery needed to introduce the vector, perhaps related to a concurrent but transient inflammation. This strategy may be useful in managing other types of rejection in the retina.

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Kitts, P. A., X. Li, D. W. Piston, R. Chervenak, and S. R. Kain. "Properties and Applications of EGFP, Enhanced Color Variants of GFP, and Unstable Derivatives of GFP." Microscopy and Microanalysis 4, S2 (July 1998): 1000–1001. http://dx.doi.org/10.1017/s1431927600025125.

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The green fluorescent protein (GFP) has great potential as a tool for biologists because it can be used as an in vivo real time reporter of protein localization and gene expression in a variety of experimental systems. Wild type GFP, however, has several undesirable properties including low brightness, a significant lag in the development of fluorescence, complex photoisomerization, inefficient protein folding at 37°C, and poor expression in several species. To improve upon these qualities, we have combined an ultra-bright variant of GFP, GFPmutl, with a synthetic gene sequence containing codons preferentially found in highly expressed human proteins. The combination of improved fluorescence intensity and higher expression levels yields an enhanced variant (EGFP) (Table 1) that greatly increases the sensitivity of this reporter.The natural green emission of GFP can conveniently be monitored by optics designed to detect fluorescein. There are, however, many potential applications for GFP that require additional emission colors.

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PRACHAIYO, P., and L. A. McLANDSBOROUGH. "A Microscopic Method to Visualize Escherichia coli Interaction with Beef Muscle." Journal of Food Protection 63, no. 4 (April 1, 2000): 427–33. http://dx.doi.org/10.4315/0362-028x-63.4.427.

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The genetic determinant for enhanced green fluorescent protein (EGFP) was introduced into Escherichia coli JM109 (ATCC 53323) and E. coli O157:H7 (ATCC 43895) on plasmid EGFP. The expression of EGFP did not change the growth kinetics or surface properties tested (hydrophobicity and electrophoretic mobility). Microscope slides were modified to allow for optimal viewing of thick meat samples with an inverted microscope. Two fluorescent dyes, nile red and Cy3 were used to stain for lipid and protein portions of beef muscle, respectively. Laser scanning confocal microscopy was used to observe interaction of the EGFP-expressing E. coli strains and the fluorescently stained muscle components without changing the spatial and temporal environment of the organisms.

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Siggins, Sarah, Christian Ehnholm, Matti Jauhiainen, and Vesa M. Olkkonen. "Plasma phospholipid transfer protein fused with green fluorescent protein is secreted by HepG2 cells and displays phosphatidylcholine transfer activity." Biochemistry and Cell Biology 84, no. 2 (April 1, 2006): 117–25. http://dx.doi.org/10.1139/o05-168.

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Phospholipid transfer protein (PLTP) is a serum glycoprotein with a central role in high-density lipoprotein metabolism. We created a fusion protein in which enhanced green fluorescent protein (EGFP) was fused to the carboxyl-terminus of PLTP. Stably transfected HepG2 cells, which overexpress this fusion protein, were generated. PLTP–EGFP was translocated into the ER and fluoresced within the biosynthetic pathway, showing a marked concentration in the Golgi complex. The transfected cells secreted into the growth medium phospholipid transfer activity 7-fold higher than that of the mock-transfected controls. The medium of the PLTP–EGFP - expressing cells displayed EGFP fluorescence, demonstrating that both the PLTP and the EGFP moieties had attained a biologically active conformation. However, the specific activity of PLTP–EGFP in the medium was markedly reduced as compared with that of endogenous PLTP. This suggests that the EGFP attached to the carboxyl-terminal tail of PLTP interferes with the interaction of PLTP with its substrates or with the lipid transfer process itself. Fluorescently tagged PLTP is a useful tool for elucidating the intracellular functions of PLTP and the interaction of exogenously added PLTP with cells, and will provide a means of monitoring the distribution of exogenously added PLTP between serum lipoprotein subspecies.Key words: GFP, fusion protein, Golgi apparatus, phospholipid transfer activity, protein secretion.

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Finke, Stefan, Krzysztof Brzózka, and Karl-Klaus Conzelmann. "Tracking Fluorescence-Labeled Rabies Virus: Enhanced Green Fluorescent Protein-Tagged Phosphoprotein P Supports Virus Gene Expression and Formation of Infectious Particles." Journal of Virology 78, no. 22 (November 15, 2004): 12333–43. http://dx.doi.org/10.1128/jvi.78.22.12333-12343.2004.

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ABSTRACT Rhabdoviruses such as rabies virus (RV) encode only five multifunctional proteins accomplishing viral gene expression and virus formation. The viral phosphoprotein, P, is a structural component of the viral ribonucleoprotein (RNP) complex and an essential cofactor for the viral RNA-dependent RNA polymerase. We show here that RV P fused to enhanced green fluorescent protein (eGFP) can substitute for P throughout the viral life cycle, allowing fluorescence labeling and tracking of RV RNPs under live cell conditions. To first assess the functions of P fusion constructs, a recombinant RV lacking the P gene, SAD ΔP, was complemented in cell lines constitutively expressing eGFP-P or P-eGFP fusion proteins. P-eGFP supported the rapid accumulation of viral mRNAs but led to low infectious-virus titers, suggesting impairment of virus formation. In contrast, complementation with eGFP-P resulted in slower accumulation of mRNAs but similar infectious titers, suggesting interference with polymerase activity rather than with virus formation. Fluorescence microscopy allowed the detection of eGFP-P-labeled extracellular virus particles and tracking of cell binding and temperature-dependent internalization into intracellular vesicles. Recombinant RVs expressing eGFP-P or an eGFP-P mutant lacking the binding site for dynein light chain 1 (DLC1) instead of P were used to track interaction with cellular proteins. In cells expressing a DsRed-labeled DLC1, colocalization of DLC1 with eGFP-P but not with the mutant P was observed. Fluorescent labeling of RV RNPs will allow further dissection of virus entry, replication, and egress under live-cell conditions as well as cell interactions.

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Bochenkov, Vladimir E., Ekaterina M. Lobanova, Aleksander M. Shakhov, Artyom A. Astafiev, Alexey M. Bogdanov, Vadim A. Timoshenko, and Anastasia V. Bochenkova. "Plasmon-Enhanced Fluorescence of EGFP on Short-Range Ordered Ag Nanohole Arrays." Nanomaterials 10, no. 12 (December 20, 2020): 2563. http://dx.doi.org/10.3390/nano10122563.

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Fluorescence of organic molecules can be enhanced by plasmonic nanostructures through coupling to their locally amplified electromagnetic field, resulting in higher brightness and better photostability of fluorophores, which is particularly important for bioimaging applications involving fluorescent proteins as genetically encoded biomarkers. Here, we show that a hybrid bionanosystem comprised of a monolayer of Enhanced Green Fluorescent Protein (EGFP) covalently linked to optically thin Ag films with short-range ordered nanohole arrays can exhibit up to 6-fold increased brightness. The largest enhancement factor is observed for nanohole arrays with a propagating surface plasmon mode, tuned to overlap with both excitation and emission of EGFP. The fluorescence lifetime measurements in combination with FDTD simulations provide in-depth insight into the origin of the fluorescence enhancement, showing that the effect is due to the local amplification of the optical field near the edges of the nanoholes. Our results pave the way to improving the photophysical properties of hybrid bionanosystems based on fluorescent proteins at the interface with easily fabricated and tunable plasmonic nanostructures.

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Takenaka-Uema, Akiko, Shin Murakami, Nanako Ushio, Tomoya Kobayashi-Kitamura, Masashi Uema, Kazuyuki Uchida, and Taisuke Horimoto. "Generation of a GFP Reporter Akabane Virus with Enhanced Fluorescence Intensity by Modification of Artificial Ambisense S Genome." Viruses 11, no. 7 (July 10, 2019): 634. http://dx.doi.org/10.3390/v11070634.

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We previously generated a recombinant reporter Akabane virus expressing enhanced green fluorescence protein (eGFP-AKAV), with an artificial S genome encoding eGFP in the ambisense RNA. Although the eGFP-AKAV was able to detect infected cells in in vivo histopathological study, its fluorescent signal was too weak to apply to in vivo imaging study. Here, we successfully generated a modified reporter, eGFP/38-AKAV, with 38-nucleotide deletion of the internal region of the 5′ untranslated region of S RNA. The eGFP/38-AKAV expressed higher intensity of eGFP fluorescence both in vitro and in vivo than the original eGFP-AKAV did. In addition, eGFP/38-AKAV was pathogenic in mice at a comparable level to that in wild-type AKAV. In the mice infected with eGFP/38-AKAV, the fluorescent signals, i.e., the virus-infected cells, were detected in the central nervous system using the whole-organ imaging. Our findings indicate that eGFP/38-AKAV could be used as a powerful tool to help elucidate the dynamics of AKAV in vivo.

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JAIN, Renu K., Paul B. M. JOYCE, Miguel MOLINETE, Philippe A. HALBAN, and Sven-Ulrik GORR. "Oligomerization of green fluorescent protein in the secretory pathway of endocrine cells." Biochemical Journal 360, no. 3 (December 10, 2001): 645–49. http://dx.doi.org/10.1042/bj3600645.

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Green fluorescent protein (GFP) is used extensively as a reporter protein to monitor cellular processes, including intracellular protein trafficking and secretion. In general, this approach depends on GFP acting as a passive reporter protein. However, it was recently noted that GFP oligomerizes in the secretory pathway of endocrine cells. To characterize this oligomerization and its potential role in GFP transport, cytosolic and secretory forms of enhanced GFP (EGFP) were expressed in GH4C1 and AtT-20 endocrine cells. Biochemical analysis showed that cytosolic EGFP existed as a 27kDa monomer, whereas secretory forms of EGFP formed disulphide-linked oligomers. EGFP contains two cysteine residues (Cys49 and Cys71), which could play a role in this oligomerization. Site-directed mutagenesis of Cys49 and Cys71 showed that both cysteine residues were involved in disulphide interactions. Substitution of either cysteine residue resulted in a reduction or loss of oligomers, although dimers of the secretory form of EGFP remained. Mutation of these residues did not adversely affect the fluorescence of EGFP. EGFP oligomers were stored in secretory granules and secreted by the regulated secretory pathway in endocrine AtT-20 cells. Similarly, the dimeric mutant forms of EGFP were still secreted via the regulated secretory pathway, indicating that the higher-order oligomers were not necessary for sorting in AtT-20 cells. These results suggest that the oligomerization of EGFP must be considered when the protein is used as a reporter molecule in the secretory pathway.

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Roper, Jason M., Rhonda J. Staversky, Jacob N. Finkelstein, Peter C. Keng, and Michael A. O'Reilly. "Identification and isolation of mouse type II cells on the basis of intrinsic expression of enhanced green fluorescent protein." American Journal of Physiology-Lung Cellular and Molecular Physiology 285, no. 3 (September 2003): L691—L700. http://dx.doi.org/10.1152/ajplung.00034.2003.

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The unique morphology and cell-specific expression of surfactant genes have been used to identify and isolate alveolar type II epithelial cells. Because these attributes can change during lung injury, a novel method was developed for detecting and isolating mouse type II cells on the basis of transgenic expression of enhanced green fluorescence protein (EGFP). A line of transgenic mice was created in which EGFP was targeted to type II cells under control of the human surfactant protein (SP)-C promoter. Green fluorescent cells that colocalized by immunostaining with endogenous pro-SP-C were scattered throughout the parenchyma. EGFP was not detected in Clara cell secretory protein-expressing airway epithelial cells or other nonlung tissues. Pro-SP-C immunostaining diminished in lungs exposed to hyperoxia, consistent with decreased expression and secretion of intracellular precursor protein. In contrast, type II cells could still be identified by their intrinsic green fluorescence, because EGFP is not secreted. Type II cells could also be purified from single-cell suspensions of lung homogenates using fluorescence-activated cell sorting. Less than 1% of presorted cells exhibited green fluorescence compared with >95% of the sorted population. As expected for type II cells, ultrastructural analysis revealed that the sorted cells contained numerous lamellar bodies. SP-A, SP-B, and SP-C mRNAs were detected in the sorted population, but T1α and CD31 (platelet endothelial cell adhesion molecule) were not, indicating enrichment of type II epithelial cells. This method will be invaluable for detecting and isolating mouse type II cells under a variety of experimental conditions.

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Muhamadali, Howbeer, Yun Xu, Rosa Morra, Drupad K. Trivedi, Nicholas J. W. Rattray, Neil Dixon, and Royston Goodacre. "Metabolomic analysis of riboswitch containing E. coli recombinant expression system." Molecular BioSystems 12, no. 2 (2016): 350–61. http://dx.doi.org/10.1039/c5mb00624d.

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Dewi, Raden Roro Sri Pudji Sinarni, Alimuddin Alimuddin, Agus Oman Sudrajat, Komar Sumantadinata, and Erma Primanita Hayuningtyas. "POLA EKSPRESI GEN ENHANCED GREEN FLUORESCENT PROTEIN PADA EMBRIO DAN LARVA IKAN PATIN SIAM (Pangasianodon hypophthalmus)." Jurnal Riset Akuakultur 8, no. 3 (April 6, 2016): 339. http://dx.doi.org/10.15578/jra.8.3.2013.339-346.

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<p>Penelitian ekspresi sementara (transient expression) dari transgen secara in vivo<br />menggunakan gen reporter berguna untuk mendesain konstruksi gen yang akan digunakan pada penelitian transgenesis. Gen reporter yang umum digunakan dalam penelitian ekspresi sementara transgen adalah gen GFP (green fluorescent protein). Pengamatan gen EGFP (enhanced green fluorescent protein) pada embrio dan larva ikan patin siam (Pangasianodon hypophthalmus) ditujukan untuk mendapatkan informasi mengenai kemampuan promoter -aktin ikan mas dalam mengendalikan ekspresi gen EGFP. Gen EGFP diintroduksikan ke dalam sperma ikan patin siam menggunakan metode elektroporasi. Sperma yang telah dielektroporasi digunakan untuk membuahi sel telur ikan patin siam. Pengamatan ekspresi gen EGFP dilakukan setiap enam jam dimulai dari embrio fase 2 sel sampai larva. Berdasarkan hasil penelitian, gen EGFP terekspresi pada fase embrio dan larva ikan patin siam. Puncak ekspresi gen EGFP terjadi pada fase neurula dan menurun pada fase larva. Berdasarkan penelitian ini maka ikan patin siam transgenik telah berhasil dibentuk dan promoter -aktin ikan mas terbukti aktif dalam mengarahkan ekspresi gen asing (GFP) di dalam tubuh ikan patin siam.</p>

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Velaz-Faircloth, Maria, Alison J. Cobb, Amanda L. Horstman, Stanley C. Henry, and Richard Frothingham. "Protection against Mycobacterium aviumby DNA Vaccines Expressing Mycobacterial Antigens as Fusion Proteins with Green Fluorescent Protein." Infection and Immunity 67, no. 8 (August 1, 1999): 4243–50. http://dx.doi.org/10.1128/iai.67.8.4243-4250.1999.

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ABSTRACT Mycobacterium avium causes disseminated disease in humans with AIDS, paratuberculosis in ruminants, lymphadenopathy in swine, and tuberculosis in birds. We constructed DNA vaccines expressing mycobacterial antigens as fusion proteins with enhanced green fluorescent protein (EGFP). Plasmids p65K-EGFP, p85A-EGFP, and p85B-EGFP expressed the M. avium 65-kDa antigen, theMycobacterium bovis BCG 85A antigen, and the M. avium 85B antigen, respectively, as EGFP fusion proteins. We visualized protein expression directly in cultured murine fibroblasts and intact muscle. p65K-EGFP expressed fusion protein in a diffuse cytoplasmic pattern, and p85A-EGFP and p85B-EGFP produced a speckled pattern. We vaccinated C57BL/6 mice with three doses of plasmid DNA and then challenged them intraperitoneally with M. avium. Negative controls received saline, and positive controls received one dose of BCG vaccine. Mice in all groups developed disseminated infection with a high burden of organisms. Compared to negative controls, mice vaccinated with p85A-EGFP had an eightfold reduction in spleen M. avium CFU at 4 weeks after infection and a fourfold reduction at 8 weeks, reductions similar to those generated by BCG vaccine. Mice vaccinated with p65K-EGFP had a fourfold CFU reduction at 4 weeks and no effect at 8 weeks. This is the first report of DNA vaccines expressing foreign antigens as fusion proteins with EGFP and the first report of successful DNA vaccination againstM. avium.

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Tsuboi, Setsuko, and Takashi Jin. "BRET based dual-colour (visible/near-infrared) molecular imaging using a quantum dot/EGFP–luciferase conjugate." RSC Advances 9, no. 60 (2019): 34964–71. http://dx.doi.org/10.1039/c9ra07011g.

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A bioluminescent dual-colour molecular-imaging probe was prepared to emit green and near-infrared luminescence from a conjugate between enhanced green fluorescent protein (EGFP), Renilla luciferase (RLuc) and CdSeTe/CdS quantum dot (QD).

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Mishra, Anurag, Sambandam Ravikumar, Young Ho Song, Nadarajan Saravanan Prabhu, Hyunuk Kim, Soon Ho Hong, Seyeon Cheon, Jaegeun Noh, and Ki-Whan Chi. "A new arene–Ru based supramolecular coordination complex for efficient binding and selective sensing of green fluorescent protein." Dalton Trans. 43, no. 16 (2014): 6032–40. http://dx.doi.org/10.1039/c3dt53186d.

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Bierhuizen, Marti F. A., Yvonne Westerman, Trudi P. Visser, Wati Dimjati, Albertus W. Wognum, and Gerard Wagemaker. "Enhanced Green Fluorescent Protein as Selectable Marker of Retroviral-Mediated Gene Transfer in Immature Hematopoietic Bone Marrow Cells." Blood 90, no. 9 (November 1, 1997): 3304–15. http://dx.doi.org/10.1182/blood.v90.9.3304.

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Abstract The further improvement of gene transfer into hematopoietic stem cells and their direct progeny will be greatly facilitated by markers that allow rapid detection and efficient selection of successfully transduced cells. For this purpose, a retroviral vector was designed and tested encoding a recombinant version of the Aequorea victoria green fluorescent protein that is enhanced for high-level expression in mammalian cells (EGFP). Murine cell lines (NIH 3T3, Rat2) and bone marrow cells transduced with this retroviral vector demonstrated a stable green fluorescence signal readily detectable by flow cytometry. Functional analysis of the retrovirally transduced bone marrow cells showed EGFP expression in in vitro clonogenic progenitors (GM-CFU), day 13 colony-forming unit-spleen (CFU-S), and in peripheral blood cells and marrow repopulating cells of transplanted mice. In conjunction with fluorescence-activated cell sorting (FACS) techniques EGFP expression could be used as a marker to select for greater than 95% pure populations of transduced cells and to phenotypically define the transduced cells using antibodies directed against specific cell-surface antigens. Detrimental effects of EGFP expression were not observed: fluorescence intensity appeared to be stable and hematopoietic cell growth was not impaired. The data show the feasibility of using EGFP as a convenient and rapid reporter to monitor retroviral-mediated gene transfer and expression in hematopoietic cells, to select for the genetically modified cells, and to track these cells and their progeny both in vitro and in vivo.

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Zhang, Ping, Yuehua Hu, Ruili Ma, Ling Li, and Jun Lu. "Enhanced green fluorescence protein/layered double hydroxide composite ultrathin films: bio-hybrid assembly and potential application as a fluorescent biosensor." Journal of Materials Chemistry B 5, no. 1 (2017): 160–66. http://dx.doi.org/10.1039/c6tb02638a.

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This article reports the fabrication and application of enhanced green fluorescent protein/layered double hydroxide nanosheet (EGFP/LDH)_n ultrathin films via layer-by-layer assembly technique based on electrostatic and hydrogen-bond interactions.

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Tallini, Yvonne N., Bo Shui, Kai Su Greene, Ke-Yu Deng, Robert Doran, Patricia J. Fisher, Warren Zipfel, and Michael I. Kotlikoff. "BAC transgenic mice express enhanced green fluorescent protein in central and peripheral cholinergic neurons." Physiological Genomics 27, no. 3 (December 2006): 391–97. http://dx.doi.org/10.1152/physiolgenomics.00092.2006.

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The peripheral nervous system has complex and intricate ramifications throughout many target organ systems. To date this system has not been effectively labeled by genetic markers, due largely to inadequate transcriptional specification by minimum promoter constructs. Here we describe transgenic mice in which enhanced green fluorescent protein (eGFP) is expressed under the control of endogenous choline acetyltransferase (ChAT) transcriptional regulatory elements, by knock-in of eGFP within a bacterial artificial chromosome (BAC) spanning the ChAT locus and expression of this construct as a transgene. eGFP is expressed in ChATBAC-eGFP mice in central and peripheral cholinergic neurons, including cell bodies and processes of the somatic motor, somatic sensory, and parasympathetic nervous system in gastrointestinal, respiratory, urogenital, cardiovascular, and other peripheral organ systems. Individual epithelial cells and a subset of lymphocytes within the gastrointestinal and airway mucosa are also labeled, indicating genetic evidence of acetylcholine biosynthesis. Central and peripheral neurons were observed as early as 10.5 days postcoitus in the developing mouse embryo. ChATBAC-eGFP mice allow excellent visualization of all cholinergic elements of the peripheral nervous system, including the submucosal enteric plexus, preganglionic autonomic nerves, and skeletal, cardiac, and smooth muscle neuromuscular junctions. These mice should be useful for in vivo studies of cholinergic neurotransmission and neuromuscular coupling. Moreover, this genetic strategy allows the selective expression and conditional inactivation of genes of interest in cholinergic nerves of the central nervous system and peripheral nervous system.

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Wang, Gang, Yan Fei Tan, Xing Dong Zhang, and Y. Z. Zhang. "Development of EGFP-Expressing Cell Line for Biocompatibility Evaluation of Osteoinductive CaP Ceramics." Key Engineering Materials 330-332 (February 2007): 1079–82. http://dx.doi.org/10.4028/www.scientific.net/kem.330-332.1079.

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This study investigated the utility of genetically modified cell line for fast and non-destructive cytotoxicity evaluation of biomaterials. The EGFP(enhanced green fluorescence protein)-expressing plasmid pcDNA-EGFP was constructed, and electroporated into ROS 17/28 osteoblastic cells to generate an EGFP-labeled stable cell line, ROS-EGFP. This genetically modified cell line provided two unique opportunities to qualitative and semi-quantitative evaluation of cell growth on biomaterials without destruction of samples. Firstly, utilizing the fluorescence of EGFP expressed in the cells, the viability state of cells on biomaterials was evaluated using a fluorescent light microscope. Secondly, the proliferation of cells on biomaterials, which was identified by MTT assay，was demonstrated according to the microscopically counted fluorescent cell numbers. From the results, it could be concluded that the ROS-EGFP cell line was an effective tool to trace the fate of cells on biomaterials and to evaluate the biocompatibility of biomaterials to cell growth in vitro.

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Jeshtadi, Ananya, Pierre Burgos, Christopher D. Stubbs, Anthony W. Parker, Linda A. King, Michael A. Skinner, and Stanley W. Botchway. "Interaction of Poxvirus Intracellular Mature Virion Proteins with the TPR Domain of Kinesin Light Chain in Live Infected Cells Revealed by Two-Photon-Induced Fluorescence Resonance Energy Transfer Fluorescence Lifetime Imaging Microscopy." Journal of Virology 84, no. 24 (October 13, 2010): 12886–94. http://dx.doi.org/10.1128/jvi.01395-10.

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ABSTRACT Using two-photon-induced fluorescence lifetime imaging microscopy, we corroborate an interaction (previously demonstrated by yeast two-hybrid domain analysis) of full-length vaccinia virus (VACV; an orthopoxvirus) A36 protein with the cellular microtubule motor protein kinesin. Quenching of enhanced green fluorescent protein (EGFP), fused to the C terminus of VACV A36, by monomeric red fluorescent protein (mDsRed), fused to the tetratricopeptide repeat (TPR) domain of kinesin, was observed in live chicken embryo fibroblasts infected with either modified vaccinia virus Ankara (MVA) or wild-type fowlpox virus (FWPV; an avipoxvirus), and the excited-state fluorescence lifetime of EGFP was reduced from 2.5 ± 0.1 ns to 2.1 ± 0.1 ns due to resonance energy transfer to mDsRed. FWPV does not encode an equivalent of intracellular enveloped virion surface protein A36, yet it is likely that this virus too must interact with kinesin to facilitate intracellular virion transport. To investigate possible interactions between innate FWPV proteins and kinesin, recombinant FWPVs expressing EGFP fused to the N termini of FWPV structural proteins Fpv140, Fpv168, Fpv191, and Fpv198 (equivalent to VACV H3, A4, p4c, and A34, respectively) were generated. EGFP fusions of intracellular mature virion (IMV) surface protein Fpv140 and type II membrane protein Fpv198 were quenched by mDsRed-TPR in recombinant FWPV-infected cells, indicating that these virion proteins are found within 10 nm of mDsRed-TPR. In contrast, and as expected, EGFP fusions of the IMV core protein Fpv168 did not show any quenching. Interestingly, the p4c-like protein Fpv191, which demonstrates late association with preassembled IMV, also did not show any quenching.

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FURLONG, Robert A., Yolanda NARAIN, Julia RANKIN, Andreas WYTTENBACH, and David C. RUBINSZTEIN. "α-Synuclein overexpression promotes aggregation of mutant huntingtin." Biochemical Journal 346, no. 3 (March 7, 2000): 577–81. http://dx.doi.org/10.1042/bj3460577.

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Protein aggregates are a neuropathological feature of Huntington's disease and Parkinson's disease. Mutant huntingtin exon 1 with 72 CAG repeats fused to enhanced green fluorescent protein (EGFP) forms hyperfluorescent inclusions in PC12 cells. Inclusion formation is enhanced in cells co-transfected with EGFP-huntingtin-(CAG)72 and α-synuclein, a major component of Parkinson's disease aggregates. However, α-synuclein does not form aggregates by itself, nor does it appear in huntingtin inclusions in vitro.

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46

Eggenstein, Evelyn, Antonia Richter, and Arne Skerra. "FluoroCalins: engineered lipocalins with novel binding functions fused to a fluorescent protein for applications in biomolecular imaging and detection." Protein Engineering, Design and Selection 32, no. 6 (June 2019): 289–96. http://dx.doi.org/10.1093/protein/gzz047.

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Abstract FluoroCalins represent novel bifunctional protein reagents derived from engineered lipocalins fused to a fluorescent reporter protein, here the enhanced green fluorescent protein (eGFP). We demonstrate the construction, facile bacterial production and broad applicability of FluoroCalins using two Anticalin® molecules directed against the tumor vasculature-associated extra domain B of fibronectin (ED-B) and the vascular endothelial growth factor receptor 3, a marker of tumor and lymphangiogenesis. FluoroCalins were prepared with two different spacers: (i) a short Ser3Ala linker and (ii) a long hydrophilic and conformationally unstructured PASylation® polypeptide comprising 200 Pro, Ala and Ser residues. These FluoroCalins were applied for direct target quantification in enzyme-linked immunosorbent assay as well as target detection by flow cytometry and fluorescence microscopy of live and fixed cells, respectively, demonstrating high specificity and signal-to-noise ratio. Hence, FluoroCalins offer a promising alternative to antibody-based reagents for state of the art fluorescent in vitro detection and biomolecular imaging.

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Ohneda, Mamoru, Manabu Arioka, and Katsuhiko Kitamoto. "Isolation and Characterization of Aspergillus oryzae Vacuolar Protein Sorting Mutants." Applied and Environmental Microbiology 71, no. 8 (August 2005): 4856–61. http://dx.doi.org/10.1128/aem.71.8.4856-4861.2005.

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ABSTRACT The vacuolar protein sorting (vps) system in the filamentous fungus Aspergillus oryzae, which has unique cell polarity and the ability to secrete large amounts of proteins, was evaluated by using mutants that missort vacuolar proteins into the medium. Vacuolar carboxypeptidase Y (CPY) fused with enhanced green fluorescent protein (EGFP) was used as a vacuolar marker. Twenty dfc (dim EGFP fluorescence in conidia) mutants with reduced intracellular EGFP fluorescence in conidia were isolated by fluorescence-activated cell sorting from approximately 20,000 UV-treated conidia. Similarly, 22 hfm (hyper-EGFP fluorescence released into the medium) mutants with increased extracellular EGFP fluorescence were isolated by using a fluorescence microplate reader from approximately 20,000 UV-treated conidia. The dfc and hfm mutant phenotypes were pH dependent, and missorting of CPY-EGFP could vary by 10- to 40-fold depending on the ambient pH. At pH 5.5, the dfc-14 and hfm-4 mutants had an abnormal hyphal morphology that is consistent with fragmentation of vacuoles and defects in cell polarity. In contrast, the hyphal and vacuolar morphology of the dfc-14 and hfm-4 mutants was normal at pH 8.0, although CPY-EGFP accumulated in perivacuolar dot-like structures similar to the class E compartments in Saccharomyces cerevisiae vps mutants. In hfm-21, CPY-EGFP localized at the Spitzenkörper when the mutant was grown at pH 8.0 but not in vacuoles, suggesting that hfm-21 may transport CPY-EGFP via a novel pathway that involves the Spitzenkörper. Correlations between vacuolar protein sorting, pH response, and cell polarity are reported for the first time for filamentous fungi.

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Xu, Q., D. J. Milner, and M. B. Wheeler. "144 Use of the CRISPR/CAS 9 system to produce porcine adipose-derived stem cells expressing enhanced green fluorescent protein." Reproduction, Fertility and Development 33, no. 2 (2021): 180. http://dx.doi.org/10.1071/rdv33n2ab144.

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The goal of our project is to produce porcine adipose-derived stem cells (ASCs) stably expressing enhanced green fluorescent protein (eGFP) by using the clustered regularly interspaced short palindromic repeats (CRIPSR) technique. Fluorescent stem cells can facilitate the tracing and visualisation of stem cell migration, fusion, and participation in tissue regeneration after stem cell injection therapy, and represent a useful tool for tissue engineering research. The production of stem cells containing eGFP from ASCs using the CRISPR gene editing technique is able to reduce the time and labour requirement necessary for harvesting fluorescent cells from transgenic pigs. To generate fluorescent, edited cells, we utilised the ROSA 26 locus of pigs for insertion of the eGFP gene by homology-directed repair of Cas9-cleaved DNA at the ROSA 26 locus. The critical steps of producing stem cells expressing eGFP are (1) cloning of guide oligos into a Cas9 cutting vector and producing a repair template vector to insert GFP; (2) transfecting porcine stem cells with CRISPR plasmids; (3) cell sorting with flow cytometry to isolate colonies expressing GFP. A Rosa 26 Cas9-gRNA cutting vector was produced by cloning a guide RNA sequence into the vector backbone of plasmid pX458-GFP, and the donor vector was produced by the combination of the eGFP gene flanked with ROSA 26 genomic DNA inserted into plasmid pUC57. To isolate cells edited to contain the eGFP gene inserted into the ROSA-26 locus, we transfected 250,000 cells with a 1:1 mass mixture of Cas9-gRNA and eGFP repair plasmid using Lipofectamine STEM reagent (Invitrogen) in three trials. GFP+ cells were isolated by fluorescence-activated cell sorting, plated in 96-well plates, and monitored for colony growth and GFP expression. These trials produced an average of ∼70 colonies from sorting, and ∼1% GFP+ colonies. As pX458 drives expression of GFP as a marker for transfection, we hypothesised that we would potentially isolate more GFP+ edited colonies if we utilised a Cas9-gRNA cutting vector expressing mCherry and sorted for cells expressing both mCherry and GFP. This would allow enrichment of edited cells expressing GFP early after transfection, without interference of cells expressing GFP from the Cas9-gRNA vector alone. Utilising this method, we again obtained an average of ∼70 colonies from sorting, and 3% GFP+ colonies. Results were subjected to Student’s t-test. The comparisons were colonies/cell sorted and GFP+ colonies/cell sorted. All data were expressed as quadratic means+mean SE. When we compared groups, no differences were found for colonies/cell sorted: P=0.53 (1.11 E-03±9.16E-04 and 5.39 E-04±3.77 E-04, respectively, for green-green or red-green) and for GFP+ colonies/cell sorted: P=0.44 (1.94 E-05±2.15E-05 and 4.59 E-05±2.46 E-05, respectively, for green-green or red-green). In conclusion, our attempts to isolate ASC edited to express GFP have been successful, and our initial results suggest that utilising a dual fluorescent label sorting strategy does not enhance the number of GFP+ ASC colonies isolated. Future studies will verify that our GFP+ ASC retain normal stem cell properties.

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49

Xu, Q., D. J. Milner, and M. B. Wheeler. "144 Use of the CRISPR/CAS 9 system to produce porcine adipose-derived stem cells expressing enhanced green fluorescent protein." Reproduction, Fertility and Development 33, no. 2 (2021): 180. http://dx.doi.org/10.1071/rdv33n2ab144.

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Abstract:

The goal of our project is to produce porcine adipose-derived stem cells (ASCs) stably expressing enhanced green fluorescent protein (eGFP) by using the clustered regularly interspaced short palindromic repeats (CRIPSR) technique. Fluorescent stem cells can facilitate the tracing and visualisation of stem cell migration, fusion, and participation in tissue regeneration after stem cell injection therapy, and represent a useful tool for tissue engineering research. The production of stem cells containing eGFP from ASCs using the CRISPR gene editing technique is able to reduce the time and labour requirement necessary for harvesting fluorescent cells from transgenic pigs. To generate fluorescent, edited cells, we utilised the ROSA 26 locus of pigs for insertion of the eGFP gene by homology-directed repair of Cas9-cleaved DNA at the ROSA 26 locus. The critical steps of producing stem cells expressing eGFP are (1) cloning of guide oligos into a Cas9 cutting vector and producing a repair template vector to insert GFP; (2) transfecting porcine stem cells with CRISPR plasmids; (3) cell sorting with flow cytometry to isolate colonies expressing GFP. A Rosa 26 Cas9-gRNA cutting vector was produced by cloning a guide RNA sequence into the vector backbone of plasmid pX458-GFP, and the donor vector was produced by the combination of the eGFP gene flanked with ROSA 26 genomic DNA inserted into plasmid pUC57. To isolate cells edited to contain the eGFP gene inserted into the ROSA-26 locus, we transfected 250,000 cells with a 1:1 mass mixture of Cas9-gRNA and eGFP repair plasmid using Lipofectamine STEM reagent (Invitrogen) in three trials. GFP+ cells were isolated by fluorescence-activated cell sorting, plated in 96-well plates, and monitored for colony growth and GFP expression. These trials produced an average of ∼70 colonies from sorting, and ∼1% GFP+ colonies. As pX458 drives expression of GFP as a marker for transfection, we hypothesised that we would potentially isolate more GFP+ edited colonies if we utilised a Cas9-gRNA cutting vector expressing mCherry and sorted for cells expressing both mCherry and GFP. This would allow enrichment of edited cells expressing GFP early after transfection, without interference of cells expressing GFP from the Cas9-gRNA vector alone. Utilising this method, we again obtained an average of ∼70 colonies from sorting, and 3% GFP+ colonies. Results were subjected to Student’s t-test. The comparisons were colonies/cell sorted and GFP+ colonies/cell sorted. All data were expressed as quadratic means+mean SE. When we compared groups, no differences were found for colonies/cell sorted: P=0.53 (1.11 E-03±9.16E-04 and 5.39 E-04±3.77 E-04, respectively, for green-green or red-green) and for GFP+ colonies/cell sorted: P=0.44 (1.94 E-05±2.15E-05 and 4.59 E-05±2.46 E-05, respectively, for green-green or red-green). In conclusion, our attempts to isolate ASC edited to express GFP have been successful, and our initial results suggest that utilising a dual fluorescent label sorting strategy does not enhance the number of GFP+ ASC colonies isolated. Future studies will verify that our GFP+ ASC retain normal stem cell properties.

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50

Ueta, Yoichi, Hiroaki Fujihara, Ryota Serino, Govindan Dayanithi, Hitoshi Ozawa, Ken-ichi Matsuda, Mitsuhiro Kawata, et al. "Transgenic Expression of Enhanced Green Fluorescent Protein Enables Direct Visualization for Physiological Studies of Vasopressin Neurons and Isolated Nerve Terminals of the Rat." Endocrinology 146, no. 1 (January 1, 2005): 406–13. http://dx.doi.org/10.1210/en.2004-0830.

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We have generated transgenic rats expressing an arginine vasopressin (AVP)-enhanced green fluorescent protein (eGFP) fusion gene. The expression of the eGFP gene and strong fluorescence were observed in the supraoptic nucleus (SON), the paraventricular nucleus (PVN), and the suprachiasmatic nucleus (SCN) in transgenic rats. The hypothalamo-neurohypophyseal tract, isolated SON neurons, and isolated axon terminals in the neurohypophysis also showed robust eGFP fluorescence. Water deprivation for 2 d increased the fluorescence of the eGFP in the SON and the PVN but not the SCN. The whole-cell patch-clamp technique was then used to record the electrical activities specifically identifying eGFP-expressing SON, PVN, and SCN AVP neurons in in vitro brain slice preparations. The AVP-eGFP transgenic rats are a unique new tool with which to study the physiological role of AVP-secreting neurons in the central nervous system and the dynamics of the regulation of AVP secretion in the living neurons and their axon terminals.

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Journal articles on the topic 'EGFP (enhanced green fluorescent protein)'

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