Improved Fluorescence and Dual Color Detection with Enhanced Blue and Green Variants of the Green Fluorescent Protein

Yang, Te Tuan; Sinai, Parisa; Green, Gisele; Kitts, Paul; Chen, Yih Tai; Lybarger, Lonnie; Chervenak, Robert; Patterson, George H.; Piston, David W.; Kain, Steven R.

doi:10.1074/jbc.273.14.8212

Cited by 139 publications

(84 citation statements)

References 25 publications

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“…pEGFP-C1 and pEGFP-N1 GFP plasmid vectors (#6084-1 and #6085-1, respectively, enhanced GFP (EGFP) plasmid vectors, Clontech Laboratories Inc., Palo Alto, CA) were used. EGFP carries a red-shifted variant of wild-type GFP that contains two amino acid substitutions that has been optimized for brighter green fluorescence and higher expression in mammalian cells (10,11). GLUT1 cDNA (12) was recovered from pHepG2 using Bam H1 restriction digest or PCR.…”

Section: Methodsmentioning

confidence: 99%

Acute Effectors of GLUT1 Glucose Transporter Subcellular Targeting in CIT3 Mouse Mammary Epithelial Cells

2008

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Lactogenic hormones cause intracellular targeting of glucose transporter 1 (GLUT1) for transport of glucose to the site of lactose synthesis in mammary glands. Our aim was to study the intracellular trafficking mechanisms involved in GLUT1 targeting and recycling in CIT 3 mouse mammary epithelial cells. Fusion proteins of GLUT1 and enhanced green fluorescent protein (EGFP) were expressed in CIT 3 cells maintained in growth medium (GM), or exposed to secretion medium (SM), containing prolactin. Agents acting on Golgi and related subcellular compartments and on GLUT1 and GLUT4 targeting in muscle and fat cells were studied. Wortmannin and staurosporine effects on internalization of GLUT1 were not specific, supporting a basal constitutive GLUT1 membranerecycling pathway between an intracellular pool and the cell surface in CIT 3 cells, which targets most GLUT1 to the plasma membrane in GM. Upon exposure to prolactin in SM, GLUT1 was specifically targeted intracellularly to a brefeldin A-sensitive compartment. Arrest of endosomal acidification by bafilomycin A1 disrupted this prolactin-induced GLUT1 intracellular trafficking with central coalescence of GLUT1-EGFP signal, suggesting that it is via endosomal pathways. This machinery offers another level of regulation of lactose synthesis by altering GLUT1 targeting within minutes to hours. (Pediatr Res 63: 56-61, 2008)

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Section: Methodsmentioning

confidence: 99%

Acute Effectors of GLUT1 Glucose Transporter Subcellular Targeting in CIT3 Mouse Mammary Epithelial Cells

2008

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“…Recently, GFP variants such as EGFP (enhanced green fluorescence protein), ECFP (enhanced cyan fluorescence protein) and EYFP (enhanced yellow fluorescence protein) with enhanced fluorescence intensity, stability and removal of all three TTA codons, had been developed (Brendan et al, 1996). These variants exhibit brighter fluorescence and high levels of gene expression in Streptomyces (Beavis and Kalejta, 1999;Yang et al, 1998).…”

Section: Introductionmentioning

confidence: 99%

Spatio-temporal expression of the pathway-specific regulatory gene redD in S. coelicolor

Zhou

Yq²,

Wu³

2005

J Zheijang Univ Sci B

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Confocal laser scanning microscopy was used to observe the spatio-temporal expression of the pathway-specific gene redD during S. coelicolor cell cultivation. The corresponding mutant S. coelicolor lyqRY1522 carrying redD::eyfp in the chromosome was constructed. The temporal expression results of the fusion protein during submerged cultivation demonstrated that expression of redD began in the transition phase, continuing through the exponential growth phase to the stationary phase, and reached maximum in the stationary phase. On the other hand, redD was expressed only in substrate mycelia during solid-state culture, while aerial mycelia remained essentially non-fluorescent throughout culture. Results demonstrated that the expression pattern of redD coincides with that of the biosynthesis of the antibiotics during culture, revealing a direct correlation between the spatio-temporal distribution of regulatory gene expression and second metabolism.

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“…23 Based on spectral analysis, fluorescence of eGFP is 35-fold higher than that of wild-type GFP. 5,24,25 This makes eGFP an attractive marker for identifying adoptively transferred cells in situ. We show in this article that the repopulation of an adoptive host with GFP ϩ cells from eGFP-tg B6 donor mice can be readily followed.…”

Section: Discussionmentioning

confidence: 99%

In Situ Characterization of Genetically Targeted (Green Fluorescent) Single Cells and Their Microenvironment in an Adoptive Host

Leithäuser

Trobonjača

Reimann

et al. 2001

The American Journal of Pathology

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Stable expression of transgene-encoded enhanced green fluorescence protein (eGFP) was used as a sensitive and specific marker to detect in situ donor cells engrafted into different tissues of adoptive hosts. eGFP ؉ lymphoid or myeloid cells (eg, CD4 ؉ T cells or bone marrow-derived dendritic cells) from eGFPtransgenic C57BL/6 donor mice were injected into congenic, immunodeficient RAG1 ؊/؊ C57/BL6 hosts. eGFP ؉ cells were detected in the adoptive host from 2 days to 4 weeks after transfer using an optimized method of fixed cryopreservation to process the tissue. This allowed the simple, sensitive, and specific detection of eGFP ؉ donor cells in histological sections of transplanted hosts. We further demonstrate that this technique can be combined with other estab- have low sensitivity (often caused by high background reactivity of the tissue of interest), and may have limited specificity because of cross-reactivities with host cells that are difficult to control. The availability of monoclonal antibodies (mAbs) suitable for immunohistology in the murine system is limited. Hence, simple and specific techniques to identify in situ engrafted cells and to simultaneously determine their differentiation, activation, and viability status would greatly facilitate many studies in vivo.Green fluorescent protein (GFP) is a bioluminescent protein from the jellyfish Aequorea victoria. 3,4 Mutant variants of this protein have been developed that show enhanced fluorescence. A commonly used variant with enhanced fluorescence is GFPmut1 (designated eGFP in this article). 5 eGFP has been used as a reporter gene in prokaryotic and eukaryotic cells in vitro. A transgenic C57BL/6 mouse line has been constructed that expresses eGFP under the chicken ␤-actin promoter control. 6 -10 These mice have been used to study hemopoietic and lymphoid development, 11-13 spermatogenesis, 14 and maternal cell transmission to offspring through placenta during pregnancy and by breast-feeding after birth. 15 These studies required the isolation of viable cells from the host and their subsequent flow cytometric identification and characterization. Because of the lack of optimized techniques to detect eGFP-expressing cells in situ, an analysis of cell migration, proliferation, and differentiation by immunohistological techniques was difficult to perform in this attractive model.We transferred eGFP ϩ cells of well-defined lymphoid or myeloid subsets, ie, splenic CD4 ϩ T cells or bone marrow-derived dendritic cells (DCs), from eGFP-transgenic (eGFP-tg) C57/BL6 (B6) donor mice into congenic, immunodeficient RAG1 Ϫ/Ϫ B6 hosts. We used the stable expression of eGFP as a sensitive and highly specific

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Improved Fluorescence and Dual Color Detection with Enhanced Blue and Green Variants of the Green Fluorescent Protein

Cited by 139 publications

References 25 publications

Acute Effectors of GLUT1 Glucose Transporter Subcellular Targeting in CIT3 Mouse Mammary Epithelial Cells

Acute Effectors of GLUT1 Glucose Transporter Subcellular Targeting in CIT3 Mouse Mammary Epithelial Cells

Spatio-temporal expression of the pathway-specific regulatory gene redD in S. coelicolor

In Situ Characterization of Genetically Targeted (Green Fluorescent) Single Cells and Their Microenvironment in an Adoptive Host

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