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At least 127 records · Page 7Linked to original sources
PubMed2003

Real-time imaging of individual fluorescent-protein color-coded metastatic colonies in vivo.

We have established stable, bright green fluorescent protein (GFP)- or red fluorescent protein (RFP)-expressing HT-1080 human fibrosarcoma clones. These cell lines showed similar cell proliferation rates and high-frequency experimental lung metastasis. The HT-1080-GFP and -RFP clones enable simultaneous real-time dual-color imaging in the live animal. HT-1080 cells were transduced with retroviral vectors containing GFP or RFP and the neomycin resistance gene. Stable transformants were selected stepwise with G418 up to 800 microl/ml. Subsequently, high GFP- or RFP-expressing clones, HT-1080-GFP or HT-1080-RFP, respectively, were selected. 3 x 10(6) cells from each clone were mixed and injected into the tail vein of SCID mice. The cells seeded the lung at high frequency with subsequent formation of pure green and pure red colonies as well as mixed yellow colonies with different patterns visualized directly on excised lungs. The lung metastases were also visualized by external fluorescence imaging in live animals through skin-flap windows over the chest wall. Lung metastases were observed on the lung surface of all mice. SCID mice well tolerated multiple surgical procedures for direct-view imaging via skin-flap windows. Real-time metastatic growth of the two different colored clones in the same lung was externally imaged with resolution and quantification of green, red, or yellow colonies in live animals. The color coding enabled determination of whether the colonies grew clonally or were seeded as a mixture with one cell type eventually dominating, or whether the colonies grew as a mixture. The simultaneous real-time dual-color imaging of metastatic colonies described in this report gives rise to the possibility of color-coded imaging of clones of cancer cells carrying various forms of gene of interest.

Animals↗
PubMed2003

Dual labeling of Pseudomonas putida with fluorescent proteins for in situ monitoring of conjugal transfer of the TOL plasmid.

We describe here a dual-labeling technique involving the green fluorescent protein (GFP) and the red fluorescent protein (DsRed) for in situ monitoring of horizontal gene transfer via conjugation. A GFPmut3b-tagged derivative of narrow-host-range TOL plasmid (pWWO) was delivered to Pseudomonas putida KT2442, which was chromosomally labeled with dsRed by transposon insertion via biparental mating. Green and red fluorescent proteins were coexpressed in donor P. putida cells. Cells expressing both fluorescent proteins were smaller in size than cells expressing GFP alone. Donors and transconjugants in mixed culture or sludge samples were discriminated on the basis of their fluorescence by using confocal laser scanning microscopy. Conjugal plasmid transfer frequencies on agar surfaces and in sludge microcosms were determined microscopically without cultivation. This method worked well for in situ monitoring of horizontal gene transfer in addition to tracking the fate of microorganisms released into complex environments. To the best of our knowledge, this is the first study that discusses the coexpression of GFP and DsRed for conjugal gene transfer studies.

Biofilms↗
PubMed2003

Development of transgenic fish for ornamental and bioreactor by strong expression of fluorescent proteins in the skeletal muscle.

In the present study, new applications of the transgenic technology in developing novel varieties of ornamental fish and bioreactor fish were explored in a model fish, the zebrafish (Danio rerio). Three "living color" fluorescent proteins, green fluorescent protein (GFP), yellow fluorescent protein (YFP), and red fluorescent protein (RFP or dsRed), were expressed under a strong muscle-specific mylz2 promoter in stable lines of transgenic zebrafish. These transgenic zebrafish display vivid fluorescent colors (green, red, yellow, or orange) visible to unaided eyes under both daylight and ultraviolet light in the dark. The level of foreign protein expression is estimated between 3% and 17% of total muscle proteins, equivalent to 4.8-27.2mg/g wet muscle tissue. Thus, the fish muscle may be explored as another useful bioreactor system for production of recombinant proteins. In spite of the high level of foreign protein expression, the expression of endogenous mylz2 mRNAs was not negatively affected. Furthermore, compared to the wild-type fish, these fluorescent transgenic fish have no advantage in survival and reproduction.

Animals↗
PubMed2006

Autofluorescent proteins for monitoring the intracellular distribution of glycosyltransferases.

To analyze the Golgi compartmentalization of glycosyltransferases (GTs), we generated versions of several enzymes fused to either the enhanced green fluorescent protein (EGFP) or the red fluorescent protein from Discosoma sp. reef coral (DsRed2) and examined their intracellular distribution by confocal fluorescence microscopy in living cells. In a previous work, we have shown that the N-terminal peptides of GTs, encompassing the cytosolic and the transmembrane domains (CTDs), can serve as Golgi-targeting signals to localize the enzymes to their corresponding compartments within the Golgi apparatus (Zerfaoui et al., 2002). Using sialyl-Lewis x synthesis and selectin binding as functional assays, we show here that by swapping CTDs between GTs, it is possible to mislocalize an enzyme from a Golgi compartment to another, thereby altering the overall cellular glycosylation. On the other hand, we demonstrate that the use of an autofluorescent tag such as EGFP offers numerous advantages including the possibility of (1) facilitating sorting by fluorescence-activated cell sorter (FACS) of stably transfected polyclonal cell population, (2) constantly monitoring the expression of the enzymes in live cells, (3) establishing a direct relationship between the fluorescence intensity and the enzyme activities in vivo and in vitro, (4) establishing a visual relationship between function and intracellular distribution of a given GT, as well as co-localization with cognate protein acceptors by confocal microscopy, and (5) detecting proteins on blots with highly sensitive commercially available antibodies.

Glycosyltransferases↗
PubMed2006

Identification of RNA-binding proteins that regulate FGFR2 splicing through the use of sensitive and specific dual color fluorescence minigene assays.

We have developed a series of fluorescent splicing reporter minigenes for the establishment of cell-based screens to identify splicing regulatory proteins. A key technical advance in the application of these reporters was the use of two different fluorescent proteins: EGFP and monomeric Red Fluorescent Protein (mRFP). Through establishment of stable cell lines expressing such dual color fluorescent reporters, these minigenes can be used to perform enhanced screens for splicing regulatory proteins. As an example of such applications we generated fluorescent minigenes that can be used to determine the splicing of mutually exclusive FGFR2 exons IIIb and IIIc by flow cytometry. One minigene contained a coding sequence for EGFP whose translation was dependent on splicing of exon IIIb, whereas a second minigene required exon IIIc splicing for translation of an mRFP coding sequence. Stable incorporation of both minigenes into cells that express endogenous FGFR2-IIIb or FGFR2-IIIc resulted in EGFP or mRFP fluorescence, respectively. Cells stably transfected with both minigenes were used to screen a panel of cDNAs encoding known splicing regulatory proteins, and several were identified that induced a switch in splicing that could be detected specifically by an increase in green, but not red, fluorescence. We further demonstrated additional minigenes that can be used in dual color fluorescent screens for identification of splicing regulatory proteins that function through specific intronic splicing enhancer elements (ISEs). The methods and minigene designs described here should be adaptable for broader applications in identification of factors and mechanisms involved in alternative splicing of numerous other gene transcripts.

Alternative Splicing↗
PubMed2002

A protease assay for two-photon crosscorrelation and FRET analysis based solely on fluorescent proteins.

GFP and the red fluorescent protein, DsRed, have been combined to design a protease assay that allows not only for fluorescence resonance energy transfer (FRET) studies but also for dual-color crosscorrelation analysis, a single-molecule-based method that selectively probes the concomitant movement of two distinct tags. The measurement principle is based on a spectrally resolved detection of single molecules diffusing in and out of a diffraction-limited laser focus. Double-labeled substrate molecules are separated into two single-labeled products by specific cleavage at a protease cleavage site between the two flanking tags, DsRed and GFP, thus disrupting joint fluctuations in the two detection channels and terminating FRET between the two labels. In contrast to enzyme assays based solely on FRET, this method of dual-color crosscorrelation is not limited to a certain range of distances between the fluorophores and is much more versatile with respect to possible substrate design. To simplify the measurement setup, two-photon excitation was used, allowing for simultaneous excitation of both tags with a single infrared laser wavelength. The general concept was experimentally verified with a GFP-peptide-DsRed construct containing the cleavage site for tobacco etch virus protease. Two-photon excitation in the infrared and the use of cloneable tags make this assay easily adaptable to intracellular applications. Moreover, the combination of FRET and crosscorrelation analysis in a single-molecule-based approach promises exciting perspectives for miniaturized high-throughput screening based on fluorescence spectroscopy.

Amino Acid Sequence↗
PubMed2002

Reporter genes lucFF, luxCDABE, gfp, and dsred have different characteristics in whole-cell bacterial sensors.

The selection of a genetic reporter can be difficult because of the wide range of genes available. In order to reduce the selection, we compared the performance of different reporter genes: firefly luciferase (Photinus pyralis lucFF), bacterial luciferase operon (Photorhabdus luminescens luxCDABE), green fluorescent protein (Aequorea victoria gfp), and red fluorescent protein (Discosoma sp. dsred) in whole-cell bacterial sensors. Escherichia coli sensor bacteria were engineered to contain a reporter plasmid that carries the reporter gene under the control of mercury- (mer from Tn21) or arsenite- (ars from R773) responsive regulatory units. Characteristics of the strains were studied by using different arsenite or mercury concentrations and incubation times. The lowest detectable concentration of analytes and the fastest responses were achieved with lucFF or luxCDABE as reporter genes. The fluorescent proteins, GFP and DsRed, gave responses at higher analyte concentrations and after significantly longer incubation times. The results indicate that luciferases are better reporters in whole-cell sensor bacteria.

Animals↗
PubMed2004

Expression analysis of ABC transporters reveals differential functions of tandemly duplicated genes in Caenorhabditis elegans.

We have previously identified 60 predicted ABC transporter genes in the Caenorhabditis elegans genome and classified them into eight groups. As an initial step towards understanding how these putative ABC genes work in worms, we generated promoter-fluorescent protein fusions for the entire family to address when and where these genes are turned on in vivo. Both Aequoria green fluorescent protein (GFP) and Discosoma red fluorescent protein (RFP) were used as reporters in our transgenic assay. Observable expression is more frequently seen from fusions to genes in subfamilies B, C, D and E than those in subfamilies A and G. Sixteen worm ABC genes are found in tandem duplications, forming two four-gene clusters and four two-gene clusters. Fifteen out of the 16 duplicated gene promoters drove different or partially overlapping expression patterns, suggesting active functions for these duplicated genes. Furthermore, our results suggest that an internal promoter can cause differential expression of genes within an operon. Finally, our observations suggest that it is possible for coding sequences to function as a regulatory region for a neighbouring gene.

ATP-Binding Cassette Transporters↗
PubMed2004

Efficient eukaryotic expression of fluorescent scFv fusion proteins directed against CD antigens for FACS applications.

Two sets of expression vectors were constructed that permitted the efficient expression of single-chain Fv fragments (scFvs) fused N-terminally to an enhanced mutant of the green fluorescent protein GFP+ or the red fluorescent protein DsRed in insect and mammalian cells. The vectors allowed rapid cloning of scFv fragments and secretion of the fusion proteins in a native conformation. Fluorescent scFv fusion proteins directed against a series of cluster of differentiation (CD) antigens were efficiently secreted by transiently transfected mammalian cells and insect cells infected with baculoviral expression constructs. Yields of the secreted proteins varied from 100 microg/l to 3 mg/l. The purified proteins were functionally active in flow cytometry, immunofluorescent microscopy, and competition binding experiments performed to delineate the epitopes recognized by different monoclonal antibodies against the same polypeptide. The use of two different scFv fragments fused with red and green fluorescent proteins and reacting with T- and B-cell lineage markers (CD7 and CD19), respectively, allowed a simplified quantitation of both subsets in two-color flow cytometry experiments with mixed populations of T- and B-lymphoid cells. Due to the lack of Fc domains in the scFv proteins, the fluorescent fusion proteins showed more than 20-fold reduced background fluorescence compared with whole antibodies of the same specificity in experiments with effector cells expressing the high affinity FcgammaRI receptor CD64. Thus, for a number of analytical applications, fluorescent scFv fusion proteins offer advantages over the use of complete primary antibodies and chemically labeled fluorescent secondary antibodies.

Animals↗
PubMed2006

In vivo dynamics of enterovirus protease revealed by fluorescence resonance emission transfer (FRET) based on a novel FRET pair.

An in vivo protease assay suitable for analysis by fluorescence resonance energy transfer (FRET) was developed on the basis of a novel FRET pair. The specifically designed fusion substrate consists of green fluorescent protein 2 (GFP2)-peptide-red fluorescent protein 2 (DsRed2), with a cleavage motif for the enterovirus 2A protease (2Apro) embedded within the peptide region. FRET can be readily visualized in real-time from cells expressing the fusion substrate until a proteolytic cleavage by 2Apro from the input virus. The level of FRET decay is a function of the amount and infection duration of the inoculated virus as measured by a fluorometer assay. The FRET biosensor also responded well to other related enteroviruses but not to a phylogenetically distant virus. Western blot analysis confirmed the physical cleavage of the fusion substrate upon the infections. The study provides proof of principle for applying the FRET technology to diagnostics, screening procedures, and cell biological research.

Blotting, Western↗
PubMed2001

Expression of fluorescently tagged connexins: a novel approach to rescue function of oligomeric DsRed-tagged proteins.

A novel, brilliantly red fluorescent protein, DsRed has become available recently opening up a wide variety of experimental opportunities for double labeling and fluorescence resonance electron transfer experiments in combination with green fluorescent protein (GFP). Unlike in the case of GFP, proteins tagged with DsRed were often found to aggregate within the cell. Here we report a simple method that allows rescuing the function of an oligomeric protein tagged with DsRed. We demonstrate the feasibility of this approach on the subunit proteins of an oligomeric membrane channel, gap junction connexins. Additionally, DsRed fluorescence was easily detected 12-16 h post transfection, much earlier than previously reported, and could readily be differentiated from co-expressed GFP. Thus, this approach can eliminate the major drawbacks of this highly attractive autofluorescent protein.

Animals↗
PubMed2004

Transcriptional analysis of mutacin I (mutA) gene expression in planktonic and biofilm cells of Streptococcus mutans using fluorescent protein and glucuronidase reporters.

Streptococcus mutans is implicated as the primary pathogen involved in the development of dental caries. The production of specific bacteriocins (called mutacins) by S. mutans is one of the major virulence factors which facilitate the dominance of the bacterium within dental plaque. While much has been revealed about the biochemical structures of mutacins, little is known about the expression and regulation of mutacin genes, largely due to the lack of proper methods to monitor mutacin gene expression, especially under biofilm conditions. In this study, a set of reporter systems with the green fluorescent protein (gfp), the monomeric red fluorescent protein (mrfp1), and the glucuronidase (gusA) are introduced to S. mutans to study the transcriptional activities of the mutacin I gene (mutA). Although the mutA-reporter fusions are in single copy on the chromosome, these reporter systems display strong signals that allow us to effectively monitor mutA gene expression in S. mutans. Using these reporter systems, we show that mutA is expressed in both planktonic and biofilm cells, even though mutacin activities are normally detected only in biofilm cells. Furthermore, we confirm that mutR, the gene upstream of the mutacin operon, is required for mutacin I gene expression. The success of this study validates the feasibility of using these reporter systems to study gene expression and regulation in S. mutans.

Artificial Gene Fusion↗
PubMed2005

Micropatterning proteins on polyhydroxyalkanoate substrates by using the substrate binding domain as a fusion partner.

A novel strategy for micropatterning proteins on the surface of polyhydroxyalkanoate (PHA) biopolymer by microcontact printing (microCP) is described. The substrate binding domain (SBD) of the Pseudomonas stutzeri PHA depolymerase was used as a fusion partner for specifically immobilizing proteins on PHA substrate. Enhanced green fluorescent protein (EGFP) and red fluorescent protein (RFP) fused to the SBD could be specifically immobilized on the micropatterns of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). Laser scanning confocal microscopic studies suggested that two fusion proteins were micropatterned in their functionally active forms. Also, antibody binding assay by surface plasmon resonance suggested that protein-protein interaction studies could be carried out using this system.

Adsorption↗
PubMed2005

RNA polymerase II mediated transcription from the polymerase III promoters in short hairpin RNA expression vector.

RNA polymerase III promoters of human ribonuclease P RNA component H1, human U6, and mouse U6 small nuclear RNA genes are commonly used in short hairpin RNA (shRNA) expression vectors due their precise initiation and termination sites. During transient transfection of shRNA vectors, we observed that H1 or U6 promoters also express longer transcripts enough to express several reporter genes including firefly luciferase, green fluorescent protein EGFP, and red fluorescent protein JRed. Expression of such longer transcripts was augmented by upstream RNA polymerase II enhancers and completely inhibited by downstream polyA signal sequences. Moreover, the transcription of firefly luciferase from human H1 promoter was sensitive to RNA polymerase II inhibitor alpha-amanitin. Our findings suggest that commonly used polymerase III promoters in shRNA vectors are also prone to RNA polymerase II mediated transcription, which may have negative impacts on their targeted use.

Animals↗
PubMed2006

Characterization of proteinase activation dynamics by capillary electrophoresis conjugating with fluorescent protein-based probe.

In this paper, a novel strategy was reported to characterize dynamics of proteinase activation based on capillary electrophoresis (CE), using caspase-2 as the model system. A fusion protein conjugating an amino acid sequence VDVAD with two fluorescent proteins enhanced cyan fluorescence protein (ECFP) and red fluorescence protein (DsRed), ECFP-VDVAD-DsRed, was specially designed and expressed in HeLa cells as the substrate of proteinase caspase-2. In this substrate, the sequence VDVAD could be specifically recognized and cleaved by caspase-2 as soon as its activation was initiated with treatment of a certain dose of cisplatin to HeLa cells, which led to a break of the substrate into two fragments. Analyses of the cell lysates using CE in a time course of the apoptosis illustrated the dynamics of caspase-2 activation. It showed that the employment of fusion fluorescent protein greatly facilitated both CE separation and identification of the analytes. This result from cell colony by CE was compared with that from single cell achieved by optical imaging.

Electrophoresis, Capillary↗
PubMed2006

Selective labeling of central and peripheral sensory neurons in the developing zebrafish using P2X(3) receptor subunit transgenes.

The two paralogous P2X receptor subunit genes p2rx3.1 and p2rx3.2 are selectively expressed in overlapping, but unique, patterns of sensory neurons in the developing zebrafish. We constructed a series of transgenes derived from both genes using the recombineering technique. Transgenes utilizing either enhanced green fluorescent protein or monomeric red fluorescent protein-1 were shown to be expressed with the same spatial and temporal patterns as the native genes. The p2rx3.1-derived transgenes labeled the vast majority of the Rohon-Beard neurons in the spinal cord and neurons of the trigeminal ganglia. The p2rx3.2-derived transgene labeled fewer Rohon-Beard and trigeminal neurons than what was observed for the p2rx3.1-derived transgenes, but was also detected in neurons of the epibranchial ganglia. Three distinct populations of sensory neurons were detected: those expressing only one or the other paralog, and those expressing both paralogs. The fluorescent proteins encoded by the transgenes allowed for visualization of the neuronal somas as well as their peripheral and central projections. These reagents should prove extremely useful in providing the basis for future studies aimed at elucidating the developmental and physiological attributes of sensory neurons.

Animals↗
PubMed2002

Biolistic transfection and morphological analysis of cultured sympathetic neurons.

We have developed a protocol for introducing foreign genes into postmitotic peripheral neurons and their subsequent analysis by computer-assisted morphometry. The application of the biolistic approach results in transfection of approximately 10% of dissociated sympathetic neurons. Gold particles coated with plasmid DNA are propelled into sympathetic neuronal nuclei using a hand-held 'gene gun'. Transfection efficiency is optimized by choosing a microcarrier loading quantity of 0.7 mg of gold per experiment and a DNA loading ratio of 2 microg DNA per mg of gold. The gene gun is modified to minimize pressure-induced cell damage. Neurons are transfected with plasmids encoding enhanced green fluorescent protein (EGFP) or red fluorescent protein (DsRed), which serve as suitable markers for the cell body, axonal processes and growth cones including lamellipodia and filopodia. A detailed routine for quantitative morphometric image analysis using METAMORPH software is described. The protocol is demonstrated by comparing the effects of nerve growth factor (NGF) and dibutyryl cyclic adenosine monophosphate (dbcAMP) on neurite outgrowth of transfected sympathetic neurons plated on glass floor dishes.

Animals↗
PubMed2005

Actin depolymerization disrupts tight junctions via caveolae-mediated endocytosis.

The tight junction (TJ) determines epithelial barrier function. Actin depolymerization disrupts TJ structure and barrier function, but the mechanisms of this effect remain poorly understood. The goal of this study was to define these mechanisms. Madin-Darby canine kidney (MDCK) cells expressing enhanced green fluorescent protein-, enhanced yellow fluorescent protein-, or monomeric red fluorescent protein 1-fusion proteins of beta-actin, occludin, claudin-1, ZO-1, clathrin light chain A1, and caveolin-1 were imaged by time-lapse multidimensional fluorescence microscopy with simultaneous measurement of transepithelial electrical resistance (TER). Actin depolymerization was induced with latrunculin A (LatA). Within minutes of LatA addition TER began to fall. This coincided with occludin redistribution and internalization. In contrast, ZO-1 and claudin-1 redistribution occurred well after maximal TER loss. Occludin internalization and TER loss, but not actin depolymerization, were blocked at 14 degrees C, suggesting that membrane traffic is required for both events. Inhibition of membrane traffic with 0.4 M sucrose also blocked occludin internalization and TER loss. Internalized occludin colocalized with caveolin-1 and dynamin II, but not with clathrin, and internalization was blocked by dominant negative dynamin II (K44A), but not by Eps15Delta95-295 expression. Inhibition of caveolae-mediated endocytosis by cholesterol extraction prevented both LatA-induced TER loss and occludin internalization. Thus, LatA-induced actin depolymerization causes TJ structural and functional disruption by mechanisms that include caveolae-mediated endocytosis of TJ components.

Actins↗