Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “GFP”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 181 records · Page 10Linked to original sources
PubMed1996

Centrosome and spindle function of the Drosophila Ncd microtubule motor visualized in live embryos using Ncd-GFP fusion proteins.

The Ncd microtubule motor protein is required for meiotic and early mitotic chromosome distribution in Drosophila. Null mutant females expressing the Ncd motor fused to the Aequorea victoria green fluorescent protein (GFP), regulated by the wild-type ncd promoter, are rescued for chromosome segregation and embryo viability. Analysis of mitosis in live embryos shows cell cycle-dependent localization of Ncd-GFP to centrosomes and spindles. The distribution of Ncd-GFP in spindles during metaphase differs strikingly from that of tubulin: the tubulin staining is excluded by the chromosomes at the metaphase plate; in contrast, Ncd-GFP forms filaments along the spindle microtubules that extend across the chromosomes. The existence of Ncd-GFP fibers that cross the metaphase plate suggests that Ncd interacts functionally with chromosomes in metaphase. Differences are no longer observed in anaphase when the chromosomes have moved off the metaphase plate. A mutant form of Ncd fused to GFP also localizes to spindles in live embryos. Mutant embryos show frequent centrosome and spindle abnormalities, including free centrosomes that dissociate from interphase nuclei, precociously split centrosomes, and spindles with microtubule spurs or bridges to nearby spindles. The precociously split and free centrosomes indicate that the Ncd motor acts in cleavage stage embryos to maintain centrosome integrity and attachment to nuclei. The frequent spindle spurs of mutant embryos are associated with mis-segregating chromosomes that partially detach from the spindle in metaphase, but can be recaptured in early anaphase. This implies that the Ncd motor functions to prevent chromosome loss by maintaining chromosome attachment to the spindle in metaphase, consistent with the Ncd-GFP fibers that across the metaphase plate.

Adenosine Triphosphatases↗
PubMed1999

Endosome fusion in living cells overexpressing GFP-rab5.

CHO and BHK cells which overexpress either wild-type rab5 or rab5:Q79L, a constitutively active rab5 mutant, develop enlarged cytoplasmic vesicles that exhibit many characteristics of early endosomes including immunoreactivity for rab5 and transferrin receptor. Time-lapse video microscopy shows the enlarged endosomes arise primarily by fusion of smaller vesicles. These fusion events occur mostly by a 'bridge' fusion mechanism in which the initial opening between vesicles does not expand; instead, membrane flows slowly and continuously from the smaller to the larger endosome in the fusing pair, through a narrow, barely perceptible membranous 'bridge' between them. The unique aspect of rab5 mediated 'bridge' fusion is the persistence of a tight constriction at the site where vesicles merge and we hypothesize that this constriction results from the relatively slow disassembly of a putative docking/fusion complex. To determine the relation of rab5 to the fusion 'bridge', we used confocal fluorescence microscopy to monitor endosome fusion in cells overexpressing GFP-rab5 fusion proteins. Vesicle docking in these cells is accompanied by recruitment of the GFP-rab5 into a brightly fluorescent spot in the 'bridge' region between fusing vesicles that persists throughout the entire length of the fusion event and which often persist for minutes following endosome fusion. Other endosomal membrane markers, including FM4-64, are not concentrated in fusion 'bridges'. These results support the idea that the GFP-rab5 spots represent the localized accumulation of GFP-rab5 between fusing endosomes and not simply overlap of adjacent membranes. The idea that the GFP-rab5 spots do not represent membrane overlap is further supported by experiments using photobleaching techniques and confocal imaging which show that GFP-rab5 localized in spots between fusion couplets is resistant to diffusion while GFP-rab5 on endosomal membranes away from these spots rapidly diffuses with a rate constant of about 1.0 (+/-0.3) x10(-)(9)cm(2)/second.

Animals↗
PubMed2003

Instrumentation and methodology for quantifying GFP fluorescence in intact plant organs.

The General Fluorescence Plant Meter (GFP-Meter) is a portable spectrofluorometer that utilizes a fiber-optic cable and a leaf clip to gather spectrofluorescence data. In contrast to traditional analytical systems, this instrument allows for the rapid detection and fluorescence measurement of proteins under field conditions with no damage to plant tissue. Here we discuss the methodology of gathering and standardizing spectrofluorescence data from tobacco and canola plants expressing GFP. Furthermore, we demonstrate the accuracy and effectiveness of the GFP-Meter. We first compared GFP fluorescence measurements taken by the GFP-Meter to those taken by a standard laboratory-based spectrofluorometer, the FluoroMax-2. Spectrofluorescence measurements were taken from the same location on intact leaves. When these measurements were tested by simple linear regression analysis, we found that there was a positive functional relationship between instruments. Finally, to exhibit that the GFP-Meter recorded accurate measurements over a span of time, we completed a time-course analysis of GFP fluorescence measurements. We found that only initial measurements were accurate; however, subsequent measurements could be used for qualitative purposes.

Brassica↗
PubMed2005

[Inhibition of HBs-GFP fusion gene expression by RNA interference].

OBJECTIVE: To develop an effective report gene system to test the effect of small interfering RNA (siRNA). METHODS: HBV S gene was fused with enhanced green fluorescent protein (EGFP) gene to form HBs-GFP and the plasmid containing HBs-GFP was constructed. A vector expressing small hairpin RNA (shRNA) pAVU6 + 4sh357 was also constructed. Two plasmids were co-transfected into HepG2 cells transiently. The fluorescence of HBs-GFP was detected by fluorescence-activated cell sorting (FACS). The mRNA expression in HepG2 cells was detected by conventional RT-PCR and real-time PCR. RESULTS: siRNA inhibited the expression of HBs-GFP 72 hours post transfection. The fluorescence of HBs-GFP in HepG2 cells treated with pAVU6+4sh357 was reduced by 55.4% compared with that of controls. The HBs-GFP expression in HepG2 cells treated with pAVU6+4sh357 was reduced by 76.3% and 90% as measured with conventional RT-PCR and real-time PCR, respectively. CONCLUSION: This investigation demonstrated siRNA derived from shRNA expression vectors can inhibit the expression of HBs-GFP in HepG2 cells.

Carcinoma, Hepatocellular↗
PubMed1999

Safe sorting of GFP-transduced live cells for subsequent culture using a modified FACS vantage.

BACKGROUND: A stream-in-air cell sorter enables rapid sorting to a high purity, but it is not well suited for sorting of infectious material due to the risk of airborne spread to the surroundings. METHODS: A FACS Vantage cell sorter was modified for safe use with potentially HIV infected cells. Safety tests with bacteriophages were performed to evaluate the potential spread of biologically active material during cell sorting. Cells transduced with a retroviral vector carrying the gene for GFP were sorted on the basis of their GFP fluorescence, and GFP expression was followed during subsequent culture. RESULTS: The bacteriophage sorting showed that the biologically active material was confined to the sorting chamber. A failure mode simulating a nozzle blockage resulted in detectable droplets inside the sorting chamber, but no droplets could be detected when an additional air suction from the sorting chamber had been put on. The GFP transduced cells were sorted to 99% purity. Cells not expressing GFP at the time of sorting did not turn on the gene during subsequent culture. Un-sorted cells and cells sorted to be positive for GFP showed a decrease in the fraction of GFP positive cells during culture. CONCLUSIONS: Sorting of live infected cells can be performed safely and with no deleterious effects on vector expression using the modified FACS Vantage instrument.

Bacteriophages↗
PubMed2000

NFAT-controlled expression of GFP permits visualization and isolation of antigen-stimulated primary human T cells.

We have developed a new method that allows detection and isolation of viable, antigen-specific, human T cells from a heterogeneous pool of T cells. We have engineered a self-inactivating retroviral vector containing multiple (3 or 6) nuclear factor of activated T-cell (NFAT)-binding sites, followed by the minimal IL2 promoter and the reporter gene GFP. Jurkat cells, primary T-cell blasts, and T-cell clones were transduced with high efficiency (20%-40%). Stimulation of the transduced cells with phorbol myristate acetate (PMA) and ionomycin resulted in a high level expression of GFP that was maximal after 12 to 14 hours and remained stable for another 12 hours. Activation of T cells carrying the construct containing 6 NFAT-binding sites resulted in the highest mean fluorescence intensity. Cyclosporin-A and FK506 were able to block the activation-dependent GFP expression. Activation of transduced T-cell blasts with the superantigen staphylococcal enterotoxin B or of transduced antigen-specific T-cell clones with cognate antigen resulted in GFP expression. After an overnight stimulation of a heterogeneous T-cell bulk culture with an HLA mismatched stimulator cell (JY), the GFP expressing cells were cloned. As expected, the cloning frequency of the antigen-specific GFP(+) cells was considerably higher than that of the total T-cell population. Most of the T-cell clones were either cytolytic, or proliferative toward JY stimulator cells. Interestingly, we also isolated T-cell clones that were noncytolytic and nonproliferative toward JY cells, but specifically up-regulated GFP after an overnight stimulation with JY. (Blood. 2000;96:459-466)

Antigens↗
PubMed2001

Generation of dopaminergic neurons in the adult brain from mesencephalic precursor cells labeled with a nestin-GFP transgene.

Mesencephalic precursor cells may one day provide dopaminergic neurons for the treatment of Parkinson's disease. However, the generation of dopaminergic neurons from mesencephalic precursors has been difficult to follow, partly because an appropriate means for recognizing mesencephalic ventricular zone precursors has not been available. To visualize and isolate mesencephalic precursor cells from a mixed population, we used transgenic mice and rats carrying green fluorescent protein (GFP) cDNA under the control of the nestin enhancer. nestin-driven GFP was detected in the mesencephalic ventricular zone, and it colocalized with specific markers for neural precursor cells. In addition, data from flow-cytometry indicated that Prominin/CD133, a cell-surface marker for ventricular zone cells, was expressed specifically in these GFP-positive (GFP(+)) cells. After sorting by fluorescence-activated cell sorting, the GFP(+) cells proliferated in vitro and expressed precursor cell markers but not neuronal markers. Using clonogenic sphere formation assays, we showed that this sorted population was enriched in multipotent precursor cells that could differentiate into both neurons and glia. Importantly, many neurons generated from nestin-GFP-sorted mesencephalic precursors developed a dopaminergic phenotype in vitro. Finally, nestin-GFP(+) cells were transplanted into the striatum of a rat model of Parkinson's disease. Bromodeoxyuridine-tyrosine hydroxylase double-labeling revealed that the transplanted cells generated new dopaminergic neurons within the host striatum. The implanted cells were able to restore dopaminergic function in the host striatum, as assessed by a behavioral measure: recovery from amphetamine-induced rotation. Together, these findings indicate that precursor cells harvested from the embryonic ventral mesencephalon can generate dopaminergic neurons able to restore function to the chemically denervated adult striatum.

AC133 Antigen↗
PubMedDate not supplied

Transgenic expression of a GFP-rhodopsin COOH-terminal fusion protein in zebrafish rod photoreceptors.

To facilitate the identification and characterization of mutations affecting the retina and photoreceptors in the zebrafish, a transgene expressing green fluorescent protein (GFP) fused to the C-terminal 44 amino acids of Xenopus rhodopsin (Tam et al., 2000) under the control of the 1.3-kb proximal Xenopus opsin promoter was inserted into the zebrafish genome. GFP expression was easily observed in a ventral patch of retinal cells at 4 days postfertilization (dpf). Between 45-50% of the progeny from the F1, F2, and F3 generations expressed the transgene, consistent with a single integration event following microinjection. Immunohistochemical analysis demonstrated that GFP is expressed exclusively in rod photoreceptors and not in the UV, blue, or red/green double cones. Furthermore, GFP is localized to the rod outer segments with little to no fluorescence in the rod inner segments, rod cell bodies, or rod synapse regions, indicating proper targeting and transport of the GFP fusion protein. Application of exogenous retinoic acid (RA) increased the number of GFP-expressing cells throughout the retina, and possibly the level of expressed rhodopsin. When bred to a zebrafish rod degeneration mutant, fewer GFP-expressing rods were seen in living mutants as compared to wild-type siblings. This transgenic line will facilitate the search for recessive and dominant mutations affecting rod photoreceptor development and survival as well as proper rhodopsin expression, targeting, and transport.

Amblyopia↗
PubMed2003

[A study on the effects of rAd-gfp-bcl-X(L) on retinal degeneration in mice].

OBJECTIVE: To observe the therapeutic effect of rAd-gfp-bcl-X(L) on retinal degeneration (RD) in mice. METHODS: rAd-gfp-bcl-X(L) granules were injected into the subretinal space of right eyes of RD mice in Group A (postnatal days 10, p10) and Group B (postnatal days 22, p22) (n = 20). The morphological changes were observed by light microscopy and transmission electron microscopy at 15 - 30 days post-injection. Frozen section was used to observe the expression of GFP. The level of bcl-X(L) mRNA and bcl-X(L) protein were assayed by RT-PCR and immunohistochemistry assay. RESULTS: In frozen section, GFP was observed in the treated eyes under fluorescence microscopy, indicating that rAd-gfp-bcl-X(L) had transfected into the retinal cells successfully and that bcl-X(L) was expressed. In Group A, an increased expression of bcl-X(L) mRNA and Bcl-X(L) protein were observed in the treated eyes. The outer nuclear layer of the treated eyes in Group A was thicker than that of the control eyes at 30 days post-injection. The inner segment of treated eyes in Group A was healthier than that of control eyes, as indicated by transmission electron microscopy. No difference was found in the thickness of retina between treated eyes and control eyes in Group B. CONCLUSION: The bcl-X(L) can be expressed and have an anti-apoptosis effect when the rAd-gfp-bcl-X(L) particles are injected into the subretinal space of RD mice's eyes at an early stage. But the expression and protective effects were not observed in RD mice at a later stage. The therapy of rAd-gfp-bcl-X(L) for retinal degenerative diseases still needs further study.

Adenoviridae↗
PubMed2003

[The expression and localization of p27(KIP1)-GFP fusion gene in hepatocellular carcinoma cell lines].

AIM: To study the expression and localization of p27(KIP1)-GFP fusion protein in hepatocellular carcinoma cell line HCC-9204. METHODS: According to the encoded sequence of human p27(KIP1) gene, stop codon TAA located at 3' terminus of p27(KIP1) gene was mutated into TGT by PCR and fused with GFP gene. Then the p27(KIP1)-GFP cDNA was transfected into HCC-9204 cells. The expression of p27(KIP1)-GFP fusion protein was observed under fluorecence microscope. RESULTS: DNA sequencing demonstrated that difference between p27(KIP1) gene and that in Genbank was only one nucleoside and the encoded protein sequence is identical. The observation under fluorecence microscope reporced showed that fluorochrome was spread in entire HCC-9204 cells transfected with GFP gene, whereas green fluorescence was seen mainly in the nucleus of HCC-9204 cells transfected with p27(KIP1)-GFP gene. CONCLUSION: The human p27(KIP1)-GFP fusion protein could be expressed in HCC-9204 cells and located in the nucleus, which is similar to the expression of p27(KIP1) gene.

Carcinoma, Hepatocellular↗
PubMed2004

[Biodistribution of immune cells in adoptive immunotherapy by using GFP transgenic mice].

All of the cells and tissues from GFP transgenic mice, with the exception of erythrocytes and hair, express green fluorescent protein. Additionally, lymphokine activated killer cells induced from splenocytes of the mice (GFP-LAK) express green fluorescence under the observation by fluorescent microscopy. In the present study, we studied the biodistribution of LAK in the two adoptive immunotherapy models by injecting GFP-LAK into non-GFP expressing syngeneic mice. In peritoneal dissemination model of B16 melanoma cells, intraperitoneally injected GFP-LAK accumulated densely on the tumor. On the other hand, in the lung metastases model, intravenously injected GFP-LAK stayed scattered around the tumor, although they were observed abundantly in the spleen. Our adoptive transfer model using GFP transgenic mice is useful for understanding antitumor mechanisms induced by adoptively transferred immune cells, without any troublesome marking procedures.

Animals↗
PubMed2006

[Whole-body fluorescent imaging of the growth and metastasis of GFP-expressing bladder tumors].

OBJECTIVE: To label a human bladder cancer cell line and establish a novel human bladder cancer mouse model. METHODS: T-24 cells, a human bladder transitional cell carcinoma cell line, were transfected with GFP plasmid to screen stable GFP-expressing clones. The latter were implanted into the wall of the bladder or the subcutaneous tissue of the neck of nude mice. The growth, invasion, and metastasis of the implanted tumor were observed and evaluated with whole-body optical imaging system. The findings were compared with those of HE staining on routine paraffin sections. RESULTS: GFP-labeled tumor cells displayed green fluorescence under fluorescent microscopy and showed stable GFP expression in vitro and in vivo. One week after in situ transplantation of 5 x 10(5) T24 cells, the new bladder cancer was observed and evaluated under whole-body optical imaging system. Two weeks later, the new bladder tumor could be palpated, and 4 weeks later, metastasis to regional drainage lymph nodes in the pelvic and retroperitoneal lymph nodes occurred. The growth and metastasis of the implant bladder tumor were easily observed and accurately evaluated by fluorescent microscope. CONCLUSION: GFP-labeled tumor cells display green fluorescence under fluorescent microscopy and show stable GFP expression. GFP-labeled T-24 cells and the novel human bladder cancer model described hereby provide a simple and reliable means for studying human bladder cancer in vivo.

Animals↗
PubMed2000

Microtubule organization and the effects of GFP-tubulin expression in dictyostelium discoideum.

We have labeled microtubules in living Dictyostelium amoebae by incorporation of a GFP-alpha-tubulin fusion protein. The GFP-alpha-tubulin incorporates into microtubules and, as reported by others [Neujahr et al., 1998], the labeled microtubules are highly motile. Electron microscopy (EM) analysis of the distribution and organization of microtubules in the amoebae shows that some cytoplasmic microtubules form close associations. These associations could allow motor proteins attached to one microtubule to walk along an adjacent microtubule and thus generate some of the observed motility. Protein blot analysis indicates that the GFP-alpha-tubulin incorporates into microtubules at a lower efficiency than does the endogenous alpha-tubulin. EM and immunofluorescence (IF) analyses suggest that the GFP-alpha-tubulin interferes with microtubule nucleation. We have also observed an increased sensitivity of the GFP-alpha-tubulin expressing cells to blue light, as compared to wild-type cells. These results suggest that although GFP-alpha-tubulin can be used as a marker for microtubules in living cells, the use of this marker is not recommended for certain types of studies such as assembly dynamics.

Animals↗
PubMedDate not supplied

Imaging of luciferase and GFP-transfected human tumours in nude mice.

Studies were performed to compare green fluorescent protein (GFP)-transfected and fi re fl y luciferase (Luc)-transfected MCF-7 human breast tumour cells both in vitro and in vivo. For in vitro studies, cells were serially diluted in 96-well microplates and analysed using a NightOwl LB 981 Molecular Light Imager and a Victor multilabel reader. For in vivo studies, nude mice were injected either intraperitoneally, intravenously or subcutaneously with transfected cells and then imaged using the NightOwl Imager after intraperitoneal injection of d-luciferin for Luc tumours, or excitation at 470 nm for GFP tumours. In vitro imaging studies revealed that both GFP and Luc transfectants were quantifiable. However, the Luc-transfected cells were detectable at a significantly lower concentration compared to GFP transfectants. In vivo studies demonstrated that GFP-transfected tumours were detectable as subcutaneous and intraperitoneal tumours but not as deep tissue lesions, whereas Luc-transfected tumours were detectable as subcutaneous and intraperitoneal tumours and as deep tissue lesions resulting from intraperitoneal or intravenous inoculation. These findings demonstrate that GFP-transfected cells may be useful for imaging studies of superficial tumours where both excitation and emission wavelengths are able to penetrate tissues, whereas luciferase-transfected cells appear superior for imaging studies of primary and metastatic tumours in distant sites and deep tissues.

Animals↗
PubMed2004

Green fluorescent protein-propidium iodide (GFP-PI) based assay for flow cytometric measurement of bacterial viability.

BACKGROUND: Several staining protocols have been developed for flow cytometric analysis of bacterial viability. One promising method is dual staining with the LIVE/DEAD BacLight bacterial viability kit. In this procedure, cells are treated with two different DNA-binding dyes (SYTO9 and PI), and viability is estimated according to the proportion of bound stain. SYTO9 diffuses through the intact cell membrane and binds cellular DNA, while PI binds DNA of damaged cells only. This dual-staining method allows effective separation between viable and dead cells, which is far more difficult to achieve with single staining. Although SYTO9-PI dual staining is practical for various bacterial viability analyses, the method has a number of disadvantages. Specifically, the passage of SYTO9 through the cell membrane is a slow process, which is significantly accelerated when the integrity of the cell membrane is disrupted. As a result, SYTO9 binding to DNA is considerably enhanced. PI competes for binding sites with SYTO9 and may displace the bound dye. These properties diminish the reliability of the LIVE/DEAD viability kit. In this study, we investigate an alternative method for measuring bacterial viability using a combination of green fluorescent protein (GFP) and PI, with a view to improving data reliability. METHODS: Recombinant Escherichia coli cells with a plasmid containing the gene for jellyfish GFP were stained with PI, and green and red fluorescence were measured by FCM. For comparison, cells containing the plasmid from which gfp was removed were stained with SYTO9 and PI, and analyzed by FCM. Viability was estimated according to the proportion of green and red fluorescence. In addition, bioluminescence and plate counting (other methods to assess viability) were used as reference procedures. RESULTS: SYTO9-PI dual staining of bacterial cells revealed three different cell populations: living, compromised, and dead cells. These cell populations were more distinct when the GFP-PI combination was used instead of dual staining. No differences in sensitivity were observed between the two methods. However, substitution of SYTO9 with GFP accelerated the procedure. Bioluminescence and plate counting results were in agreement with flow cytometric viability data. CONCLUSIONS: In bacterial viability analyses, the GFP-PI combination provided better distinction between current viability stages of E. coli cells than SYTO9-PI dual staining. Additionally, the overall procedure was more rapid. No marked differences in sensitivity were observed.

Escherichia coli↗
PubMed2004

Inducible expression of a MAP kinase phosphatase-3-GFP chimera specifically blunts fibroblast growth and ras-dependent tumor formation in nude mice.

The p42/p44 mitogen activated protein kinase (MAPK) pathway participates in a wide range of cellular programs including proliferation, migration, differentiation, and survival. Specific pharmacological inhibitors, like PD98059 and U0126, are often used to inhibit p42/p44 MAPK signaling. However, these inhibitors are not appropriate to study the function of these kinases in whole organisms. We thus developed an inducible system designed to inhibit p42/p44 MAPK activity through the expression of a phosphatase specific for these two kinases, the MAPK phosphatase 3 (MKP-3). A fibroblast cell line was established in which MKP-3 expression is controlled by tetracycline. Tetracycline-induced MKP-3 resulted in partial de-phosphorylation of p42/p44 MAPKs in serum-stimulated cells. However, we could improve MKP-3 stability and thereby the rate of MAPK de-phosphorylation, when the C-terminal end of MKP-3 was fused to the green fluorescent protein (GFP). Importantly, the fusion of GFP to MKP-3 did not alter the specificity of the phosphatase towards its MAPK substrates. We further show that conditional expression of MKP-3-GFP in this fibroblast cell line results in the inhibition of: (a) the phosphorylation of the p42/p44 MAPK substrates Elk1 and HIF-1alpha, (b) vascular endothelial growth factor (VEGF), cyclin D1, and c-fos gene transcription in response to MAPK pathway activation, and (c) cell proliferation. Finally, the MKP-3-GFP inducible cell line was transformed by Ha-ras and injected into nude mice. Treatment of mice with the tetracycline analog doxycycline resulted in a large delay in tumor emergence and growth as compared to the untreated control group, indicating that MKP-3-GFP activity is maintained in vivo. Altogether, these results show that inducible expression of MKP-3-GFP constitutes a valuable tool to study the role of p42/p44 MAPKs in various cellular responses in both cultured cell and animal models, a tool that may also be used to block unwanted cell growth in pathological conditions.

Animals↗
PubMed2005

Monitoring of skeletal progression of prostate cancer by GFP imaging, X-ray, and serum OPG and PTHrP.

BACKGROUND: Prostate cancers (PCas) produce factors that can serve as biomarkers for tumor metastasis and bone progression. Transduced GFP expression by cancer cells can be imaged to monitor therapy. We exploited both concepts by developing a GFP-expressing PCa cell line that expresses PTHrP and studying it in an animal model of malignancy with methods that assess the skeletal progression of this tumor. METHODS: We developed a GFP-producing PCa cell line by stable transduction of PC-3 PCa cells. This PC-3 variant was used to study tumor progression in an immunocompromised mouse model. Skeletal progression of the PCa cells and the effects of pamidronate administration were evaluated radiologically, fluorometrically, and by measurement of serum tumor markers. RESULTS: The PC-3 cells produced extensive bone lesions when injected into the tibia of immunocompromised mice. The skeletal progression of the PC-3 cells could be monitored by GFP optical imaging, X-ray, and by measurements of tumor products in serum, notably PTHrP and OPG. Pamidronate treatment reduced tumor burden as assessed at autopsy by imaging and biomarkers. CONCLUSIONS: Pamidronate treatment exhibited anti-tumor effects that were reflected by decreases in serum PTHrP, OPG, and by GFP and radiological imaging procedures. Imaging of GFP expression enables real-time monitoring of tumor growth in the bone. PTHrP and OPG may be useful as tumor biomarkers for PCa that has metastasized to bone. This novel human PCa model can be used to study the clinical potential of diagnostic and therapeutic modalities in the skeletal progression of PCas.

Adenocarcinoma↗
PubMed1997

GFP-moesin illuminates actin cytoskeleton dynamics in living tissue and demonstrates cell shape changes during morphogenesis in Drosophila.

Moesin, ezrin, and radixin (MER) are components of the cortical actin cytoskeleton and membrane processes such as filopodia and microvilli. Their C-terminal tails contain an extended region that is predicted to be helical, an actin binding domain, and a region(s) that participates in self-association. We engineered an in vivo fluorescent actin binding protein (GFP-moe) by joining sequences that encode the jellyfish green fluorescent protein (GFP) to sequences that encode the C-terminal end of the sole Drosophila MER homolog, moesin [Moesin-like gene product, referred to previously as the D17 MER-like protein; Edwards et al., 1994, Proc. Natl. Acad. Sci. USA 91, 4589], and Dmoesin [McCartney and Fehon, 1996, J. Cell Biol. 133, 843]. Transgenic flies expressing this fusion protein under control of the hsp70 promoter were generated and used for analysis of cell shape changes during morphogenesis of various developmental stages and tissues. Following heat shock, high levels of stable fusion protein are produced by all somatic tissues. GFP-moe localizes to the cortical actin cytoskeleton, providing a strong in vivo marker for cell shape and pattern during epithelial morphogenesis. The protein also becomes highly enriched in pseudopods, microvilli, axons, denticles, the border cell process, and other membrane projections, potentially by binding to endogenous moesin as well as actin. We show that GFP-moe can be used to examine the development and behavior of these dynamic structures in live specimens. We observe a bright green fluorescent, presumably actin-rich, polar cell proboscis that inserts itself into the forming micropyle and appears to maintain an opening for sperm passage around which the chorion is formed. We also confirm the existence of an actin-rich purse string at the leading edge of the lateral epidermis and provide a dynamic analysis of its behavior as it migrates during dorsal closure. Observations of embryos, larvae, and pupae show that GFP-moe is also useful for labeling the developing nervous system and will be a good general marker of dynamic cell behavior during morphogenesis in live tissues and demonstrate that fusion of a subcellular localization signal to GFP greatly increases its utility as a cell marker.

Amino Acid Sequence↗