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At least 235 records · Page 13Linked to original sources
PubMed1999

Enrichment of transiently transfected mesangial cells by cell sorting after cotransfection with GFP.

Early passage mesangial cells, like many other nonimmortalized cultured cells, can be difficult to transfect. We devised a simple method to improve the efficiency of transient protein expression under the transcriptional control of promoters in conventional plasmid vectors in rat mesangial cells. We used a vector encoding modified green fluorescent protein (GFP) and sterile fluorescence-activated cell sorting (FACS) to select a population consisting of >90% GFP-expressing cells from passaged nonimmortalized cultures transfected at much lower efficiency. Only 10% transfection efficiency was noted with a beta-galactosidase expression vector alone, but cotransfection with GFP followed by FACS and replating of GFP+ cells yielded greater than fivefold enrichment of cells with detectable beta-galactosidase activity. To demonstrate the expression of a properly oriented and processed membrane protein, we cotransfected GFP with a natriuretic peptide clearance receptor (NPR-C) expression vector. Plasmid-dependent cell surface NPR-C density was enhanced by 89% after FACS, though expression remained lower in selected mesangial cells than in the CHO cell line transfected with the same vector. We conclude that cotransfection of rat mesangial cells with GFP, followed by FACS, results in improvement in transient transfection efficiencies to levels that should suffice for many applications.

Adenoviridae↗
PubMed2002

GFP-FRNK disrupts focal adhesions and induces anoikis in neonatal rat ventricular myocytes.

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase involved in adhesion-dependent signal transduction. FAK is highly expressed in cultured neonatal rat ventricular myocytes (NRVMs) and undergoes tyrosine autophosphorylation in response to cell adhesion, stretch, and growth factor stimulation. We previously showed that inhibition of FAK phosphorylation by adenovirally mediated overexpression of FRNK (the autonomously expressed C-terminal domain of FAK) prevented endothelin-1 (ET)-induced NRVM hypertrophy. One question raised by these studies was whether FRNK localized to focal adhesions and displaced FAK from sites required for downstream signaling. Therefore, we constructed a replication-defective adenovirus encoding a GFP-FRNK fusion protein (Adv-GFP-FRNK) and examined its effects on NRVM cytoarchitecture and signaling. Uninfected NRVMs contained small amounts of endogenous FRNK. NRVMs infected with Adv-GFP-FRNK expressed much larger amounts of a 66-/68-kDa protein that localized to costameres and focal adhesions. GFP-FRNK overexpression suppressed basal and ET-induced FAK phosphorylation and also inhibited ET-induced phosphorylation of PYK2, the other member of the FAK family of nonreceptor protein tyrosine kinases. In contrast, GFP-FRNK overexpression did not prevent ET-induced ERK, JNK, or p70S6K phosphorylation. Furthermore, GFP-FRNK resulted in the loss of detectable FAK and paxillin in focal adhesions, which was accompanied by reduced levels of total paxillin and, ultimately, cell detachment and apoptosis. We conclude that FRNK functions as a dominant-negative inhibitor of adhesion-dependent signaling by displacing FAK from focal adhesions and interfering with the anchorage of NRVMs that is necessary for cell survival, a process known as anoikis.

Adenoviridae↗
PubMed2005

Analysis of thrombocyte development in CD41-GFP transgenic zebrafish.

Thrombocytes are the nucleated equivalent of platelets in nonmammalian vertebrates such as the zebrafish, Danio rerio. We have cloned zebrafish CD41 cDNA (alpha(IIb), glycoprotein IIb [GPIIb]) and its promoter and have generated transgenic zebrafish lines with green fluorescent protein (GFP)-tagged thrombocytes. CD41 mRNA transcripts appeared 42 hours after fertilization (hpf) by reverse-transcriptase-polymerase chain reaction (RT-PCR) and at 48 hpf in circulating hematopoietic cells. Flow sorting of thrombocytes from the mesonephros of adult CD41-GFP zebrafish showed a GFP(high) subset, which had the morphologic appearance of mature thrombocytes, and a GFP(low) subset with an immature appearance, suggesting that they may be thrombocyte precursors. Confocal laser microscopy of embryos 40 and 48 hpf also showed a nonmobile population of GFP+ cells in a discrete area between the dorsal aorta and caudal vein. Production of circulating thrombocytes was inhibited by the injection of antisense morpholinos for the stem-cell transcription factor scl and c-mpl, the receptor for thrombopoietin. The nonmobile pool of GFP+ cells was abolished by scl knockdown and partially inhibited by c-mpl knockdown. These studies have shown that it is possible to identify thrombocytes, thrombocyte precursors, and, possibly, early hematopoietic stem cells in zebrafish embryos and track their proliferation and maturation.

Animals↗
PubMed2005

Imaging plant cell death: GFP-Nit1 aggregation marks an early step of wound and herbicide induced cell death.

BACKGROUND: A great deal is known about the morphological endpoints of plant cell death, but relatively little is known about its sequence of events and/or its execution at the biochemical level. Live cell imaging using GFP-tagged markers is a powerful way to provide dynamic portraits of a cellular process that can in turn provide a descriptive foundation valuable for future biochemical and genetic investigations. RESULTS: While characterizing a collection of random GFP-protein fusion markers we discovered that mechanical wounding induces rapid aggregation of a GFP-Nitrilase 1 fusion protein in Arabidopsis cells directly abutting wound sites. Time-lapse imaging of this response shows that the aggregation occurs in cells that subsequently die 30-60 minutes post-wounding, indicating that GFP-Nit1 aggregation is an early marker of cell death at wound sites. Time-lapse confocal imaging was used to characterize wound-induced cell death using GFP-Nit1 and markers of the nucleus and endoplasmic reticulum. These analyses provide dynamic portraits of well-known death-associated responses such as nuclear contraction and cellular collapse and reveal novel features such as nuclear envelope separation, ER vesiculation and loss of nuclear-lumen contents. As a parallel system for imaging cell death, we developed a chemical method for rapidly triggering cell death using the herbicides bromoxynil or chloroxynil which cause rapid GFP-Nit1 aggregation, loss of nuclear contents and cellular collapse, but not nuclear contraction, separating this response from others during plant cell death. CONCLUSION: Our observations place aggregation of Nitrilase 1 as one of the earliest events associated with wound and herbicide-induced cell death and highlight several novel cellular events that occur as plant cells die. Our data create a detailed descriptive framework for future investigations of plant cell death and provide new tools for both its cellular and biochemical analysis.

Aminohydrolases↗
PubMed1997

Microtubule-dependent transport of secretory vesicles visualized in real time with a GFP-tagged secretory protein.

Biosynthetic transport from the trans-Golgi network (TGN) to the plasma membrane (PM) is mediated by secretory vesicles. We analyzed secretory vesicle transport in real time using a GFP-tagged secretory protein, hCgB-GFP, consisting of human chromogranin B (hCgB) and green fluorescent protein (GFP). The fusion protein was expressed transiently in Vero cells or in a stable clone after induction with butyrate. After arrest of the biosynthetic protein transport at 20 degrees C, fluorescent hCgB-GFP colocalized with TGN38, a marker of the TGN. Subsequent release of the secretion block at 37 degrees C led to the formation of green fluorescent vesicles. Confocal analysis revealed that these vesicles were devoid of TGN38 and of Texas Red-coupled transferrin and cathepsin D, markers of the endosomal/lysosomal pathway. As determined by fluorometry and metabolic labelling hCgB-GFP was secreted from the TGN to the PM with a t(1/2) of 20-30 minutes. Video-microscope analysis of green fluorescent vesicles showed brief periods of rapid directed movement with maximal velocities of 1 microm/second. Vesicle movement occurred in all directions, centrifugal, centripetal and circumferential, and 50% of the vesicles analyzed reversed their direction of movement at least once within an observation period of 45 seconds. In the presence of nocodazole the movement of fluorescent vesicles ceased. Concomitantly, secretion of hCgB-GFP was slowed but not completely blocked. We suggest that microtubules (MT) facilitate the delivery of secretory vesicles to the PM by a stochastic transport, thereby increasing the probability for a vesicle/target membrane encounter.

Animals↗
PubMed2004

Effect of epsilon toxin-GFP on MDCK cells and renal tubules in vivo.

Epsilon toxin (epsilon-toxin), produced by Clostridium perfringens types B and D, causes fatal enterotoxemia, also known as pulpy kidney disease, in livestock. Recombinant epsilon-toxin-green fluorescence protein (epsilon-toxin-GFP) and epsilon-prototoxin-GFP were successfully expressed in Escherichia coli. MTT assays on MDCK cells confirmed that recombinant epsilon-toxin-GFP retained the cytotoxicity of the native toxin. Direct fluorescence analysis of MDCK cells revealed a homogeneous peripheral pattern that was temperature sensitive and susceptible to detergent. epsilon-Toxin-GFP and epsilon-prototoxin-GFP bound to endothelia in various organs of injected mice, especially the brain. However, fluorescence mainly accumulated in kidneys. Mice injected with epsilon-toxin-GFP showed severe kidney alterations, including hemorrhagic medullae and selective degeneration of distal tubules. Moreover, experiments on kidney cryoslices demonstrated specific binding to distal tubule cells of a range of species. We demonstrate with new recombinant fluorescence tools that epsilon-toxin binds in vivo to endothelial cells and renal tubules, where it has a strong cytotoxic effect. Our binding experiments indicate that an epsilon-toxin receptor is expressed on renal distal tubules of mammalian species, including human.

Animals↗
PubMed2006

The gas-phase absorption spectrum of a neutral GFP model chromophore.

We have studied the gas-phase absorption properties of the green fluorescent protein (GFP) chromophore in its neutral (protonated) charge state in a heavy-ion storage ring. To accomplish this we synthesized a new molecular chromophore with a charged NH(3) group attached to a neutral model chromophore of GFP. The gas-phase absorption cross section of this chromophore molecule as a function of the wavelength is compared to the well-known absorption profile of GFP. The chromophore has a maximum absorption at 415 +/- 5 nm. When corrected for the presence of the charged group attached to the GFP model chromophore, the unperturbed neutral chromophore is predicted to have an absorption maximum at 399 nm in vacuum. This is very close to the corresponding absorption peak of the protein at 397 nm. Together with previous data obtained with an anionic GFP model chromophore, the present data show that the absorption of GFP is primarily determined by intrinsic chromophore properties. In other words, there is strong experimental evidence that, in terms of absorption, the conditions in the hydrophobic interior of this protein are very close to those in vacuum.

Animals↗
PubMed2006

In vivo direct molecular imaging of early tumorigenesis and malignant progression induced by transgenic expression of GFP-Met.

The tyrosine kinase receptor Met and its ligand, hepatocyte growth factor/scatter factor (HGF/SF), play an important role in normal developmental processes, as well as in tumorigenicity and metastasis. We constructed a green fluorescent protein (GFP) Met chimeric molecule that functions similarly to the wild-type Met receptor and generated GFP-Met transgenic mice. These mice ubiquitously expressed GFP-Met in specific epithelial and endothelial cells and displayed enhanced GFP-Met fluorescence in sebaceous glands. Thirty-two percent of males spontaneously developed adenomas, adenocarcinomas, and angiosarcomas in their lower abdominal sebaceous glands. Approximately 70% of adenocarcinoma tumors metastasized to the kidneys, lungs, or liver. Quantitative subcellular-resolution intravital imaging revealed very high levels of GFP-Met in tumor lesions and in single isolated cells surrounding them, relative to normal sebaceous glands. These single cells preceded the formation of local and distal metastases. Higher GFP-Met levels correlated with earlier tumor onset and aggressiveness, further demonstrating the role of Met-HGF/SF signaling in cellular transformation and acquisition of invasive and metastatic phenotypes. Our novel mouse model and high-resolution intravital molecular imaging create a powerful tool that enables direct real-time molecular imaging of receptor expression and localization during primary events of tumorigenicity and metastasis at single-cell resolution.

Animals↗
PubMed2002

Cell sorting of formalin-treated pathogenic Mycobacterium paratuberculosis expressing GFP.

GFP is widely used as a molecular tool for the study of microbial pathogens. However, the manipulation of these pathogenic microorganisms poses a health threat to the laboratory worker, requiring biosafety level II or III containment. Although the GFPfluorophore is tolerant toformalin, a thorough analysis of this treatment on fluorescent output in prokaryotic systems has not been described. In addition, the analysis of microorganisms expressing GFP often depends on specialized equipment, which may not be housed in biosafety level II or III laboratories. Therefore, we sought to develop a safe and effective method for manipulating the GFP-expressing pathogenic bacterium Mycobacterium avium subsp, paratuberculosis (M. paratuberculosis) utilizing a formalin treatment that would permit the analysis of GFP fluorescence without requiring stringent biosafety containment. We demonstrate that formalin-treated M. paratuberculosis expresses 50% less fluorescence than viable cells, but this reduction is still compatible with spectrofluorometry and cell sorting. Furthermore, plasmid DNA that expresses GFP can be recovered efficiently from nonviable, sorted fluorescent cells. This approach is flexible, provides an additional margin of safety for laboratory personnel, and can be easily applied to other pathogenic microorganisms expressing GFP.

Blotting, Southern↗
PubMed2001

Role of cytoskeletal elements in the recruitment of Cx43-GFP and Cx26-YFP into gap junctions.

Cytoskeletal elements may be important in connexin transport to the cell surface, cell surface gap junction plaque formation and/or gap junction internalization. In this study, fluorescence recovery after photobleaching was used to examine the role of microfilaments and microtubules in the recruitment and coalescence of green fluorescent protein-tagged Cx43 (Cx43-GFP) or yellow fluorescent tagged-Cx26 (Cx26-YFP) into gap junctions in NRK cells. In untreated cells, both Cx26-YFP and Cx43-GFP were recruited into gap junctions within photobleached areas of cell-cell contact within 2 hrs. However, disruption of microfilaments with cytochalasin B inhibited the recruitment and assembly of both Cx26-YFP and Cx43-GFP into gap junctions within photobleached areas. Surprisingly, disruption of microtubules with nocodazole inhibited the recruitment of Cx43-GFP into gap junctions but had limited effect on the transport and clustering of Cx26-YFP into gap junctions within the photobleached regions of cell-cell contact. These results suggest that the recruitment of Cx43-GFP and Cx26-YFP to the cell surface or their lateral clustering into gap junctions plaques is dependent in part on the presence of intact actin microfilaments while Cx43-GFP was more dependent on intact microtubules than Cx26-YFP.

Actin Cytoskeleton↗
PubMed2004

Intracerebral xenotransplantation of GFP mouse bone marrow stromal cells in intact and stroke rat brain: graft survival and immunologic response.

The present study characterized survival and immunologic response of bone marrow stromal cells (BMSCs) following transplantation into intact and stroke brains. In the first study, intrastriatal transplantation of BMSC (60,000 in 3 microl) or vehicle was performed in normal adult Sprague-Dawley male rats that subsequently received daily cyclosporin A (CsA, 10 mg/kg, IP in 3 ml) or vehicle (olive oil, similar volume) starting on day of surgery up to 3 days posttransplantation. Animals were euthanized at 3 or 30 days posttransplantation and brains were processed either for green fluorescent protein (GFP) microscopy or flow cytometry (FACS). Both GFP epifluorescence and FACS scanning revealed GFP+ BMSCs in both groups of transplanted rats with or without CsA, although significantly increased (1.6- to 3-fold more) survival of GFP+ BMSCs was observed in the immunosuppressed animals. Further histologic examination revealed widespread dispersal of BMSCs away from the graft core accompanied by many long outgrowth processes in non-CsA-transplanted animals, whereas a very dense graft core, with cells expressing only sporadic short outgrowth processes, was observed in CsA-transplanted animals. There were no detectable GFP+ BMSCs in nontransplanted rats that received CsA or vehicle. Immunologic response via FACS analysis revealed a decreased presence of cytotoxic cells, characterized by near complete absence of CD8+ cells, and lack of activation depicted by low CD69 expression in CsA-treated transplanted animals. In contrast, elevated levels of CD8+ cells and increased activation of CD69 expression were observed in transplanted animals that received vehicle alone. CD4+ helper cells were almost nondetectable in transplanted rats that received CsA, but also only minimally elevated in transplanted rats that received vehicle. Nontransplanted rats that received either CsA or vehicle displayed very minimal detectable levels of all three lymphocyte markers. In the second study, a new set of male Sprague-Dawley rats initially received bilateral stereotaxic intrastriatal transplantation of BMSCs and 3 days after were subjected to unilateral transient occlusion of middle cerebral artery. The animals were allowed to survive for 3 days after stroke without CsA immunosuppression. Epifluorescence microscopy revealed significantly higher (5-fold more) survival of transplanted GFP+ BMSCs in the stroke striatum compared with the intact striatum. The majority of the grafts remained within the original dorsal striatal transplant site, characterized by no obvious migration in intact striatum, but with long-distance migration along the ischemic penumbra in the stroke striatum. Moreover, FACS scanning analyses revealed low levels of immunologic response of grafted BMSCs in both stroke and intact striata. These results, taken together, suggest that xenotransplantation of mouse BMSCs into adult rats is feasible. Immunosuppression therapy can enhance xenograft survival and reduce graft-induced immunologic response; however, in the acute phase posttransplantation, BMSCs can survive in intact and stroke brain, and may even exhibit long-distance migration and increased outgrowth processes without immunosuppression.

Animals↗
PubMed2001

Efficient intracellular delivery of an exogenous protein GFP with genetically fused basic oligopeptides.

Several oligopeptides, derived from certain proteins, translocate as a form fused to small molecules or exogenous proteins across the plasma membrane into cells. Some of these oligopeptides, the so-called protein-transduction domains (PTDs), contain a high proportion of basic residues. The translocation of some of these basic PTDs, such as oligoarginines, has been studied as chemically fused forms to other organic compounds. In this study, we also tested to determine whether or not oligoarginines, when fused genetically to an exogenous protein such as GFP, are also able to translocate efficiently across the plasma membrane. The oligoarginine Rn (n = 5,6,7,8,9)-GFP fusion proteins were translocated quite efficiently, and the transduction efficiency increased in proportion to the number of arginine residues. However, the cellular uptake of the oligolysine-GFP fusion proteins was less efficient than that of the corresponding oligoarginine-GFP fusion proteins. When fused to GFP, the translocation efficiency of R5 was similar to that of Tat(49-57)(RKKRRQRRR). This finding suggests that the arginine homo-oligopeptide is more efficient than other PTDs which contain a mixture of basic residues. On the other hand, both the K9- and Tat(49-57)-GFP fusion proteins were transduced with similar efficiencies. It appears that basic oligopeptides may be useful for the efficient translocation of diverse exogenous proteins as genetically fused forms.

Amino Acid Sequence↗
PubMed2001

GFP-centrin as a marker for centriole dynamics in the human breast cancer cell line MCF-7.

Centrosome duplication plays an important role in genomic stability through bipolar spindle formation and equal chromosome segregation during mitosis. Defects in centrosome duplication and centrosome amplification correlate with aggressive tumors and aneuploidy. Cyclin-dependent cell cycle regulators play a key role in signaling centrosome duplication and the tumor suppressor genes p53, BRCA1 and BRCA2 are suspected to function at mitotic checkpoints that monitor centrosome duplication. The relationship between loss of hormone dependence in breast cancer, and signaling of centrosome duplication in tumor progression is not known. We have developed a MCF-7 cell line expressing GFP-centrin that allows direct visualization of centriole duplication during the cell cycle in living cells. GFP-centrin is expressed and selectively incorporated into the structure of both centrioles making them clearly visible in living cells. Our studies demonstrate three important aspects of recombinant GFP-centrin incorporation into centrioles. 1) GFP-centrin transfected cells grow normally in culture and show no adverse effect associated with GFP-centrin expression; 2) newly duplicated centrioles incorporate centrin during their genesis; and 3) GFP-centrin incorporation into centrioles does not grossly affect cell cycle progression, or centrosome function.

Aneuploidy↗
PubMed2003

Visualization of microtubule growth in cultured neurons via the use of EB3-GFP (end-binding protein 3-green fluorescent protein).

Several microtubule binding proteins, including CLIP-170 (cytoplasmic linker protein-170), CLIP-115, and EB1 (end-binding protein 1), have been shown to associate specifically with the ends of growing microtubules in non-neuronal cells, thereby regulating microtubule dynamics and the binding of microtubules to protein complexes, organelles, and membranes. When fused to GFP (green fluorescent protein), these proteins, which collectively are called +TIPs (plus end tracking proteins), also serve as powerful markers for visualizing microtubule growth events. Here we demonstrate that endogenous +TIPs are present at distal ends of microtubules in fixed neurons. Using EB3-GFP as a marker of microtubule growth in live cells, we subsequently analyze microtubule dynamics in neurons. Our results indicate that microtubules grow slower in neurons than in glia and COS-1 cells. The average speed and length of EB3-GFP movements are comparable in cell bodies, dendrites, axons, and growth cones. In the proximal region of differentiated dendrites approximately 65% of EB3-GFP movements are directed toward the distal end, whereas 35% are directed toward the cell body. In more distal dendritic regions and in axons most EB3-GFP dots move toward the growth cone. This difference in directionality of EB3-GFP movements in dendrites and axons reflects the highly specific microtubule organization in neurons. Together, these results suggest that local microtubule polymerization contributes to the formation of the microtubule network in all neuronal compartments. We propose that similar mechanisms underlie the specific association of CLIPs and EB1-related proteins with the ends of growing microtubules in non-neuronal and neuronal cells.

Animals↗
PubMed2004

Controversial aspect of using GFP as a marker for the production of transgenic cattle.

The objective of this study was to examine the feasibility of identification and selection of cattle embryos based on green fluorescence (GFP-positive) in order to obtain calves carrying an integrated transgene. The construct used (pbLGTNF-EGFP) contained the human tumor necrosis factor alpha (hTNFalpha) gene fused to the bovine beta-lactoglobulin promoter (bLG) in plasmid vector pCX-EGFP. In four experiments, 76 zygotes were injected; eight of them developed to the morulae/blastocysts stage of which only five were GFP positive (one of them 100%, one-50%, three- 25%). All of the GFP positive embryos were transferred to recipients. Two calves were born: one after transfer of the 100% GFP positive embryo and the other after transfer of one of the 25% GFP positive embryos. Both animals were healthy with normal weight when compared to two control calves. The integration of pbLGTNF-EGFP in the host genome could not be detected in either of the calves, suggesting that GFP is an unreliable marker for preimplantation screening of embryos.

Animals↗
PubMed2004

FADDdel-GFP modified mouse insulinoma cells counteract the cytotoxicity of reactive T cells.

IDDM results from pancreatic beta cell destruction by islet-reactive T cells, a process that involves beta cell apoptosis. Fas-FasL pathway plays a major role in pancreatic beta cell death. Fas-associated death domain protein (FADD), the component of the tumor necrosis factor receptor type 1 (TNFR1) and Fas signaling complexes, is involved in TNFR1- and Fas-induced apoptosis. Inhibiting the function of FADD will lead to blocking downstream apoptosis signal, which protects pancreatic beta cells from destruction by Fas-FasL pathway. In this study we constructed eukaryotic expressing vector of fusional protein FADDdel-GFP named pFADDdel-GFP. After pFADDdel-GFP was transfected into NIT, the expression of FADDdel-GFP in NIT was detected by fluorescence microscopy and the resistance of NIT transfected with pFADDdel-GFP to cytotoxicity mediated by special T cells was detected by FACS and MTT. The results showed that NIT modified by pFADDdel-GFP obviously resisted cytotoxicity mediated by special T cells. Therefore, it may be useful in the prevention or treatment of IDDM by intervening Fas-FasL pathway.

Adaptor Proteins, Signal Transducing↗
PubMed2006

[Expression vector for the inhibitor of growth-1 gene is constructed and the NLS-GFP fusion protein expresses in MRC-5 cells].

OBJECTIVE: To construct the NLS(ING1)-GFP vector, transfer it into MRC-5 cells and establish a cell model expressing NLS (ING1)-GFP fusion protein. METHODS: Firstly, cDNA fragment of nuclear locating sequence (NLS) of inhibitor of growth-1 gene (ING1) was gained by RT-PCR and inserted into multi-clone site of pEGFP-C1 to construct the NLS (ING1)-GFP expression vector. Then the vector was used to transfect the MRC-5 cells to observe the subcellular signal localization of green fluorescence protein (GFP). RESULTS: We successfully constructed the expressing vector of NLS (ING1)-GFP fusion protein. After transferring the fusion expressing vector into MRC-5 cells, we observed that green fluorescence signal located in the cell nucleus. However, the green fluorescence signal located in the cytoplasm in MRC-5 cells transfected with pEGFP-C1 control only expressing GFP. CONCLUSION: In living cells, physiologically p33 ING1b locates absolutely in nucleus. The p33(ING1b) NLS plays a decisive role in the transporting process of subcellular localization.

Base Sequence↗
PubMed1996

Rapid generation and flow cytometric analysis of stable GFP-expressing cells.

Expression of green fluorescent protein (GFP) represents a unique method for the fluorescent labeling of viable mammalian cells, with many potential applications. The studies detailed in this report examine the detection of GFP expression in murine cells using flow cytometry. Direct comparison of NIH 3T3 cells transiently expressing GFP or GFPS65T, a mutant form of GFP, showed that GFPS65T fluorescence (using 488 nm excitation) was detected more readily than fluorescence from wildtype GFP. Efficient generation of cell lines that stably expression GFPS65T was achieved using a plasmid vector that encoded a hygromycin phosphotransferase/GFPS65T fusion protein. Flow cytometric detection of NIH 3T3 cells expressing this fusion protein was improved using a 510/20 band pass filter instead of the standard filter setup for fluorescein detection. Additionally, staining the surface of these cells with phycoerythrin, RED 670, or allophycocyanin did not interfere with the detection of GFPS65T fluorescence, indicating that multiparameter analyses using GFPS65T fluorescence are possible. Importantly, we also observed that GFPS65T expression could be detected in NIH 3T3, BW5147, or freshly cultured Thy1lo CD3- murine bone marrow cells transduced with a retroviral vector encoding the fusion protein, suggesting that the potential applications of this system may be quite broad.

3T3 Cells↗