Green fluorescent protein: A novel viability assay for cryobiological applications

Assessment of tissue viability following the application of a freezing protocol is challenging due to the paucity of viability assays that can be used dynamically, in situ. Cells transfected with a green fluorescent protein (GFP) vector actively produce GFP, which is retained intracellularly. Because of its constitutive and heritable expression, GFP fluorescence of transfected cells may have significant utility as a viability assay for cells within tissues. As a first step toward application to tissues, this work seeks to establish the validity of this GFP-based assay in cell suspensions by comparing the results to other accepted measures of viability. To the authors' knowledge, this is the first use of GFP in cryobiology applications. Intracellular GFP fluorescence was evaluated following slow freezing. Nontransfected and GFP-transfected rat 3230 adenocarcinoma (R3230AC) cells were frozen at 1 degrees C/min to minimum temperatures between -5 and -30 degrees C and then immediately thawed in a 37 degrees C water bath. Samples were assayed using the common viability indicators trypan blue and ethidium bromide (EtBr). A regression analysis of recovery measured with the GFP assay as a function of recovery measured with a trypan blue assay gave a correlation coefficient of 0.97. A similar correlation coefficient, 0.95, was determined for recovery assessed by the GFP assay as a function of recovery measured by an EtBr assay. Nontransfected and GFP-transfected cells responded similarly to slow freezing, indicating that GFP transfection did not significantly alter the response of cells to typical freezing conditions. The excellent correlation of GFP assay results with those of two common viability assays suggests that the GFP-based assay is valid for cells and that further development of a tissue viability assay based on transfection is appropriate.     

Green fluorescent protein as a novel tool to measure apoptosis and necrosis

BACKGROUND: Several apoptosis-detecting methods are currently available. Many of them are work intensive and require the additional use of antibodies, dyes, specific substrates, or enzymatic reactions. A simple, fast, and reliable method was developed to test for apoptosis or necrosis using mouse and human cell lines (e.g., Jurkat, A20.2J, and PB3c cells) stably transfected with a vector coding for green fluorescent protein (GFP) as indicator cells. METHODS: Apoptosis in GFP-transfected cell lines was induced either by soluble Fas-Ligand (sFasL), recombinant human TRAIL (rhTRAIL), or interleukin-3 (IL-3) deprivation. Necrosis was induced by polyclonal anti-A20 and complement treatment of GFP-transfected A20. Cells were analyzed by flow cytometry for GFP fluorescence. Propidium iodide and Annexin V staining were used to confirm the results obtained with the GFP-method. RESULTS: Live GFP-transfected cells show a strong fluorescence intensity, which is significantly diminished upon induction of apoptosis, whereas necrotic GFP-transfected cells almost completely lose their GFP-associated fluorescence. Apoptosis but not necrosis of GFP-transfected cells was blocked by the use of a caspase inhibitor. The results are highly comparable to conventional apoptosis-detecting methods. CONCLUSIONS: The advantage of our GFP-based assay compared with other methods is the analysis of apoptosis or necrosis without the necessity for additional staining or washing steps, making it an ideal tool for screening apoptotic or necrotic stimuli.     

Green fluorescent protein as a novel indicator of antimicrobial susceptibility in Aureobasidium pullulans.

Presently there is no method available that allows noninvasive and real-time monitoring of fungal susceptibility to antimicrobial compounds. The green fluorescent protein (GFP) of the jellyfish Aequoria victoria was tested as a potential reporter molecule for this purpose. Aureobasidium pullulans was transformed to express cytosolic GFP using the vector pTEFEGFP (A. J. Vanden Wymelenberg, D. Cullen, R. N. Spear, B. Schoenike, and J. H. Andrews, BioTechniques 23:686-690, 1997). The transformed strain Ap1 gfp showed bright fluorescence that was amenable to quantification using fluorescence spectrophotometry. Fluorescence levels in Ap1 gfp blastospore suspensions were directly proportional to the number of viable cells determined by CFU plate counts (r(2) > 0.99). The relationship between cell viability and GFP fluorescence was investigated by adding a range of concentrations of each of the biocides sodium hypochlorite and 2-n-octylisothiozolin-3-one (OIT) to suspensions of Ap1 gfp blastospores (pH 5 buffer). These biocides each caused a rapid (< 25-min) loss of fluorescence of greater than 90% when used at concentrations of 150 microg of available chlorine ml(-1) and 500 microg ml(-1), respectively. Further, loss of GFP fluorescence from A. pullulans cells was highly correlated with a decrease in the number of viable cells (r(2) > 0.92). Losses of GFP fluorescence and cell viability were highly dependent on external pH; maximum losses of fluorescence and viability occurred at pH 4, while reduction of GFP fluorescence was absent at pH 8.0 and was associated with a lower reduction in viability. When A. pullulans was attached to the surface of plasticized poly(vinylchloride) containing 500 ppm of OIT, fluorescence decreased more slowly than in cell suspensions, with > 95% loss of fluorescence after 27 h. This technique should have broad applications in testing the susceptibility of A. pullulans and other fungal species to antimicrobial compounds.     

绿色荧光蛋白及其应用

绿色荧光蛋白是在水母中发现的新型报告分子,能在多种生物体内表达并发出荧光。对GFP中一些特定氨基酸进行突变可以产生多种类型的突变体,有利于研究蛋白之间或细胞器之间的相互作用。目前,GFP已经用于基因表达的报告、细胞动态的研究、活细胞内蛋白的定位及westernbloting检测中。GFP美好的应用前景也促进了有关GFP的研究,特别是寻找新的突变体并将之运用到细胞生物学和分子生物学的各个领域。     

A novel fluorescent probe for the detection of nitric oxide in vitro and in vivo

Fluorescence imaging of nitric oxide (NO) in vitro and in vivo is essential to developing our understanding of the role of nitric oxide in biology and medicine. Current probes such as diaminofluorescein depend on reactions with oxidized NO products, but not with nitric oxide directly, and this limits their applicability. Here we report the formation of an imaging probe for nitric oxide by coordinating the highly fluorescent chemical 4-methoxy-2-(1H-naphtho[2,3-d]imidazol-2-yl)phenol (MNIP) with Cu(II). The coordination compound MNIP-Cu reacts rapidly and specifically with nitric oxide to generate a product with blue fluorescence that can be used in vitro and in vivo. In the present study MNIP-Cu was used to reveal nitric oxide produced by inducible nitric oxide synthase in lipopolysaccharide (LPS)-activated macrophages (Raw 264.7 cells) and by endothelial nitric oxide synthase in endothelial cells (HUVEC). MNIP-Cu was also used to evaluate the distribution of nitric oxide synthesis in a model of acute liver injury induced by LPS and d-galactosamine in mice. The results demonstrate that MNIP-Cu can act as a novel fluorescent probe for nitric oxide and has many potential applications in biomedical research.     

A novel, high-affinity, fluorescent progesterone receptor antagonist. Synthesis and in vitro studies

The present paper describes the chemical synthesis and in vitro characterization of a novel, high-affinity, fluorescent progesterone receptor (PR) antagonist. The three-step synthesis was carried out starting from mifepristone. After demethylation with calcium oxide, the methylamino group was alkylated with 6-bromohexanol, and the resulting compound was reacted with fluorescein 5-isothiocyanate, yielding the fluorescein-mifepristone conjugate. Interaction of the conjugate as well as of its precursors with PR was determined in cell culture (alkaline phosphatase assay and transactivation assay). Antiprogestagenic activity of the intermediates were comparable to that of the parent compound. Even after attachment of the bulky fluorescein moiety, considerable antiprogestagenic activity was maintained. Microscopic studies revealed that fluorescence of the conjugate was almost confined to the nuclei of steroid hormone receptor-positive cells, whereas the nuclei of steroid hormone receptor-negative cells remained unstained. To our knowledge, this is the first report on a fluorescent ligand for PR suitable for studies in living cells. It is proposed that the present fluorescent PR antagonist might serve as a lead compound for the development of contrast agents for PR imaging, e.g., by near-infrared optical imaging.     

Dendrimer-based fluorescent indicators: in vitro and in vivo applications

BackgroundThe development of fluorescent proteins and synthetic molecules whose fluorescence properties are controlled by the environment makes it possible to monitor physiological and pathological events in living systems with minimal perturbation. A large number of small organic dyes are available and routinely used to measure biologically relevant parameters. Unfortunately their application is hindered by a number of limitations stemming from the use of these small molecules in the biological environment.ConclusionWe believe that the proposed architecture can represent a useful and novel tool in fluorescence imaging that can be widely applied in conjunction with a broad range of sensing dyes and experimental setups.     

Green fluorescent protein: an in vivo reporter of plant gene expression

Summary Protoplasts were isolated from H89, an embryogenic sweet orange (Citrus sinensis (L.) Osbeck cv. Hamlin) suspension culture, and electroporated with p35S-GFP, a plasmid carrying the gene for the green fluorescent protein (GFP) from the bioluminescent jellyfish Aequorea victoria. p35S-GFP was constructed by replacing the GUS coding sequence of pBI221 with a functional GFP gene, thereby placing the GFP gene under the control of the CaMV 35S promoter. Protoplasts were viewed by incident-light fluorescence microscopy twentyfour h after electroporation. 20–60% of the protoplasts emitted an intense green light when illuminated with blue (450–490 nm) light.Abbreviations GUS -glucuronidase - LUC luciferase - NPTII neomycinphosphotransferase - CaMV cauliflower mosaic virus - MUG 4-methylumbelliferyl -D-glucuronide     

Green fluorescent protein as an all-purpose reporter in Petunia

Two critical attributes of a reporter gene are ease of scoring for activity and capacity for expression in all cell types. We have examined a variant of the gene encoding green fluorescent protein,mgfp5, for its ability to meet these criteria in petunia. Under regulation of the Cauliflower Mosaic Virus (CaMV) 35S promoter, GFP was detectable in all vegetative and most floral cell types. Promoters from petuniaadhl andadh2 allowed for production of GFP in those few cell types lacking GFP production from the CaMV 35S promoter, verifying its capacity for expression in all cell types. With the appropriate promoter, GFP fluorescence was thus readily detectable throughout the plant. A potential complication is the green autofluorescence exhibited by some plant tissues. This auto-fluorescence is for the most part distinguishable from that contributed by GFP, but under-scores the need for appropriate controls in GFP-reporter-based experiments. An erratum to this article is available at .     

Green fluorescent protein as a marker in transgenic mice

Green fluorescent protein (GFP) found in Aequorea victoria absorbs blue light and emits green fluorescence without exogenous substrates or co-factors. We studied the possibility of using the GFP as a marker in mammals. Transgenic mice were produced using the GFP coding sequence, ligated with the chicken beta-actin promoter. Green fluorescence was observed in muscle, pancreas, kidney, heart and other organs in all the three transgenic mouse lines. Detection of the transgenic mouse was possible by observing a tail or fingers of new born pups under a fluorescent microscope. The marker also enabled us to detect localized expression of the transgene in intact tissues without preliminary steps. It was also demonstrated that the GFP expression could be quantified by measuring the fluorescence in tissue extracts.     

Green fluorescent protein as a molecular marker in microbiology

Molecular markers such as: lacZ (b-galactosidase), xylE (catechol 2,3-dioxygenase), lux (bacterial luciferase), luc (insect luciferase), phoA (alkaline phosphatase), gusA and gurA (beta-glucuronidase), gfp (green fluorescent protein), bla (beta-lactamase) and other antibiotic resistance markers, heavy metals resistance genes are commonly used in environmental microorganisms research (Errampaii et al., 1998; Kohler et al., 1999). Most of these markers require one or more substrates, complex media and/or expensive equipment for detection. The gfp gene is widely used as a marker because of its very useful properties such as high stability, minimal toxicity, non-invasive detection and the ability to generate the green light without addition of external cofactors and without application of expensive equipment. Various applications of that reporter gene were showed starting from monitoring of microorganism's survival in complex biological systems such as activated sludge to biodegradation of chemical compounds in soil. GFP allowed the detection, determination of spatial location and enumeration of bacterial cells from diverse environmental samples such as biofilm and water. The gfp as a biomarker was very useful in monitoring of gene expression and protein localisation in bacterial cells, too. The techniques with using gfp marker promise to supply a better understanding of environmental processes. It can make possible to use that knowledge in designing more effective and more efficient methods of biodegradation of toxic compounds from different environments.     

Green fluorescent protein-transgenic rat: a tool for organ transplantation research

The purpose of this study is to evaluate green fluorescent protein (GFP) transgenic rats for use as a tool for organ transplantation research. The GFP gene construct was designed to express ubiquitously. By flow cytometry, the cells obtained from the bone marrow, spleen, and peripheral blood of the GFP transgenic rats consisted of 77, 91, and 75% GFP-positive cells, respectively. To examine cell migration of GFP-positive cells after organ transplantation, pancreas graft with or without spleen transplantation, heart graft with or without lung transplantation, auxiliary liver and small bowel transplantation were also performed from GFP transgenic rat to LEW (RT1(1)) rats under a 2-week course of 0.64 mg/kg tacrolimus administration. GFP-positive donor cells were detected in the fully allogenic LEW rats after organ transplantation. These results showed that GFP transgenic rat is a useful tool for organ transplantation research such as cell migration study after organ transplantation without donor cell staining.