Spatial and temporal patterns of green fluorescent protein (GFP) fluorescence during leaf canopy development in transgenic oilseed rape, Brassica napus L.

ABSTRACT 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 jellyfishAequoria 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 Ap1gfp 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 μg of available chlorine ml−1 and 500 μg ml−1, respectively. Further, loss of GFP fluorescence from A. pullulanscells 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. pullulanswas 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.

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Non-viral genetic transfection of rat Schwann cells with FuGENE HD© lipofection and AMAXA© nucleofection is feasible but impairs cell viability

Neuron Glia Biology ◽

10.1017/s1740925x11000056 ◽

2010 ◽

Vol 6 (4) ◽

pp. 225-230 ◽

Cited By ~ 4

Author(s):

Armin Kraus ◽

Joachim Täger ◽

Konrad Kohler ◽

Max Haerle ◽

Frank Werdin ◽

...

Keyword(s):

Cell Culture ◽

Green Fluorescent Protein ◽

Cell Viability ◽

Schwann Cells ◽

Fluorescent Protein ◽

Transfection Efficiency ◽

Cell Counting ◽

Gfp Fluorescence ◽

Green Fluorescent ◽

Viral Transfection

Purpose:To determine transfection efficiency of FuGENE HD© lipofection and AMAXA© nucleofection on rat Schwann cells (SC).Methods:The ischiadic and median nerves of 6-8 week old Lewis rats were cultured in modified melanocyte-growth medium. SCs were genetically transfected with green fluorescent protein (GFP) as reporter gene using FuGENE HD© lipofection and AMAXA© nucleofection. Transfection rates were determined by visualization of GFP fluorescence under fluorescence microscopy and cell counting. Transfected cell to non-transfected cell relation was determined.Results:Purity of Schwann cell culture was 88% as determined by immunohistologic staining. Transfection rate of FuGENE HD© lipofection was 2%, transfection rate of AMAXA© nucleofection was 10%. With both methods, Schwann cells showed pronounced aggregation behavior which made them unfeasible for further cultivation. Settling of Schwann cells on laminin and poly-l-ornithine coated plates was compromised by either method.Conclusion:Non-viral transfection of rat SC with FuGENE HD© lipofection and AMAXA© nucleofection is basically possible with a higher transfection rate for nucleofection than for lipofection. As cell viability is compromised by either method however, viral transfection is to be considered if higher efficiency is required.

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Predictive and Interpretive Simulation of Green Fluorescent Protein Expression in Reporter Bacteria

Journal of Bacteriology ◽

10.1128/jb.183.23.6752-6762.2001 ◽

2001 ◽

Vol 183 (23) ◽

pp. 6752-6762 ◽

Cited By ~ 129

Author(s):

Johan H. J. Leveau ◽

Steven E. Lindow

Keyword(s):

Growth Rate ◽

Green Fluorescent Protein ◽

Green Fluorescent Protein Expression ◽

Fluorescent Protein ◽

Proteolytic Degradation ◽

Bacterial Cells ◽

Prior Testing ◽

Gfp Fluorescence ◽

Green Fluorescent ◽

Best Fit

ABSTRACT We have formulated a numerical model that simulates the accumulation of green fluorescent protein (GFP) in bacterial cells from a generic promoter-gfp fusion. The model takes into account the activity of the promoter, the time it takes GFP to mature into its fluorescent form, the susceptibility of GFP to proteolytic degradation, and the growth rate of the bacteria. From the model, we derived a simple formula with which promoter activity can be inferred easily and quantitatively from actual measurements of GFP fluorescence in growing bacterial cultures. To test the usefulness of the formula, we determined the activity of the LacI-repressible promoter P A1/O4/O3 in response to increasing concentrations of the inducer IPTG (isopropyl-β-d-thiogalactopyranoside) and were able to predict cooperativity between the LacI repressors on each of the two operator sites within P A1/O4/O3 . Aided by the model, we also quantified the proteolytic degradation of GFP[AAV], GFP[ASV], and GFP[LVA], which are popular variants of GFP with reduced stability in bacteria. Best described by Michaelis-Menten kinetics, the rate at which these variants were degraded was a function of the activity of the promoter that drives their synthesis: a weak promoter yielded proportionally less GFP fluorescence than a strong one. The degree of disproportionality is species dependent: the effect was more pronounced in Erwinia herbicola than in Escherichia coli. This phenomenon has important implications for the interpretation of fluorescence from bacterial reporters based on these GFP variants. The model furthermore predicted a significant effect of growth rate on the GFP content of individual bacteria, which if not accounted for might lead to misinterpretation of GFP data. In practice, our model will be helpful for prior testing of different combinations of promoter-gfpfusions that best fit the application of a particular bacterial reporter strain, and also for the interpretation of actual GFP fluorescence data that are obtained with that reporter.

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A method for quantifying the gene flow that results from a single bumblebee visit using transgenic oilseed rape,Brassica napus L. cv. Westar

Transgenic Research ◽

10.1007/bf01974092 ◽

1994 ◽

Vol 3 (2) ◽

pp. 134-137 ◽

Cited By ~ 19

Author(s):

James E. Cresswell

Keyword(s):

Gene Flow ◽

Brassica Napus ◽

Oilseed Rape ◽

Brassica Napus L ◽

Transgenic Oilseed Rape

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A Rapid Method to Determine the Stress Status of Saccharomyces cerevisiae by Monitoring the Expression of a Hsp12:Green Fluorescent Protein (GFP) Construct under the Control of the Hsp12 Promoter

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057104273485 ◽

2005 ◽

Vol 10 (3) ◽

pp. 253-259 ◽

Cited By ~ 9

Author(s):

Robert J. Karreman ◽

George G. Lindsey

Keyword(s):

Saccharomyces Cerevisiae ◽

Heat Stress ◽

Green Fluorescent Protein ◽

Fluorescence Microscopy ◽

Fusion Protein ◽

Expression Vector ◽

Fluorescent Protein ◽

Time Dependent ◽

Gfp Fluorescence ◽

Green Fluorescent

The gene for the green fluorescent protein (GFP) was fused in-frame to the 3′ end of HSP12. This construct was regulated by the HSP12 promoter in a pYES2 yeast expression vector. No fluorescence was observed in yeast growing exponentially in glucose-containing medium, but fluorescence was observed when the yeast entered the stationary phase. Fluorescence microscopy indicated that the fusion protein was localized to the peripheral regions of the cell as well as to the cytoplasm and the tonoplast. Subjecting the yeast to a variety of stresses known to induce HSP12 transcription, including salt, osmotic, ethanol, and heat stress, resulted in a time-dependent increase in GFP fluorescence. The use of this system as a method to assess the general stress status of yeast growing in an industrial application is proposed.

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Comparison of the performance of bromoxynil-resistant and susceptible near-isogenic populations of oilseed rape

Canadian Journal of Plant Science ◽

10.4141/p00-115 ◽

2001 ◽

Vol 81 (3) ◽

pp. 367-372 ◽

Cited By ~ 11

Author(s):

Janice L. Cuthbert ◽

Peter B. E. McVetty ◽

Georges Freyssinet ◽

Martine Freyssinet

Keyword(s):

Brassica Napus ◽

Oilseed Rape ◽

Herbicide Resistance ◽

Negative Effects ◽

Brassica Napus L ◽

Comparative Performance ◽

Positive Effects ◽

Biological Cost ◽

Transgenic Oilseed Rape ◽

The University

Bromoxynil herbicide resistance is the newest type of broad-spectrum, non-selective herbicide resistance to be introduced into oilseed rape (Brassica napus L.). This herbicide resistance is conferred by a single transgene (the oxy gene), taken from a soil bacterium, which confers the ability to metabolize hydroxybenzonitrile herbicides such as bromoxynil. The level of resistance to bromoxynil herbicide in oilseed rape is high, but it is not known whether there are any performance changes associated with the oxy transgene or with the derived herbicide resistance. To determine if there are changes in performance related to the oxy transgene, or the derived herbicide resistance, two near-isogenic transgenic bromoxynil-resistant populations, with two different forms of the oxy gene, Westar 235 and Westar 237, were developed at the University of Manitoba, grown in the field in Manitoba for several years and evaluated for comparative performance. Westar 235 and Westar 237 near-isogenic populations, either sprayed with bromoxynil at 280 g a.i. ha–1 or left unsprayed, were compared with the non-transgenic near-isogenic population cultivar, Westar, in Winnipeg, Carman, and Portage la Prairie from 1994 to 1997. There were no consistent differences in the performance of the sprayed or unsprayed transgenic near-isogenic populations in comparison to Westar for any trait. The few significant and minimal differences that were found were evenly divided between positive effects and negative effects of the oxy gene and derived herbicide resistance. There were no significant effects of spraying bromoxynil herbicide detected in paired comparisons of Westar 235 and Westar 237 sprayed versus Westar 235 and Westar 237 unsprayed, respectively. The effects of the oxy transgenes and the effects of bromoxynil spraying of herbicide-resistant lines were negligible, indicating that there is little, if any biological cost associated with the bromoxynil resistance transgenes or related herbicide resistance. Key words: Transgenic oilseed rape, Brassica napus, biological cost

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