A High-Throughput Approach to Promoter Study Using Green Fluorescent Protein

2004 ◽  
Vol 20 (6) ◽  
pp. 1634-1640 ◽  
Author(s):  
C. Lu ◽  
W.E. Bentley ◽  
G. Rao
Keyword(s):  
Green Fluorescent Protein ◽  
High Throughput ◽  
Fluorescent Protein ◽  
Green Fluorescent ◽  
Promoter Study

scholarly journals Neutralization Assay Using a Modified Vaccinia Virus Ankara Vector Expressing the Green Fluorescent Protein Is a High-Throughput Method To Monitor the Humoral Immune Response against Vaccinia Virus

2004 ◽  
Vol 11 (2) ◽  
pp. 406-410 ◽  
Author(s):  
Antonio Cosma ◽  
Silja Bühler ◽  
Rashmi Nagaraj ◽  
Caroline Staib ◽  
Anna-Lena Hammarin ◽  
...  
Keyword(s):  
Immune Response ◽  
Green Fluorescent Protein ◽  
Vaccinia Virus ◽  
High Throughput ◽  
Neutralizing Antibodies ◽  
Fluorescent Protein ◽  
Safety Concern ◽  
Neutralization Assay ◽  
Modified Vaccinia Virus Ankara ◽  
Green Fluorescent

ABSTRACT Vaccination against smallpox is again considered in order to face a possible bioterrorist threat, but the nature and the level of the immune response needed to protect a person from smallpox after vaccination are not totally understood. Therefore, simple, rapid, and accurate assays to evaluate the immune response to vaccinia virus need to be developed. Neutralization assays are usually considered good predictors of vaccine efficacy and more informative with regard to protection than binding assays. Currently, the presence of neutralizing antibodies to vaccinia virus is measured using a plaque reduction neutralization test, but this method is time-consuming and labor-intensive and has a subjective readout. Here, we describe an innovative neutralization assay based on a modified vaccinia virus Ankara (MVA) vector expressing the green fluorescent protein (MVA-gfp). This MVA-gfp neutralization assay is rapid and sensitive and has a high-throughput potential. Thus, it is suitable to monitor the immune response and eventually the efficacy of a large campaign of vaccination against smallpox and to study the vector-specific immune response in clinical trials that use genetically engineered vaccinia viruses. Most importantly, application of the highly attenuated MVA eliminates the safety concern in using the replication-competent vaccinia virus in the standard clinical laboratory.

scholarly journals Recombinant Lassa Virus Expressing Green Fluorescent Protein as a Tool for High-Throughput Drug Screens and Neutralizing Antibody Assays

Viruses ◽  
2018 ◽  
Vol 10 (11) ◽  
pp. 655 ◽  
Author(s):  
Yíngyún Caì ◽  
Masaharu Iwasaki ◽  
Brett Beitzel ◽  
Shuīqìng Yú ◽  
Elena Postnikova ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
High Throughput ◽  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Neutralizing Antibody ◽  
Quantitative Detection ◽  
Lassa Virus ◽  
Wild Type ◽  
Green Fluorescent

Lassa virus (LASV), a mammarenavirus, infects an estimated 100,000–300,000 individuals yearly in western Africa and frequently causes lethal disease. Currently, no LASV-specific antivirals or vaccines are commercially available for prevention or treatment of Lassa fever, the disease caused by LASV. The development of medical countermeasure screening platforms is a crucial step to yield licensable products. Using reverse genetics, we generated a recombinant wild-type LASV (rLASV-WT) and a modified version thereof encoding a cleavable green fluorescent protein (GFP) as a reporter for rapid and quantitative detection of infection (rLASV-GFP). Both rLASV-WT and wild-type LASV exhibited similar growth kinetics in cultured cells, whereas growth of rLASV-GFP was slightly impaired. GFP reporter expression by rLASV-GFP remained stable over several serial passages in Vero cells. Using two well-characterized broad-spectrum antivirals known to inhibit LASV infection, favipiravir and ribavirin, we demonstrate that rLASV-GFP is a suitable screening tool for the identification of LASV infection inhibitors. Building on these findings, we established a rLASV-GFP-based high-throughput drug discovery screen and an rLASV-GFP-based antibody neutralization assay. Both platforms, now available as a standard tool at the IRF-Frederick (an international resource), will accelerate anti-LASV medical countermeasure discovery and reduce costs of antiviral screens in maximum containment laboratories.

High Throughput Studies of Gene Expression Using Green Fluorescent Protein-Oxidative Stress Promoter Probe Constructs The Potential for Living Chips

2001 ◽  
Vol 6 (6) ◽  
pp. 421-428
Author(s):  
C. Renee Albano ◽  
Canghai Lu ◽  
William E. Bentley ◽  
Govind Rao
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Green Fluorescent Protein ◽  
High Throughput ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Chip Technology ◽  
Great Specificity ◽  
Green Fluorescent ◽  
Stress Promoter

Green fluorescent protein fusions were constructed with several oxidative stress promoters from Escherichia coli. These promoters were chosen for their induction by reactive oxygen species (ROS) such as superoxide, hydrogen peroxide, and hydroxyl radicals. When exposed to various free radical insults, the cells fluoresced with great specificity based on the corresponding ROS. In this work, we propose a way in which these constructs could be used to study the mode of action of a variety of antitumor drugs. This approach offers the possibility of complementing gene chip technology by the creation of living chips for high throughput screening as well as studying differential gene expression.

scholarly journals Development of a Keratinocyte-Based Screening Model for Antipsoriatic Drugs Using Green Fluorescent Protein Under the Control of an Endogenous Promoter

2002 ◽  
Vol 7 (4) ◽  
pp. 325-332 ◽  
Author(s):  
Arno Pol ◽  
Fred Van Ruissen ◽  
Joost Schalkwijk
Keyword(s):  
Green Fluorescent Protein ◽  
Fusion Protein ◽  
Cell Line ◽  
Reporter Gene ◽  
High Throughput ◽  
Fluorescent Protein ◽  
Endogenous Promoter ◽  
Keratinocyte Cell ◽  
Egfp Fusion Protein ◽  
Green Fluorescent

Inflamed epidermis (psoriasis, wound healing, ultraviolet-irradiated skin) harbors keratinocytes that are hyperproliferative and display an abnormal differentiation program. A distinct feature of this so-called regenerative maturation pathway is the expression of proteins such as the cytokeratins CK6, CK16, and CK17 and the antiinflammatory protein SKALP/elafin. These proteins are absent in normal skin but highly induced in lesional psoriatic skin. Expression of these genes can be used as a surrogate marker for psoriasis in drug-screening procedures of large compound libraries. The aim of this study was to develop a keratinocyte cell line that contained a reporter gene under the control of a psoriasis-associated endogenous promoter and demonstrate its use in an assay suitable for screening. We generated a stably transfected keratinocyte cell line that expresses enhanced green fluorescent protein (EGFP), under the control of a 0.8-kb fragment derived from the promoter of the SKALP/elafin gene, which confers high levels of tissue-specific expression at the mRNA level. Induction of the SKALP promoter by tumor necrosis factor-ca resulted in increased expression levels of the secreted SKALP-EGFP fusion protein as assessed by direct readout of fluorescence and fluorescence polarization in 96-well cell culture plates. The fold stimulation of the reporter gene was comparable to that of the endogenous SKALP gene as assessed by enzyme-linked immunosorbent assay. Although the dynamic range of the screening system is limited, the small standard deviation yields a Z factor of 0.49. This indicates that the assay is suitable as a high-throughput screen, and provides proof of the concept that a secreted EGFP fusion protein under the control of a physiologically relevant endogenous promoter can be used as a fluorescence-based high-throughput screen for differentiation-modifying or antiinflammatory compounds that act via the keratinocyte.

scholarly journals Green fluorescent protein-based assays for high-throughput functional characterization and ligand-binding studies of biotin protein ligase

2016 ◽  
Vol 8 (2) ◽  
pp. 418-424 ◽  
Author(s):  
Samuel P. Askin ◽  
Thomas E. H. Bond ◽  
Patrick M. Schaeffer
Keyword(s):  
Green Fluorescent Protein ◽  
Ligand Binding ◽  
High Throughput ◽  
Fluorescent Protein ◽  
Functional Characterization ◽  
Binding Studies ◽  
Biotin Protein Ligase ◽  
Green Fluorescent

Rapid functional characterization of GFP-tagged biotin protein ligase (BirA-GFP) with a high-throughput DSF-GTP assay.

scholarly journals Improved Green Fluorescent Protein Reporter Gene-Based Microplate Screening for Antituberculosis Compounds by Utilizing an Acetamidase Promoter

2003 ◽  
Vol 47 (12) ◽  
pp. 3682-3687 ◽  
Author(s):  
Chartchai Changsen ◽  
Scott G. Franzblau ◽  
Prasit Palittapongarnpim
Keyword(s):  
Green Fluorescent Protein ◽  
High Throughput ◽  
High Throughput Screening ◽  
Antimicrobial Agents ◽  
Fluorescent Protein ◽  
Signal To Noise Ratio ◽  
Enhanced Fluorescence ◽  
Signal To Noise ◽  
Green Fluorescent ◽  
Fluorescent Protein Reporter

ABSTRACT The green fluorescent protein (GFP) gene offers many advantages as a viability reporter for high-throughput antimicrobial drug screening. However, screening for antituberculosis compounds by using GFP driven by the heat shock promoter, hsp60, has been of limited utility due to the low signal-to-noise ratio. Therefore, an alternative promoter was evaluated for its enhanced fluorescence during microplate-based culture and its response to 18 established antimicrobial agents by using a green fluorescent protein microplate assay (GFPMA). Mycobacterium tuberculosis strains H37Rv, H37Ra, and Erdman were transformed with pFPCA1, which contains a red-shifted gfp gene driven by the acetamidase promoter of M. smegmatis mc2155. The pFPCA1 transformants achieved higher levels of GFP-mediated fluorescence than those carrying the hsp60 construct, with signal-to-noise ratios of 20.6 to 27.8 and 3.8 to 4.5, respectively. The MICs of 18 established antimicrobial agents for all strains carrying pFPCA1 in the GFPMA were within 1 to 2 twofold dilutions of those determined by either the fluorometric or the visual microplate Alamar Blue assay (MABA). No significant differences in MICs were observed between wild-type and pFPCA1 transformants by MABA. The optimized GFPMA is sufficiently simple, robust, and inexpensive (no reagent costs) to be used for routine high-throughput screening for antituberculosis compounds.

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