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1
Sasuga, Shintaro, and Toshiya Osada. "The Reporter System for GPCR Assay with the Fission YeastSchizosaccharomyces pombe." Scientifica 2012 (2012): 1–11. http://dx.doi.org/10.6064/2012/674256.
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G protein-coupled receptors (GPCRs) are associated with a great variety of biological activities. Yeasts are often utilized as a host for heterologous GPCR assay. We engineered the intense reporter plasmids for fission yeast to produce green fluorescent protein (GFP) through its endogenous GPCR pathway. As a control region of GFP expression on the reporter plasmid, we focused on seven endogenous genes specifically activated through the pathway. When upstream regions of these genes were used as an inducible promoter in combination with LPI terminator, themam2upstream region produced GFP most rapidly and intensely despite the high background. Subsequently, LPI terminator was replaced with the corresponding downstream regions. The SPBC4.01 downstream region enhanced the response with the low background. Furthermore, combining SPBC4.01 downstream region with the sxa2 upstream region, the signal to noise ratio was obviously better than those of other regions. We also evaluated the time- and dose-dependent GFP productions of the strains transformed with the reporter plasmids. Finally, we exhibited a model of simplified GPCR assay with the reporter plasmid by expressing endogenous GPCR under the control of the foreign promoter.
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Collins, L. A., M. N. Torrero, and S. G. Franzblau. "Green Fluorescent Protein Reporter Microplate Assay for High-Throughput Screening of Compounds againstMycobacterium tuberculosis." Antimicrobial Agents and Chemotherapy 42, no. 2 (February 1, 1998): 344–47. http://dx.doi.org/10.1128/aac.42.2.344.
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ABSTRACT An optimal assay for high-throughput screening for new antituberculosis agents would combine the microplate format and low cost of firefly luciferase reporter assays and redox dyes with the ease of kinetic monitoring inherent in the BACTEC system. The green fluorescent protein (GFP) of the jellyfish Aequorea victoria is a useful reporter molecule which requires neither substrates nor cofactors due to the intrinsically fluorescent nature of the protein. The gene encoding a red-shifted, higher-intensity GFP variant was introduced by electroporation into Mycobacterium tuberculosis H37Ra and M. tuberculosisH37Rv on expression vector pFPV2. A microplate-based fluorescence assay (GFP microplate assay [GFPMA]) was developed and evaluated by determining the MICs of existing antimycobacterial agents. The MICs of isoniazid, rifampin, ethambutol, streptomycin, amikacin, ofloxacin, ethionamide, thiacetazone, and capreomycin, but not cycloserine, determined by GFPMA were within 1 log2dilution of those determined with the BACTEC 460 system and were available in 7 days. Equivalent MICs of antituberculosis agents in the BACTEC 460 system for both the reporter and parent strains suggested that introduction of pFPV2 did not influence drug susceptibility, in general. GFPMA provides a unique tool with which the dynamic response of M. tuberculosis to the existing and potential antituberculosis agents can easily, rapidly, and inexpensively be monitored.
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Beck, Verena, Angelika Pfitscher, and Alois Jungbauer. "GFP-reporter for a high throughput assay to monitor estrogenic compounds." Journal of Biochemical and Biophysical Methods 64, no. 1 (July 2005): 19–37. http://dx.doi.org/10.1016/j.jbbm.2005.05.001.
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Simonen, Marjo, Yvonne Ibig-Rehm, Gabriele Hofmann, Johann Zimmermann, Genevieve Albrecht, Maxime Magnier, Valerie Heidinger, and Daniela Gabriel. "High-Content Assay to Study Protein Prenylation." Journal of Biomolecular Screening 13, no. 6 (July 2008): 456–67. http://dx.doi.org/10.1177/1087057108318757.
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The mevalonate pathway leads to synthesis of cholesterol and isoprenoid lipids. Prenyltransferases attach the isoprenoid lipids to the C-terminus of several small guanosine triphosphate—binding proteins. The prenyl groups are essential for the biological activity of these proteins. The prenyltransferases and other components of the mevalonate pathway are either present or potential drug targets for cancer, osteoporosis, restenosis, or high serum cholesterol level. Until recently, cellular assays to study protein prenylation have been tedious, low-throughput assays. The authors have developed a high-content imaging-based assay to study protein prenylation. The assay is based on a green fluorescent protein (GFP) reporter, which is tagged with the prenylation motif of human H-Ras. The C-terminus of H-Ras targets GFP to the plasma membrane. When protein prenylation is inhibited, the tagged GFP cannot be localized to plasma membrane but is soluble in the cells. The localization of the GFP reporter can be analyzed in the 96- or 384-well format using automated microscopy and automated image analysis. Information about cell number and nuclear intensity can be obtained from the same images. In compound screening, these readouts provide valuable information about the toxicity of the compounds. The authors have validated their assay using several inhibitors of the mevalonate pathway as well as siRNA against farnesyl pyrophosphate synthase, a critical enzyme in the synthesis of the isoprenoid lipids. ( Journal of Biomolecular Screening 2008:456-467)
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Brandsma, Inger, Nynke Moelijker, Remco Derr, and Giel Hendriks. "Aneugen Versus Clastogen Evaluation and Oxidative Stress-Related Mode-of-Action Assessment of Genotoxic Compounds Using the ToxTracker Reporter Assay." Toxicological Sciences 177, no. 1 (July 3, 2020): 202–13. http://dx.doi.org/10.1093/toxsci/kfaa103.
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Abstract Understanding the mode-of-action (MOA) of genotoxic compounds and differentiating between direct DNA interaction and indirect genotoxicity is crucial for their reliable safety assessment. ToxTracker is a stem cell-based reporter assay that detects activation of various cellular responses that are associated with genotoxicity and cancer. ToxTracker consists of 6 different GFP reporter cell lines that can detect the induction of DNA damage, oxidative stress, and protein damage in a single test. The assay can thereby provide insight into the MOA of compounds. Genotoxicity is detected in ToxTracker by activation of 2 independent GFP reporters. Activation of the Bscl2-GFP reporter is associated with induction of DNA adducts and subsequent inhibition of DNA replication and the Rtkn-GFP reporter is activated following the formation of DNA double-strand breaks. Here, we show that the differential activation of these 2 genotoxicity reporters could be used to further differentiate between a DNA reactive and clastogenic or a non-DNA-reactive aneugenic MOA of genotoxic compounds. For further classification of aneugenic and clastogenic compounds, the ToxTracker assay was extended with cell cycle analysis and aneuploidy assessment. The extension was validated using a selection of 16 (genotoxic) compounds with a well-established MOA. Furthermore, indirect genotoxicity related to the production of reactive oxygen species was investigated using the DNA damage and oxidative stress ToxTracker reporters in combination with different reactive oxygen species scavengers. With these new extensions, ToxTracker was able to accurately classify compounds as genotoxic or nongenotoxic and could discriminate between DNA-reactive compounds, aneugens, and indirect genotoxicity caused by oxidative stress.
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Standage-Beier, Kylie, Stefan J. Tekel, Nicholas Brookhouser, Grace Schwarz, Toan Nguyen, Xiao Wang, and David A. Brafman. "A transient reporter for editing enrichment (TREE) in human cells." Nucleic Acids Research 47, no. 19 (August 20, 2019): e120-e120. http://dx.doi.org/10.1093/nar/gkz713.
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Abstract Current approaches to identify cell populations that have been modified with deaminase base editing technologies are inefficient and rely on downstream sequencing techniques. In this study, we utilized a blue fluorescent protein (BFP) that converts to green fluorescent protein (GFP) upon a C-to-T substitution as an assay to report directly on base editing activity within a cell. Using this assay, we optimize various base editing transfection parameters and delivery strategies. Moreover, we utilize this assay in conjunction with flow cytometry to develop a transient reporter for editing enrichment (TREE) to efficiently purify base-edited cell populations. Compared to conventional cell enrichment strategies that employ reporters of transfection (RoT), TREE significantly improved the editing efficiency at multiple independent loci, with efficiencies approaching 80%. We also employed the BFP-to-GFP conversion assay to optimize base editor vector design in human pluripotent stem cells (hPSCs), a cell type that is resistant to genome editing and in which modification via base editors has not been previously reported. At last, using these optimized vectors in the context of TREE allowed for the highly efficient editing of hPSCs. We envision TREE as a readily adoptable method to facilitate base editing applications in synthetic biology, disease modeling, and regenerative medicine.
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Liu, Yan Qun, and Xiong Bing Lu. "Establishing a Assay for Detection of Nonylphenol Estrogenic Effects." Applied Mechanics and Materials 71-78 (July 2011): 3003–6. http://dx.doi.org/10.4028/www.scientific.net/amm.71-78.3003.
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Environmental estrogen could mimic natural estrogens thereby disrupting the endocrine systems of human and animals. The actions of such endocrine disruptors have been studied mainly on reproduction and development. To explore the estrogenic effects of NP by reporter genebased assays we developed. pERE-GFP plamid was generated by inserting estrogen response element fragment into pGADD153-GFP. the recombinant was confirmed by restriction enzyme map and transfected into SPC-A1 cells to ensure the expression of green fluorescent protein．The assay we established in usful, NP could induce the estrogenic activities at any of the tested concentrations.
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Hughes, Chris, Adam Rabinowitz, Matthew Tate, Louise Birrell, Jodie Allsup, Nicholas Billinton, and Richard M. Walmsley. "Development of a High-Throughput Gaussia Luciferase Reporter Assay for the Activation of the GADD45a Gene by Mutagens, Promutagens, Clastogens, and Aneugens." Journal of Biomolecular Screening 17, no. 10 (July 10, 2012): 1302–15. http://dx.doi.org/10.1177/1087057112453312.
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Exposure to genotoxic carcinogens leads to increased expression of the GADD45a gene in mammalian cells. This signature of genotoxic hazard has previously been exploited in the GreenScreen HC assay, in which GADD45a expression is linked to green fluorescent protein (GFP) expression in the human TK6 lymphoblastoid cell line. This article describes the development and validation of an alternative assay (“BlueScreen HC”), in which expression is linked to Gaussia luciferase (GLuc) expression, yielding a luminescent reporter, the preferred optical output in high-throughput screening. The coelentrazine substrate of GLuc is relatively unstable, and a new buffer is reported that improves its stability. A more sensitive method is demonstrated for the measurement of cell densities in the assay, using the fluorescent cyanine dye thiazole orange. A protocol amendment also allows the assessment of pro-genotoxicity using S9 liver extracts. Compounds from the European Centre for the Validation of Alternative Methods (ECVAM) recommended list for the assessment of new or improved genotoxicity assays were evaluated with and without S9 in the new assay. The new GLuc assay was as effective as the GFP assay in producing positive results for all classes of genotoxic carcinogen and negative results for all nongenotoxins tested.
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Bui, Van Ngoc, Thi Thu Huyen Nguyen, Yvan Bettarel, Thi Hoai Thu Nguyen, Thuy Linh Pham, Thi Yen Hoang, Vu Thanh Thanh Nguyen, Ngoc Minh Nghiem, and Stefan Wölfl. "Genotoxicity of Chemical Compounds Identification and Assessment by Yeast Cells Transformed With GFP Reporter Constructs Regulated by the PLM2 or DIN7 Promoter." International Journal of Toxicology 34, no. 1 (January 2015): 31–43. http://dx.doi.org/10.1177/1091581814566870.
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Yeast cells transformed with high-copy number plasmids comprising a green fluorescent protein (GFP)-encoding gene optimized for yeast under the control of the new DIN7 or PLM2 and the established RNR2 and RAD54 promoters were used to assess the genotoxic potential of chemical compounds. The activity of potential DNA-damaging agents was investigated by genotoxicity assays and by OxoPlate assay in the presence of various test compounds. The fluorescence signal generated by GFP in response to DNA damage was related to the different concentrations of analytes and the analyte-dependent GFP synthesis. The use of distinct DNA damage-inducible promoters presents alternative genotoxicity testing strategies by selective induction of promoters in response to DNA damage. The new DIN7 and PLM2 systems show higher sensitivity than the RNR2 and RAD54 systems in detecting 4-nitroquinoline- N-oxide and actinomycin D. Both DIN7 and PLM2 systems are able to detect camptothecin while RNR2 and RAD54 systems are not. Automated laboratory systems with assay performance on 384-well microplates provide for cost-effective high-throughput screening of DNA-damaging agents, reducing compound consumption to about 53% as compared with existing eukaryotic genotoxicity bioassays.
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Changsen, Chartchai, Scott G. Franzblau, and Prasit Palittapongarnpim. "Improved Green Fluorescent Protein Reporter Gene-Based Microplate Screening for Antituberculosis Compounds by Utilizing an Acetamidase Promoter." Antimicrobial Agents and Chemotherapy 47, no. 12 (December 2003): 3682–87. http://dx.doi.org/10.1128/aac.47.12.3682-3687.2003.
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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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Jugder, Bat-Erdene, Jeffrey Welch, Nady Braidy, and Christopher P. Marquis. "Construction and use of aCupriavidus necatorH16 soluble hydrogenase promoter (PSH) fusion togfp(green fluorescent protein)." PeerJ 4 (July 26, 2016): e2269. http://dx.doi.org/10.7717/peerj.2269.
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Hydrogenases are metalloenzymes that reversibly catalyse the oxidation or production of molecular hydrogen (H2). Amongst a number of promising candidates for application in the oxidation of H2is a soluble [Ni–Fe] uptake hydrogenase (SH) produced byCupriavidus necatorH16. In the present study, molecular characterisation of the SH operon, responsible for functional SH synthesis, was investigated by developing a green fluorescent protein (GFP) reporter system to characterise PSHpromoter activity using several gene cloning approaches. A PSHpromoter-gfp fusion was successfully constructed and inducible GFP expression driven by the PSHpromoter under de-repressing conditions in heterotrophic growth media was demonstrated in the recombinantC. necatorH16 cells. Here we report the first successful fluorescent reporter system to study PSHpromoter activity inC. necatorH16. The fusion construct allowed for the design of a simple screening assay to evaluate PSHactivity. Furthermore, the constructed reporter system can serve as a model to develop a rapid fluorescent based reporter for subsequent small-scale process optimisation experiments for SH expression.
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Sugiyama, Kei-ichi, Hiroko Furusawa, Petr Grúz, and Masamitsu Honma. "Detection of epigenetic mutagens including anthracene-derived compounds using yeast FLO1 promoter GFP reporter gene assay." Mutagenesis 32, no. 4 (April 20, 2017): 429–35. http://dx.doi.org/10.1093/mutage/gex009.
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Lyu, Pin, Kyung Whan Yoo, Manish Kumar Yadav, Anthony Atala, Annemieke Aartsma-Rus, Maaike van Putten, Dongsheng Duan, and Baisong Lu. "Sensitive and reliable evaluation of single-cut sgRNAs to restore dystrophin by a GFP-reporter assay." PLOS ONE 15, no. 9 (September 24, 2020): e0239468. http://dx.doi.org/10.1371/journal.pone.0239468.
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Caì, Yíngyún, Masaharu Iwasaki, Brett Beitzel, Shuīqìng Yú, Elena Postnikova, Beatrice Cubitt, Lisa DeWald, et al. "Recombinant Lassa Virus Expressing Green Fluorescent Protein as a Tool for High-Throughput Drug Screens and Neutralizing Antibody Assays." Viruses 10, no. 11 (November 20, 2018): 655. http://dx.doi.org/10.3390/v10110655.
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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.
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Koeppel, Martin B., Jana Glaser, Tobias Baumgartner, Stefanie Spriewald, Roman G. Gerlach, Benedikt von Armansperg, John M. Leong, and Bärbel Stecher. "Scalable Reporter Assays to Analyze the Regulation of stx2 Expression in Shiga Toxin-Producing Enteropathogens." Toxins 13, no. 8 (July 29, 2021): 534. http://dx.doi.org/10.3390/toxins13080534.
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Stx2 is the major virulence factor of EHEC and is associated with an increased risk for HUS in infected patients. The conditions influencing its expression in the intestinal tract are largely unknown. For optimal management and treatment of infected patients, the identification of environmental conditions modulating Stx2 levels in the human gut is of central importance. In this study, we established a set of chromosomal stx2 reporter assays. One system is based on superfolder GFP (sfGFP) using a T7 polymerase/T7 promoter-based amplification loop. This reporter can be used to analyze stx2 expression at the single-cell level using FACSs and fluorescence microscopy. The other system is based on the cytosolic release of the Gaussia princeps luciferase (gluc). This latter reporter proves to be a highly sensitive and scalable reporter assay that can be used to quantify reporter protein in the culture supernatant. We envision that this new set of reporter tools will be highly useful to comprehensively analyze the influence of environmental and host factors, including drugs, small metabolites and the microbiota, on Stx2 release and thereby serve the identification of risk factors and new therapies in Stx-mediated pathologies.
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Grant, Barth, and David Hirsh. "Receptor-mediated Endocytosis in the Caenorhabditis elegans Oocyte." Molecular Biology of the Cell 10, no. 12 (December 1999): 4311–26. http://dx.doi.org/10.1091/mbc.10.12.4311.
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The Caenorhabditis elegans oocyte is a highly amenable system for forward and reverse genetic analysis of receptor-mediated endocytosis. We describe the use of transgenic strains expressing a vitellogenin::green fluorescent protein (YP170::GFP) fusion to monitor yolk endocytosis by theC. elegans oocyte in vivo. This YP170::GFP reporter was used to assay the functions of C. eleganspredicted proteins homologous to vertebrate endocytosis factors using RNA-mediated interference. We show that the basic components and pathways of endocytic trafficking are conserved between C. elegans and vertebrates, and that this system can be used to test the endocytic functions of any new gene. We also used the YP170::GFP assay to identify rme(receptor-mediated endocytosis) mutants. We describe a new member of the low-density lipoprotein receptor superfamily, RME-2, identified in our screens for endocytosis defective mutants. We show that RME-2 is the C. elegans yolk receptor.
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Aviat, Florence, Leyla Slamti, Gustavo M. Cerqueira, Kristel Lourdault, and Mathieu Picardeau. "Expanding the Genetic Toolbox for Leptospira Species by Generation of Fluorescent Bacteria." Applied and Environmental Microbiology 76, no. 24 (October 29, 2010): 8135–42. http://dx.doi.org/10.1128/aem.02199-10.
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ABSTRACT Our knowledge of the genetics and molecular basis of the pathogenesis associated with Leptospira, in comparison to those of other bacterial species, is very limited. An improved understanding of pathogenic mechanisms requires reliable genetic tools for functional genetic analysis. Here, we report the expression of gfp and mRFP1 genes under the control of constitutive spirochetal promoters in both saprophytic and pathogenic Leptospira strains. We were able to reliably measure the fluorescence of Leptospira by fluorescence microscopy and a fluorometric microplate reader-based assay. We showed that the expression of the gfp gene had no significant effects on growth in vivo and pathogenicity in L. interrogans. We constructed an expression vector for L. biflexa that contains the lacI repressor, an inducible lac promoter, and gfp as the reporter, demonstrating that the lac system is functional in Leptospira. Green fluorescent protein (GFP) expression was induced by the addition of isopropyl-β-d-thiogalactopyranoside (IPTG) in L. biflexa transformants harboring the expression vector. Finally, we showed that GFP can be used as a reporter to assess promoter activity in different environmental conditions. These results may facilitate further advances for studying the genetics of Leptospira spp.
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Hendriks, Giel, Mirna Atallah, Bruno Morolli, Fabienne Calléja, Nienke Ras-Verloop, Ilse Huijskens, Martine Raamsman, Bob van de Water, and Harry Vrieling. "The ToxTracker Assay: Novel GFP Reporter Systems that Provide Mechanistic Insight into the Genotoxic Properties of Chemicals." Toxicological Sciences 125, no. 1 (October 14, 2011): 285–98. http://dx.doi.org/10.1093/toxsci/kfr281.
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Ngai, Siew Ching, Rozita Rosli, Akram Al Abbar, and Syahril Abdullah. "DNA Methylation and Histone Modifications Are the Molecular Lock in Lentivirally Transduced Hematopoietic Progenitor Cells." BioMed Research International 2015 (2015): 1–11. http://dx.doi.org/10.1155/2015/346134.
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Stable introduction of a functional gene in hematopoietic progenitor cells (HPCs) has appeared to be an alternative approach to correct genetically linked blood diseases. However, it is still unclear whether lentiviral vector (LV) is subjected to gene silencing in HPCs. Here, we show that LV carrying green fluorescent protein (GFP) reporter gene driven by cytomegalovirus (CMV) promoter was subjected to transgene silencing after transduction into HPCs. This phenomenon was not due to the deletion of proviral copy number. Study using DNA demethylating agent and histone deacetylase (HDAC) inhibitor showed that the drugs could either prevent or reverse the silencing effect. Using sodium bisulfite sequencing and chromatin immunoprecipitation (ChIP) assay, we demonstrated that DNA methylation occurred soon after LV transduction. At the highest level of gene expression, CMV promoter was acetylated and was in a euchromatin state, while GFP reporter gene was acetylated but was strangely in a heterochromatin state. When the expression declined, CMV promoter underwent transition from acetylated and euchromatic state to a heterochromatic state, while the GFP reporter gene was in deacetylated and heterochromatic state. With these, we verify that DNA methylation and dynamic histone modifications lead to transgene silencing in HPCs transduced with LV.
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Giddings, Angela M., and Rangan Maitra. "A Disease-Relevant High-Content Screening Assay to Identify Anti-Inflammatory Compounds for Use in Cystic Fibrosis." Journal of Biomolecular Screening 15, no. 10 (October 13, 2010): 1204–10. http://dx.doi.org/10.1177/1087057110384612.
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Chronic lung inflammation caused by bacterial pathogenesis through activation of nuclear factor kappa B (NFκB)–responsive proinflammatory genes is a major hurdle in the management of lung disease in cystic fibrosis (CF) patients. The authors generated a disease-relevant cell-based high-content screen to identify novel anti-inflammatory compounds for treating lung inflammation in CF. The human bronchial epithelial cell line KKLEB, harboring the most common form of mutation that causes CF, was modified to express an NFκB-responsive green fluorescent protein (GFP) reporter. After creation, the cell line was tested for its ability to respond to disease-relevant inflammatory stimuli elicited by treatment of cells with filtrates of Pseudomonas aeruginosa isolated from the airways of a CF patient. P. aeruginosa filtrates potently activated NFκB-responsive GFP reporter expression in cells. Subsequently, the assay was optimized for high-throughput screening (HTS) through generation of a Z factor (~0.5) and by testing its tolerance to the commonly used solvents ethanol and DMSO. A pilot library of clinically approved compounds was screened for assay validation. Several compounds with known NFκB inhibitory activity were identified, including several steroidal compounds that have been clinically tested in CF. Thus, the assay can be used in a broader HTS campaign to find anti-inflammatory agents for use in CF.
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Campelo, Ana Belén, Ana Rodríguez, and Beatriz Martínez. "Use of Green Fluorescent Protein To Monitor Cell Envelope Stress in Lactococcus lactis." Applied and Environmental Microbiology 76, no. 3 (November 30, 2009): 978–81. http://dx.doi.org/10.1128/aem.02177-09.
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ABSTRACT A Lactococcus lactis reporter system suitable to detect cell envelope stress in high-throughput settings was developed by fusing the CesR-regulated promoter of llmg0169 to the gfpuv gene. A dot blot assay allowed fast detection of green fluorescent protein (GFP) fluorescence even at low production levels. Unexpectedly, this promoter was also induced by mitomycin C via CesR.
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Ozawa, Makoto, Sylvia T. Victor, Andrew S. Taft, Shinya Yamada, Chengjun Li, Masato Hatta, Subash C. Das, et al. "Replication-incompetent influenza A viruses that stably express a foreign gene." Journal of General Virology 92, no. 12 (December 1, 2011): 2879–88. http://dx.doi.org/10.1099/vir.0.037648-0.
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A biologically contained influenza A virus that stably expresses a foreign gene can be effectively traced, used to generate a novel multivalent vaccine and have its replication easily assessed, all while satisfying safety concerns regarding pathogenicity or reversion. This study generated a PB2-knockout (PB2-KO) influenza virus that harboured the GFP reporter gene in the coding region of its PB2 viral RNA (vRNA). Replication of the PB2-KO virus was restricted to a cell line stably expressing the PB2 protein. The GFP gene-encoding PB2 vRNA was stably incorporated into progeny viruses during replication in PB2-expressing cells. The GFP gene was expressed in virus-infected cells with no evidence of recombination between the recombinant PB2 vRNA and the PB2 protein mRNA. Furthermore, other reporter genes and the haemagglutinin and neuraminidase genes of different virus strains were accommodated by the PB2-KO virus. Finally, the PB2-KO virus was used to establish an improved assay to screen neutralizing antibodies against influenza viruses by using reporter gene expression as an indicator of virus infection rather than by observing cytopathic effect. These results indicate that the PB2-KO virus has the potential to be a valuable tool for basic and applied influenza virus research.
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Qazi, Saara N. A., Emilie Counil, Julie Morrissey, Catherine E. D. Rees, Alan Cockayne, Klaus Winzer, Weng C. Chan, Paul Williams, and Philip J. Hill. "agr Expression Precedes Escape of InternalizedStaphylococcus aureus from the Host Endosome." Infection and Immunity 69, no. 11 (November 1, 2001): 7074–82. http://dx.doi.org/10.1128/iai.69.11.7074-7082.2001.
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ABSTRACT Staphylococcus aureus is a versatile pathogen capable of causing life-threatening infections. Many of its cell wall and exoproduct virulence determinants are controlled via the accessory gene regulator (agr). Although considered primarily as an extracellular pathogen, it is now recognized that S. aureus can be internalized by epithelial and endothelial cells. Traditional experimental approaches to investigate bacterial internalization are extremely time-consuming and notoriously irreproducible. We present here a new reporter gene method to assess intracellular growth of S. aureus in MAC-T cells that utilizes a gfp-luxABCDE reporter operon under the control of the Bacillus megaterium xylA promoter, which in S. aureus is expressed in a growth-dependent manner. This facilitates assessment of the growth of internalized bacteria in a nondestructive assay. The dual gfp-lux reporter cassette was also evaluated as a reporter of agr expression and used to monitor the temporal induction of agr during the MAC-T internalization process. The data obtained suggest thatagr induction occurs prior to endosomal lysis and thatagr-regulated exoproteins appear to be required prior to the release and replication of S. aureus within the infected MAC-T cells.
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Bilello, John P., Jennifer S. Morgan, Blossom Damania, Sabine M. Lang, and Ronald C. Desrosiers. "A Genetic System for Rhesus Monkey Rhadinovirus: Use of Recombinant Virus To Quantitate Antibody-Mediated Neutralization." Journal of Virology 80, no. 3 (February 1, 2006): 1549–62. http://dx.doi.org/10.1128/jvi.80.3.1549-1562.2006.
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ABSTRACT Rhesus monkey rhadinovirus (RRV), a simian gamma-2 herpesvirus closely related to the Kaposi sarcoma-associated herpesvirus, replicates lytically in cultured rhesus monkey fibroblasts and establishes persistence in B cells. Overlapping cosmid clones were generated that encompass the entire 130-kilobase-pair genome of RRV strain 26-95, including the terminal repeat regions required for its replication. Cloned RRV that was produced by cotransfection of overlapping cosmids spanning the entire RRV26-95 genome replicated with growth kinetics and to titers similar to those of the parental, uncloned, wild-type RRV26-95. Expression cassettes for secreted-engineered alkaline phosphatase (SEAP) and green fluorescent protein (GFP) were inserted upstream of the R1 gene, and the cosmid-based system for RRV genome reconstitution was used to generate replication-competent, recombinant RRV that expressed either the SEAP or GFP reporter gene. Using the SEAP and GFP recombinant RRVs, assays were developed to monitor RRV infection, neutralization, and replication. Heat-inactivated sera from rhesus monkeys that were naturally or experimentally infected with RRV were assayed for their ability to neutralize RRV-SEAP and RRV-GFP infectivity using rhesus monkey fibroblasts. Sera from RRV-positive monkeys, but not RRV-negative monkeys, were consistently able to neutralize RRV infectivity when assayed by the production of SEAP activity or by the ability to express GFP. The neutralizing activity was present in the immunoglobulin fraction. Of the 17 rhesus monkeys tested, sera from rhesus monkey 26-95, i.e., the monkey that yielded the RRV 26-95 isolate, had the highest titer of neutralizing activity against RRV26-95. This cosmid-based genetic system and the reporter virus neutralization assay will facilitate study of the contribution of individual RRV glycoproteins to entry into different cell types, particularly fibroblasts and B cells.
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Zhao, Yan, Robert A. Owens, and Rosemarie W. Hammond. "Use of a vector based on Potato virus X in a whole plant assay to demonstrate nuclear targeting of Potato spindle tuber viroid." Journal of General Virology 82, no. 6 (June 1, 2001): 1491–97. http://dx.doi.org/10.1099/0022-1317-82-6-1491.
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Potato spindle tuber viroid (PSTVd) is a covalently closed circular RNA molecule of 359 nucleotides that replicates within the nucleus of host cells. To determine how this small, highly structured RNA enters the nucleus, we have developed a virus-based, whole plant in vivo assay that uses green fluorescent protein (GFP) as the reporter molecule. The coding region of GFP was interrupted by insertion of an intron derived from the intervening sequence 2 of the potato ST-LS1 gene. A cDNA copy of the complete PSTVd genome was, in turn, embedded within the intron, and this construct was delivered into Nicotiana benthamiana plants via a vector based on Potato virus X. The intron-containing GFP subgenomic RNA synthesized during virus infection cannot produce a functional GFP unless the RNA is imported into the nucleus, where the intron can be removed and the spliced RNA returned to the cytoplasm. The appearance of green fluorescence in leaf tissues inoculated with constructs containing a full-length PSTVd molecule embedded in the intron indicates that nuclear import and RNA splicing events did occur.
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Simpson, Kate, Nicola Bevan, Paul Hastwell, Patrick Eidam, Poonam Shah, Elke Gogo, Steve Rees, and Andrew Brown. "The BlueScreen-384 Assay as an Indicator of Genotoxic Hazard Potential in Early-Stage Drug Discovery." Journal of Biomolecular Screening 18, no. 4 (December 20, 2012): 441–52. http://dx.doi.org/10.1177/1087057112470858.
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High-throughput cell-based techniques that permit early detection of compound-induced genotoxic damage have recently become available. Methods based on induction of the GADD45a promoter are attractive because multiple intracellular mechanisms that detect genetic damage intersect at this checkpoint gene. Consequently, assays such as GreenScreen HC, which uses p53-competant human TK6 lymphoblastoid cells and a GADD45a-GFP reporter, have been developed. GreenScreen HC allows weekly testing of dozens of compounds using 96-well microplates, with high interassay consistency. BlueScreen HC is a recent advancement, coupling GADD45a to Gaussia luciferase, with several advantages over GADD45a-GFP including the potential for miniaturization. Here we describe implementation of a 384-well BlueScreen assay. For drug discovery programs carrying out iterative analogue synthesis around a chemical lead series, these assays permit assessment of compound genotoxic potential in parallel to, rather than subsequent to, determination of activity at a therapeutic target. We demonstrate comparability of BlueScreen-384 to GreenScreen HC and illustrate the use of BlueScreen-384 to explore the structure-activity relationship around a genotoxic lead molecule to identify nongenotoxic analogues. BlueScreen-384 can reduce the need for costly and time-consuming analogue testing in more traditional genotoxicity tests, such as the Ames test.
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Chaudhuri, Tandra R., Zhihong Cao, Victor N. Krasnykh, Amanda V. Stargel, Natalya Belousova, Edward E. Partridge, and Kurt R. Zinn. "Blood-based Screening and Light Based Imaging for the Early Detection and Monitoring of Ovarian Cancer Xenografts." Technology in Cancer Research & Treatment 2, no. 2 (April 2003): 171–79. http://dx.doi.org/10.1177/153303460300200214.
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We report a novel technology for in vivo early detection, identification, and monitoring of ovarian cancer in live mice leading to better treatment outcome. A genetic dualistic reporter system that uses an adenoviral (Ad) vector to transfer the genetic reporters to the ovarian cancer is described. Infection of the cancer cells leads to expression of one reporter that is detected in blood, namely, secreted human placental alkaline phosphatase (SEAP). A second reporter, namely, enhanced green fluorescent protein (GFP) is also delivered by the Ad, leading to expression at the site of ovarian cancer. The SEAP gene under control of the cytomegalovirus (CMV) promoter element is linked to the GFP gene with an IRES element. A diagnostic adenoviral vector (Ad) encoding the SEAP and GFP (Ad5-SEAP-GFP) is produced. Efficacy of newly developed diagnostic vector is tested in cell culture and animal models. SKOV3ip.1 cells are infected with Ad5-SEAP-GFP. Over time the cells are monitored for fluorescence and SEAP is also measured in the growth media supernatant. For animal experiments, SKOV3ip.1 cells are implanted first in nude mice either subcutaneously (SC) or intraperitoneally (IP) separately. After 4–7 days, the Ad5-SEAP-GFP is administered. Control mice do not receive any Ad vector. All mice are imaged with a fluorescent stereomicroscope after 24 h, and blood is collected for SEAP analyses. Increasing green fluorescence is detected in all SKOV3ip.1 cells infected with Ad5-SEAP-GFP, while SEAP levels in growth media increase over monitoring period. Expression of GFP in both SC and IP tumors is detected by 24 h in the live mice. At this time, the SEAP blood levels are more than 2–3 fold greater than blood levels of control group. GFP fluorescence and SEAP levels continue to increase in all mice that are injected with Ad5-SEAP-GFP until termination. Control mice (both SC and IP) do not express GFP or SEAP throughout the experiment. GFP contrast is necessary to differentiate between micro-sized early stage non-palpable ovarian tumor nodules and surrounding normal tissue. While the studies are conducted in mice, it is envisioned that the dual-based approach will eventually be translated into human applications for routine diagnosis and monitoring of ovarian cancer when an ovarian cancer specific promoter will be available. Due to the thickness of the abdominal wall in human laparoscopy or laparotomy will be necessary. This system will provide gynecologic oncologists with a more effective tool for treating patients. The blood-based screening assay provides a quick test to determine the presence of the ovarian cancer at its earliest stage. The location of the ovarian cancer is afforded by the light-based imaging component, which represents a new and improving technology with tremendous advantages of sensitivity and spatial resolution to localize micro-sized tumor nodules. The novelty of the dualistic system is the linkage of blood-based reporter screening as a selection criteria for subsequent light-based imaging procedures. This combination will lead to an accurate and widely applicable method for the early detection and monitoring of ovarian cancer, especially in high-risk women.
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Choy, Jennifer S., Latt Latt Aung, and A. Wali Karzai. "Lon Protease Degrades Transfer-Messenger RNA-Tagged Proteins." Journal of Bacteriology 189, no. 18 (July 6, 2007): 6564–71. http://dx.doi.org/10.1128/jb.00860-07.
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ABSTRACT Bacterial trans translation is activated when translating ribosomes are unable to elongate or terminate properly. Small protein B (SmpB) and transfer-messenger RNA (tmRNA) are the two known factors required for and dedicated to trans translation. tmRNA, encoded by the ssrA gene, is a bifunctional molecule that acts both as a tRNA and as an mRNA during trans translation. The functions of tmRNA ensure that stalled ribosomes are rescued, the causative defective mRNAs are degraded, and the incomplete polypeptides are marked for targeted proteolysis. We present in vivo and in vitro evidence that demonstrates a direct role for the Lon ATP-dependent protease in the degradation of tmRNA-tagged proteins. In an endogenous protein tagging assay, lon mutants accumulated excessive levels of tmRNA-tagged proteins. In a reporter protein tagging assay with λ-CI-N, the protein product of a nonstop mRNA construct designed to activate trans translation, lon mutant cells efficiently tagged the reporter protein, but the tagged protein exhibited increased stability. Similarly, a green fluorescent protein (GFP) construct containing a hard-coded C-terminal tmRNA tag (GFP-SsrA) exhibited increased stability in lon mutant cells. Most significantly, highly purified Lon preferentially degraded the tmRNA-tagged forms of proteins compared to the untagged forms. Based on these results, we conclude that Lon protease participates directly in the degradation of tmRNA-tagged proteins.
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Wills, John W., Elias Halkes-Wellstead, Huw D. Summers, Paul Rees, and George E. Johnson. "Empirical comparison of genotoxic potency estimations: the in vitro DNA-damage ToxTracker endpoints versus the in vivo micronucleus assay." Mutagenesis 36, no. 4 (June 10, 2021): 311–20. http://dx.doi.org/10.1093/mutage/geab020.
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Abstract Genetic toxicology is an essential component of compound safety assessment. In the face of a barrage of new compounds, higher throughput, less ethically divisive in vitro approaches capable of effective, human-relevant hazard identification and prioritisation are increasingly important. One such approach is the ToxTracker assay, which utilises murine stem cell lines equipped with green fluorescent protein (GFP)-reporter gene constructs that each inform on distinct aspects of cellular perturbation. Encouragingly, ToxTracker has shown improved sensitivity and specificity for the detection of known in vivo genotoxicants when compared to existing ‘standard battery’ in vitro tests. At the current time however, quantitative genotoxic potency correlations between ToxTracker and well-recognised in vivo tests are not yet available. Here we use dose–response data from the three DNA-damage-focused ToxTracker endpoints and from the in vivo micronucleus assay to carry out quantitative, genotoxic potency estimations for a range of aromatic amine and alkylating agents using the benchmark dose (BMD) approach. This strategy, using both the exponential and the Hill BMD model families, was found to produce robust, visually intuitive and similarly ordered genotoxic potency rankings for 17 compounds across the BSCL2-GFP, RTKN-GFP and BTG2-GFP ToxTracker endpoints. Eleven compounds were similarly assessed using data from the in vivo micronucleus assay. Cross-systems genotoxic potency correlations for the eight matched compounds demonstrated in vitro–in vivo correlation, albeit with marked scatter across compounds. No evidence for distinct differences in the sensitivity of the three ToxTracker endpoints was found. The presented analyses show that quantitative potency determinations from in vitro data enable more than just qualitative screening and hazard identification in genetic toxicology.
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Zeng, Hongqiu, Yanwei Xie, Guoyin Liu, Yunxie Wei, Wei Hu, and Haitao Shi. "Agrobacterium-Mediated Gene Transient Overexpression and Tobacco Rattle Virus (TRV)-Based Gene Silencing in Cassava." International Journal of Molecular Sciences 20, no. 16 (August 15, 2019): 3976. http://dx.doi.org/10.3390/ijms20163976.
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Agrobacterium-mediated transient expression and virus-induced gene silencing (VIGS) are very useful in functional genomics in plants. However, whether these methods are effective in cassava (Manihot esculenta), one of the most important tropical crops, remains elusive. In this study, we used green fluorescent protein (GFP) and β-glucuronidase (GUS) as reporter genes in a transient expression assay. GFP or GUS could be detected in the infiltrated leaves at 2 days postinfiltration (dpi) and were evidenced by visual GFP and GUS assays, reverse-transcription PCR, and Western blot. In addition, phytoene desaturase (PDS) was used to show the silencing effect in a VIGS system. Both Agrobacterium GV3101 and AGL-1 with tobacco rattle virus (TRV)-MePDS-infiltrated distal leaves showed an albino phenotype at 20 dpi; in particular, the AGL-1-infiltrated plants showed an obvious albino area in the most distal leaves. Moreover, the silencing effect was validated by molecular identification. Notably, compared with the obvious cassava mosaic disease symptom infiltrated by African-cassava-mosaic-virus-based VIGS systems in previous studies, TRV-based VIGS-system-infiltrated cassava plants did not show obvious virus-induced disease symptoms, suggesting a significant advantage. Taken together, these methods could promote functional genomics in cassava.
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Yang, Jiangtao, Xujing Wang, Agula Hasi, and Zhixing Wang. "Structural and Functional Analysis of a Bidirectional Promoter from Gossypium hirsutum in Arabidopsis." International Journal of Molecular Sciences 19, no. 11 (October 23, 2018): 3291. http://dx.doi.org/10.3390/ijms19113291.
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Stacked traits have become an important trend in the current development of genomically modified crops. The bidirectional promoter can not only prevent the co-suppression of multigene expression, but also increase the efficiency of the cultivation of transgenic plants with multigenes. In Gossypium hirsutum, Ghrack1 and Ghuhrf1 are head-to-head gene pairs located on chromosome D09. We cloned the 1429-bp intergenic region between the Ghrack1 and Ghuhrf1 genes from Gossypium hirsutum. The cloned DNA fragment GhZU had the characteristics of a bidirectional promoter, with 38.7% G+C content, three CpG islands and no TATA-box. Using gfp and gus as reporter genes, a series of expression vectors were constructed into young leaves of tobacco. The histochemical GUS (Beta-glucuronidase) assay and GFP (green fluorescence protein) detection results indicated that GhZU could drive the expression of the reporter genes gus and gfp simultaneously in both orientations. Furthermore, we transformed the expression vectors into Arabidopsis and found that GUS was concentrated at vigorous growth sites, such as the leaf tip, the base of the leaves and pod, and the stigma. GFP was also mainly expressed in the epidermis of young leaves. In summary, we determined that the intergenic region GhZU was an orientation-dependent bidirectional promoter, and this is the first report on the bidirectional promoter from Gossypium hirsutum. Our findings in this study are likely to enhance understanding on the regulatory mechanisms of plant bidirectional promoters.
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Uchiyama, Taku, and Kentaro Miyazaki. "Product-Induced Gene Expression, a Product-Responsive Reporter Assay Used To Screen Metagenomic Libraries for Enzyme-Encoding Genes." Applied and Environmental Microbiology 76, no. 21 (September 10, 2010): 7029–35. http://dx.doi.org/10.1128/aem.00464-10.
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ABSTRACT A reporter assay-based screening method for enzymes, which we named product-induced gene expression (PIGEX), was developed and used to screen a metagenomic library for amidases. A benzoate-responsive transcriptional activator, BenR, was placed upstream of the gene encoding green fluorescent protein and used as a sensor. Escherichia coli sensor cells carrying the benR-gfp gene cassette fluoresced in response to benzoate concentrations as low as 10 μM but were completely unresponsive to the substrate benzamide. An E. coli metagenomic library consisting of 96,000 clones was grown in 96-well format in LB medium containing benzamide. The library cells were then cocultivated with sensor cells. Eleven amidase genes were recovered from 143 fluorescent wells; eight of these genes were homologous to known bacterial amidase genes while three were novel genes. In addition to their activity toward benzamide, the enzymes were active toward various substrates, including d- and l-amino acid amides, and displayed enantioselectivity. Thus, we demonstrated that PIGEX is an effective approach for screening novel enzymes based on product detection.
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Wright, Edward, Nigel J. Temperton, Denise A. Marston, Lorraine M. McElhinney, Anthony R. Fooks, and Robin A. Weiss. "Investigating antibody neutralization of lyssaviruses using lentiviral pseudotypes: a cross-species comparison." Journal of General Virology 89, no. 9 (September 1, 2008): 2204–13. http://dx.doi.org/10.1099/vir.0.2008/000349-0.
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Cross-neutralization between rabies virus (RABV) and two European bat lyssaviruses (EBLV-1 and -2) was analysed using lentiviral pseudotypes as antigen vectors. Glycoprotein (G-protein) cDNA from RABV challenge virus standard-11 (CVS-11) and EBLV-1 and -2 were cloned and co-expressed with human immunodeficiency virus (HIV) or murine leukemia virus (MLV) gag–pol and packageable green fluorescent protein (GFP) or luciferase reporter genes in human cells. The harvested lentiviral (HIV) vector infected over 40 % of baby hamster kidney (BHK) target cells, providing high-titre pseudotype stocks. Tests on blinded antibody-positive (n=15) and -negative (n=45) sera, predetermined by the fluorescent antibody virus neutralization (FAVN) test approved by the World Health Organization (WHO) and Office International des Epizooties (OIE), revealed that the CVS-11 pseudotype assay had 100 % concordance with FAVN and strongly correlated with neutralization titres (r 2=0.89). Cross-neutralization tests using sera from RABV-vaccinated humans and animals on pseudotypes with CVS-11, EBLV-1 and EBLV-2 envelopes showed that the relative neutralization titres correlated broadly with the degree of G-protein diversity. Pseudotypes have three major advantages over live-virus neutralization tests: (i) they can be handled in low-biohazard-level laboratories; (ii) the use of reporter genes such as GFP or β-galactosidase will allow the assay to be undertaken at low cost in laboratories worldwide; (iii) each assay requires <10 μl serum. This robust microassay will improve our understanding of the protective humoral immunity that current rabies vaccines confer against emerging lyssaviruses, and will be applicable to surveillance studies, thus helping to control the spread of rabies.
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Bagchi Bhattacharjee, Gargi, and S. M. Paul Khurana. "In VitroReporter Assays for Screening of Chemicals That Disrupt Androgen Signaling." Journal of Toxicology 2014 (2014): 1–7. http://dx.doi.org/10.1155/2014/701752.
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Endocrine disruptive chemicals (EDCs) modulate hormone signaling and cause developmental and reproductive anomalies. Today, there is a global concern regarding endocrine disruption effects, particularly those mediated by the androgen receptor (AR). Androgen or male hormones are critical for the development and maintenance of male characteristics and numerous EDCs exist in the environment with the potential to disrupt androgen action. The threat is more during critical developmental windows when there is increased sensitivity to these compounds. Timely screening and detection of the EDCs is essential to minimize deleterious effects produced by these toxic chemicals. As a first line of screening,in vitrotranscription assays are very useful due to their speed, convenience, and cost effectiveness. In this paper, recentin vitroreporter assays for detecting androgenic or antiandrogenic activity of EDCs have been reviewed. Two important cell systems used for this purpose, namely, the mammalian or yeast cell systems, have been discussed. Use of reporter genes such as bacterial luciferase (lux) and green fluorescent protein (gfp) has significantly improved speed and sensitivity of detection. Also, many of the current reporter assay systems can be used in a high throughput format allowing speedy evaluation of multiple potential EDCs at a lower price.
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Humann, Jodi L., Brenda K. Schroeder, Michael W. Mortimer, Brent L. House, Svetlana N. Yurgel, Scott C. Maloney, Kristel L. Ward, Heather M. Fallquist, Hope T. Ziemkiewicz, and Michael L. Kahn. "Construction and Expression of Sugar Kinase Transcriptional Gene Fusions by Using the Sinorhizobium meliloti ORFeome." Applied and Environmental Microbiology 74, no. 21 (September 12, 2008): 6756–65. http://dx.doi.org/10.1128/aem.01468-08.
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ABSTRACT The Sinorhizobium meliloti ORFeome project cloned 6,314 open reading frames (ORFs) into a modified Gateway entry vector system from which the ORFs could be transferred to destination vectors in vivo via bacterial conjugation. In this work, a reporter gene destination vector, pMK2030, was constructed and used to generate ORF-specific transcriptional fusions to β-glucuronidase (gusA) and green fluorescent protein (gfp) reporter genes. A total of 6,290 ORFs were successfully transferred from the entry vector library into pMK2030. To demonstrate the utility of this system, reporter plasmids corresponding to 30 annotated sugar kinase genes were integrated into the S. meliloti SM1021 and/or SM8530 genome. Expression of these genes was measured using a high-throughput β-glucuronidase assay to track expression on nine different carbon sources. Six ORFs integrated into SM1021 and SM8530 had different basal levels of expression in the two strains. The annotated activities of three other sugar kinases were also confirmed.
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Thatcher, J. D., C. Haun, and P. G. Okkema. "The DAF-3 Smad binds DNA and represses gene expression in the Caenorhabditis elegans pharynx." Development 126, no. 1 (January 1, 1999): 97–107. http://dx.doi.org/10.1242/dev.126.1.97.
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Gene expression in the pharyngeal muscles of Caenorhabditis elegans is controlled in part by organ-specific signals, which in the myo-2 gene target a short DNA sequence termed the C subelement. To identify genes contributing to these signals, we performed a yeast one-hybrid screen for cDNAs encoding factors that bind the C subelement. One clone recovered was from daf-3, which encodes a Smad most closely related to vertebrate Smad4. We demonstrated that DAF-3 binds C subelement DNA directly and specifically using gel mobility shift and DNase1 protection assays. Mutation of any base in the sequence GTCTG interfered with binding in the gel mobility shift assay, demonstrating that this pentanucleotide is a core recognition sequence for DAF-3 binding. daf-3 is known to promote formation of dauer larvae and this activity is negatively regulated by TGFbeta-like signaling. To determine how daf-3 affects C subelement enhancer activity in vivo, we examined expression a gfp reporter controlled by a concatenated C subelement oligonucleotide in daf-3 mutants and other mutants affecting the TGFbeta-like signaling pathway controlling dauer formation. Our results demonstrate that wild-type daf-3 can repress C subelement enhancer activity during larval development and, like its dauer-promoting activity, daf-3's repressor activity is negatively regulated by TGFbeta-like signaling. We have examined expression of this gfp reporter in dauer larvae and have observed no daf-3-dependent repression of C activity. These results suggest daf-3 directly regulates pharyngeal gene expression during non-dauer development.
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Andersson, Emma K., Mårten Strand, Karin Edlund, Kristina Lindman, Per-Anders Enquist, Sara Spjut, Annika Allard, Mikael Elofsson, Ya-Fang Mei, and Göran Wadell. "Small-Molecule Screening Using a Whole-Cell Viral Replication Reporter Gene Assay Identifies 2-{[2-(Benzoylamino)Benzoyl]Amino}-Benzoic Acid as a Novel Antiadenoviral Compound." Antimicrobial Agents and Chemotherapy 54, no. 9 (June 28, 2010): 3871–77. http://dx.doi.org/10.1128/aac.00203-10.
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ABSTRACT Adenovirus infections are widespread in society and are occasionally associated with severe, but rarely with life-threatening, disease in otherwise healthy individuals. In contrast, adenovirus infections present a real threat to immunocompromised individuals and can result in disseminated and fatal disease. The number of patients undergoing immunosuppressive therapy for solid organ or hematopoietic stem cell transplantation is steadily increasing, as is the number of AIDS patients, and this makes the problem of adenovirus infections even more urgent to solve. There is no formally approved treatment of adenovirus infections today, and existing antiviral agents evaluated for their antiadenoviral effect give inconsistent results. We have developed a whole cell-based assay for high-throughput screening of potential antiadenoviral compounds. The assay is unique in that it is based on a replication-competent adenovirus type 11p green fluorescent protein (GFP)-expressing vector (RCAd11pGFP). This allows measurement of fluorescence changes as a direct result of RCAd11pGFP genome expression. Using this assay, we have screened 9,800 commercially available small organic compounds. Initially, we observed approximately 400 compounds that inhibited adenovirus expression in vitro by ≥80%, but only 24 were later confirmed as dose-dependent inhibitors of adenovirus. One compound in particular, 2-{[2-(benzoylamino)benzoyl]amino}-benzoic acid, turned out to be a potent inhibitor of adenovirus replication.
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Lentini, Laura, Raffaella Melfi, Aldo Di Leonardo, Angelo Spinello, Giampaolo Barone, Andrea Pace, Antonio Palumbo Piccionello, and Ivana Pibiri. "Toward a Rationale for the PTC124 (Ataluren) Promoted Readthrough of Premature Stop Codons: A Computational Approach and GFP-Reporter Cell-Based Assay." Molecular Pharmaceutics 11, no. 3 (February 7, 2014): 653–64. http://dx.doi.org/10.1021/mp400230s.
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Apfel, Johanna, Patricia Reischmann, and Oliver Müller. "A New Fluorescence-Based Reporter Gene Vector as a Tool for Analyzing and Fishing Cells with Activated Wnt Signaling Pathway." ISRN Oncology 2013 (August 28, 2013): 1–8. http://dx.doi.org/10.1155/2013/603129.
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The dysregulated Wnt pathway is a major cause for the activation of cell proliferation and reduced differentiation in tumor cells. Therefore the Wnt signaling pathway is the on-top target in searching for new anticancer drugs or therapeutic strategies. Although the key players of the pathway are known, no specific anti-Wnt drug entered a clinical trial by now. Several screening approaches for potential compounds have been performed with a reporter gene assay using multiple T-cell factor/lymphoid enhancer factor (TCF/LEF) binding motifs as promoters which control luciferase or β-galactosidase as reporter genes. In our work, we designed a reporter gene construct which anchors the enhanced green fluorescent protein (eGFP) to the plasma membrane. HEK 293T cells, which were stably transfected with this construct, express eGFP on the outer membrane after activation with either LiCl or WNT3A protein. Thus, cells with activated Wnt pathway could be identified and fished out of a heterogeneous cell pool by the use of magnetic-labeled anti-GFP antibodies. In summary, we present a new tool to easily detect, quantify, and sort cells with activated Wnt signaling pathway in a simple, fast, and cost-effective way.
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Kachko, Ilana, Adva Maissel, Livnat Mazor, Ronit Ben-Romano, Robert T. Watson, June C. Hou, Jeffrey E. Pessin, Nava Bashan, and Assaf Rudich. "Postreceptoral Adipocyte Insulin Resistance Induced by Nelfinavir Is Caused by Insensitivity of PKB/Akt to Phosphatidylinositol-3,4,5-Trisphosphate." Endocrinology 150, no. 6 (January 29, 2009): 2618–26. http://dx.doi.org/10.1210/en.2008-1205.
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Adipocyte insulin resistance can be caused by proximal insulin signaling defects but also from postreceptor mechanisms, which in large are poorly characterized. Adipocytes exposed for 18 h to the HIV protease inhibitor nelfinavir manifest insulin resistance characterized by normal insulin-stimulated tyrosine phosphorylation of the insulin receptor and insulin receptor substrate proteins, preserved in vitro phosphatidylinositol 3-kinase (PI 3-kinase) assay activity but impaired activation of PKB/Akt and stimulation of glucose uptake. Here we aimed to assess whether impaired PKB/Akt activation is indeed rate limiting for insulin signaling propagation in response to nelfinavir and the mechanism for defective PKB/Akt activation. Nelfinavir treatment of 3T3-L1 adipocytes impaired the insulin-stimulated translocation and membrane fusion of myc-glucose transporter (GLUT)-4-green fluorescent protein (GFP) reporter. Phosphorylation of PKB/Akt substrates including glycogen synthase kinase-3 and AS160 decreased in response to nelfinavir, and this remained true, even in cells with forced generation of phosphatidylinositol-3,4,5-trisphohphate (PIP3) by a membrane-targeted active PI 3-kinase, confirming that impaired PKB/Akt activation was rate limiting for insulin signal propagation. Cells expressing a GFP-tagged pleckstrin homology domain of general receptors for phosphoinositides 1, which binds PIP3, revealed intact PIP3-mediated plasma membrane translocation of this reporter in nelfinavir-treated cells. However, expression of a membrane-targeted catalytic subunit of PI 3-kinase failed to induce myc-GLUT4-GFP translocation in the absence of insulin, as it did in control cells. Conversely, a membrane-targeted and constitutively active PKB/Akt mutant was normally phosphorylated on S473 and T308, confirming intact PKB/Akt kinases activity, and induced myc-GLUT4-GFP translocation. Collectively, nelfinavir uncovers a postreceptor mechanism for insulin resistance, caused by interference with the sensing of PIP3 by PKB/Akt, leading to impaired GLUT4 translocation and membrane fusion.
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Rodrigues, Margret S., Jeffrey R. Gonneville, Klaus Podar, David M. Weinstock, James D. Griffin, and Martin Sattler. "BCR-ABL Induces Error-Prone Single Strand Annealing in Transformed Cells." Blood 110, no. 11 (November 16, 2007): 2937. http://dx.doi.org/10.1182/blood.v110.11.2937.2937.
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Abstract Chronic myelogenous leukemia (CML) is a hematopoietic stem cell disorder, caused by the BCR-ABL tyrosine kinase oncogene. BCR-ABL kinase activity is required for all aspects of transformation, including abnormal proliferation and neoplastic expansion of stem cells, activation of signaling pathways and genomic instability. We have recently shown that BCR-ABL kinase activity also causes elevated intracellular levels of reactive oxygen species (ROS). These chronically elevated ROS levels have been implicated in the induction of DNA double-strand breaks (DSB) and therapy-related drug resistance, mainly through low- fidelity homology-directed repair (HDR) of DNA lesions. We have utilized GFP-based reporters in BCR-ABL transformed cells to measure both HDR and single-strand annealing (SSA), a mutagenic pathway of homologous repair between repetitive sequences. Repair rates of a single DSB by each of these pathways was measured in BCR-ABL expressing BaF3 cells (BaF3.p210), which have previously been shown to be an excellent system to model the induction of imatinib resistant mutations. We found that inhibition of BCR-ABL kinase activity by imatinib decreased the frequency of SSA by 73 ± 2% (n=3) and increased the frequency of HDR by 70 ± 11% (n=3) in BaF3.p210, compared to untreated cells. Treatment with imatinib did not affect HDR or SSA in BaF3 cells that do not express BCR-ABL. We also determined the fidelity of both SSA and HDR in our model system using clonal populations that stably express the repaired GFP+ reporters (n=20). As expected, sequencing of GFP+ repair products from cells containing the SSA reporter confirmed the expected sequence deletions, consistent with an error-prone mechanism. In contrast, sequencing of GFP+ repair products from the HDR reporter indicated a high-fidelity repair mechanism, without any mutations. It should be noted that this reporter assay does not account for potentially imprecise HDR, leading to a non-functional GFP. Nevertheless, point mutations at the previously reported rate were not detected. Thus, altered HDR fidelity may not be a universal mechanism for the induction of point mutations by BCR-ABL. Our data suggests a unique and alternative pathway, whereby BCR-ABL alters the balance between the SSA and HDR pathways. Moreover, in the presence of interleukin-3 at a concentration that supports not only viability but also cell growth (1ng/ml), imatinib was ineffective at protecting cells from error-prone SSA. In vivo, stromal cells may provide these additional growth signals. Our in vitro data show that stromal cell conditioned medium is not only sufficient to support growth and viability of CML cell lines in the presence of imatinib, but it can also lead to elevated levels of ROS. An abnormal increase in ROS is sufficient by itself to cause genomic mutations (transitions and/or transversions) as a result of single strand oxidative DNA lesions, even in the absence of altered DSB or single strand lesion repair mechanisms. Thus, therapy-related resistance and additional genomic abnormalities may not only be a result of BCR-ABL dependent ROS induction but may also occur within the stromal cell microenvironment through ROS induction by growth factors.
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Galovic, Vladislava, T. Rausch, and Slobodanka Grsic-Rausch. "Mature embryo-derived wheat transformation with major stress-modulated antioxidant target gene." Archives of Biological Sciences 62, no. 3 (2010): 539–46. http://dx.doi.org/10.2298/abs1003539g.
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The mature embryos of fourteen elite winter wheat cultivars have been transformed by a biolistic approach. The gene coding for ?-glutamylcysteine synthetase (EC 6.3.2.2) was used as a transgene in order to obtain stable transformants resistant to drought stress. A binary vector, pBinarUTRECS, was used. The gene was under the control of the CaMV35S promoter region. GUS::GFP gene fusion was used as a reporter system and nptII served as a selectable marker gene. A high regeneration capacity of callus tissue under the selective pressure and successful GUS assay of transformed tissue were an indication of successful insertion of a transgene into mature embryo derived wheat tissue. .
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Yin, Shixue, Mayuree Fuangthong, William P. Laratta, and James P. Shapleigh. "Use of a Green Fluorescent Protein-Based Reporter Fusion for Detection of Nitric Oxide Produced by Denitrifiers." Applied and Environmental Microbiology 69, no. 7 (July 2003): 3938–44. http://dx.doi.org/10.1128/aem.69.7.3938-3944.2003.
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ABSTRACT To determine if green fluorescent protein could be used as a reporter for detecting nitric oxide production, gfp was fused to nnrS from Rhodobacter sphaeroides 2.4.3. nnrS was chosen because its expression requires nitric oxide. The presence of the fusion in R. sphaeroides 2.4.3 resulted in a significant increase in fluorescent intensity of the cells, but only when nitrite reductase was active. Cells lacking nitrite reductase activity and consequently the ability to generate nitric oxide were only weakly fluorescent when grown under denitrification-inducing conditions. One of the R. sphaeroides strains unable to generate nitric oxide endogenously was used as a reporter to detect exogenously produced nitric oxide. Incubation of this strain with sodium nitroprusside, a nitric oxide generator, significantly increased its fluorescence intensity. Mixing of known denitrifiers with the reporter strain also led to significant increases in fluorescence intensity, although the level varied depending on the denitrifier used. The reporter was tested on unknown isolates capable of growing anaerobically in the presence of nitrate, and one of these was able to induce expression of the fusion. Analysis of the 16S rRNA gene sequence of this isolate placed it within the Thauera aromatica subgroup, which is known to contain denitrifiers. These experiments demonstrate that this green fluorescent protein-based assay provides a useful method for assessing the ability of bacteria to produce nitric oxide.
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Tadjali, Mehrdad, Sheng Zhou, and Brian P. Sorrentino. "Expression of Abcg2 in Murine Skeletal Muscle Cells Identifies Two Populations with Different Myogenic Potential." Blood 108, no. 11 (November 16, 2006): 1669. http://dx.doi.org/10.1182/blood.v108.11.1669.1669.
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Stem cells can be identified by a side population (SP) phenotype in a variety of adult and embryonic tissues. We have previously shown that expression of the Abcg2 serves as a prospective marker for isolating HSCs suggesting that Abcg2 expression may also serve as a marker for stem cell activity in other non-hematopoetic tissues. In particular, skeletal muscle SP cells have been shown to have stem cell activity in muscle reconstitution experiments and the SP population in skeletal muscle is significantly reduced in Abcg2 null mice. To investigate the possibility that Abcg2 can serve as a muscle stem cell marker, we used our mouse strain in which a GFP reporter gene was inserted into the Abcg2 locus. Skeletal muscle cells from adult Abcg2/GFP knock-in mice were isolated based on GFP expression and tested for stem cell activity. To exclude contamination by hematopoetic cells, all experiments were performed on cells gated for the CD45 −/Ter119− phenotype. Flow cytometric analysis showed that 11.6 ± 4.2 % of these muscle cells expressed the Abcg2/GFP allele. Since myogenic progenitor cells have the CD34+/ Sca-1−phenotype, GFP positive and negative cell populations were further analyzed for CD34 and Sca-1 expression. This analysis showed that 15.6 ± 5.3 % of Abcg2/GFP+ cells were CD34+/ Sca-1−. In contrast, 51.3 ±18.3 % of Abcg2/GFP− cells were CD34+/ Sca-1−. These results indicated that Abcg2/GFP− cell population may have a higher frequency of myogenic progenitor cells when compared to Abcg2/GFP+ cells. Analysis of skeletal muscle SP cells for GFP expression showed that 57.5±12 % of the SP and 10.8±0.9 % of non-SP or main population (MP) cells expressed the Abcg2/GFP allele. When SP and MP cell populations were analyzed for CD34 and Sca-1 expression, the highest percentage of CD34+/Sca-1− cells were found in MP/GFP− cell population (33.8±5.3%). Since 61.7 % of total cells were MP/GFP− cells, the greatest absolute number of cells with the myogenic phenotype were found to be located in MP/GFP− population. The growth characteristics and differentiation potential of Abcg2/GFP+ and Abcg2/GFP− cells were then assessed in a myogenic clonal culture assay. Sorted Abcg2/GFP+ and Abcg2/GFP− cells were plated in collagen-coated plates in proliferation medium. Both cell populations increased in number and formed large colonies after 7 days in culture. When these cells were then cultured in myogenic differentiation medium for 4 days, only GFP− cells differentiated into contracting myofibers. In contrast, GFP+ cells differentiated mostly into adherent fibroblast like cells. This data was further validated by DNA micro-arrays analysis of GFP+ and GFP− cell populations. We found that GFP− cells expressed skeletal muscle-specific genes such as MyoD, myf-5, myogenin and troponin whereas GFP+ population did not express any of these genes. Based on these data, we conclude that myogenic progenitor cells did not express the Abcg2/GFP allele. We are currently characterizing the Abcg2/GFP+ population for potential mesenchymal stem cell activity. Transplantation assays to determine myogenic activity of GFP+ and GFP− populations in vivo are in progress.
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Breiman, Adrien, Damien Vitour, Myriam Vilasco, Catherine Ottone, Sonia Molina, Lydiane Pichard, Chantal Fournier, et al. "A hepatitis C virus (HCV) NS3/4A protease-dependent strategy for the identification and purification of HCV-infected cells." Journal of General Virology 87, no. 12 (December 1, 2006): 3587–98. http://dx.doi.org/10.1099/vir.0.82214-0.
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As a tool for the identification and/or purification of hepatitis C virus (HCV)-infected cells, a chimeric form of the Gal4VP16 transcription factor was engineered to be activated only in the presence of the HCV NS3/4A protease and to induce different reporter genes [choramphenical acetyltransferase (CAT), green fluorescent protein (GFP) and the cell-surface marker H-2Kk] through the (Gal4)5-E1b promoter. For this, the NS5A/5B trans-cleavage motif of HCV of genotype 1a was inserted between Gal4VP16 and the N terminus of the endoplasmic reticulum (ER)-resident protein PERK, and it was demonstrated that it could be cleaved specifically by NS3/4A. Accordingly, transient transfection in tetracycline-inducible UHCV-11 cells expressing the HCV polyprotein of genotype 1a revealed the migration of the Gal4VP16 moiety of the chimera from the ER to the nucleus upon HCV expression. Activation of the chimera provoked specific gene induction, as shown by CAT assay, first in UHCV-11 cells and then in Huh-7 cells expressing an HCV replicon of genotype 1b (Huh-7 Rep). In addition, the GFP reporter gene allowed rapid fluorescence monitoring of HCV expression in the Huh-7 Rep cells. Finally, the chimera was introduced into Huh-7.5 cells infected with cell culture-generated HCV JFH1 (genotype 2a), allowing the purification of the HCV-infected cells by immunomagnetic cell sorting using H-2Kk as gene reporter. In conclusion, the Gal4VP16 chimera activation system can be used for the rapid identification and purification of HCV-infected cells.
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Atta, Ghada, Falk Schroedl, Alexandra Kaser-Eichberger, Gabriel Spitzer, Andreas Traweger, Ludwig M. Heindl, and Herbert Tempfer. "Scleraxis expressing scleral cells respond to inflammatory stimulation." Histochemistry and Cell Biology 156, no. 2 (May 8, 2021): 123–32. http://dx.doi.org/10.1007/s00418-021-01985-y.
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AbstractThe sclera is an ocular tissue rich of collagenous extracellular matrix, which is built up and maintained by relatively few, still poorly characterized fibroblast-like cells. The aims of this study are to add to the characterization of scleral fibroblasts and to examine the reaction of these fibroblasts to inflammatory stimulation in an ex vivo organotypic model. Scleras of scleraxis-GFP (SCX-GFP) mice were analyzed using immunohistochemistry and qRT-PCR for the expression of the tendon cell associated marker genes scleraxis (SCX), mohawk and tenomodulin. In organotypic tissue culture, explanted scleras of adult scleraxis GFP reporter mice were exposed to 10 ng/ml recombinant interleukin 1-ß (IL1-ß) and IL1-ß in combination with dexamethasone. The tissue was then analyzed by immunofluorescence staining of the inflammation- and fibrosis-associated proteins IL6, COX-2, iNOS, connective tissue growth factor, MMP2, MMP3, and MMP13 as well as for collagen fibre degradation using a Collagen Hybridizing Peptide (CHP) binding assay. The mouse sclera displayed a strong expression of scleraxis promoter-driven GFP, indicating a tendon cell-like phenotype, as well as expression of scleraxis, tenomodulin and mohawk mRNA. Upon IL1-ß stimulation, SCX-GFP+ cells significantly upregulated the expression of all proteins analysed. Moreover, IL1-ß stimulation resulted in significant collagen degradation. Adding the corticosteroid dexamethasone significantly reduced the response to IL1-ß stimulation. Collagen degradation was significantly enhanced in the IL1-ß group. Dexamethasone demonstrated a significant rescue effect. This work provides insights into the characteristics of scleral cells and establishes an ex vivo model of scleral inflammation.
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Qian, Hong, and Mikael Sigvardsson. "Early B-Cell Factor 2 Represents A Novel Marker of Highly Purified Messenchymal Stem-Like Cells in Mouse Bone Marrow." Blood 114, no. 22 (November 20, 2009): 252. http://dx.doi.org/10.1182/blood.v114.22.252.252.
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Abstract Abstract 252 The future therapeutic use of mesenchymal stem cells (MSCs) for human depends on the establishment of preclinical studies with other mammals such as mouse. However, purification and characterization of mouse MSCs from bone marrow (BM) remain poorly documented. The lack of MSC-specific markers for isolation and characterization has been a main obstacle to both research and clinical application with MSCs. The isolation method based on the adherence properties of MSCs in culture has proven ineffective because of large contamination of various hematopoietic lineages and potential phenotypic changes in cultured cells. Consequently, there is very little knowledge about the precise properties of a MSC in its native environment. In the present study, we have utilized a bacterial artificial chromosomes (BAC) transgenic reporter mouse line expressing enhanced green fluorescent protein (EGFP) under the control of the regulatory elements of the Ebf2 gene. Early B cell factor 2 (EBF2) , is a member of the EBF family of transcription factors and has been shown to be expressed in the endosteal niche (Kieslinger et al 2005), a region where MSCs may be defined. The Ebf2-EGFP expressing stromal cells (CD45−TER119−GFP+) are composed 0.002% of total BM mononuclear cells and could be sorted by fluorescence-activated cell sorter (FACS) from adult Tg (Ebf2-EGFP)FB58Gsat/Mmcd mice. The fidelity of GFP expression to that of the endogenous Ebf2 gene was confirmed by quantitative real time PCR providing evidences for that the reporter gene expression marked a defined population of CD45− cells. GFP+ cells could not be found in the hematopoietic cell compartments (CD45+TER119+), indicating a unique expression of EBF2 in stromal cells. In addition, a 10-fold reduction in frequency of CD45−TER119−GFP+ cells in marrow cells compared to that in bone suggested a preferential distribution of the GFP+ stromal cells in endosteal area of the bone. Colony forming unit-fibroblast (CFU-F) assay of the sorted cells revealed that the frequency of CFU-Fs in CD45−TER119−GFP+ cells reached as high as 1 out of 15 whereas only around1/4000 could be detected in CD45−TER119−GFP− cells, indicating the GFP+ EBF2 expressing cells are enriched for primitive stromal progenitor cells in BM. Morphologically, CFU-Fs derived from the GFP+ cells are mostly spindle-shaped and mononuclear. In contrast, the CFU-Fs derived from the GFP- cells are enriched with big cells containing multiple nuclei and differentiated stromal cells. In line with the function and morphological data, Microarray data obtained from two independent experiments revealed a dramatic downregulation of cell cycle genes including Cdc6, Cdca2-4, −7,−8 and Ki67, Cdk4-6) and up-regulation of Cdkis such as p57 and p21 in the GFP+ cells, compared to the GFP− cells, indicating quiescence state of GFP+ cells. Even though the GFP+ cells functionally appeared more primitive than the GFP− cells, multiple lineage associated genes specific for osteoblasts, adipocyte, chondrocyte and myocyte were relatively higher expressed in the GFP+ cell population compared to the GFP− cells, possibly indicating lineage priming events. To test the expression profiles of cell surface antigens that has been studied in the MSCs selected in culture, we performed multiple-color FACS analysis of expressions of CD34, SCA1, CD44 and CD29 within CD45−TER119−GFP+ cells. In order to exclude contamination of hematopoietic cells, we add additional markers B220/CD19 in separated channels during the FACS analysis. Consistent with the Microarray data, we found that GFP+ cells are 100% CD29+, but 100% CD44−. In addition, they express higher levels of CD34, SCA1, compared to the GFP− cells. This is in contrast to the previous studies showing that the MSCs from culture express higher level of CD44 and most of them are CD34−. Thus, using the transgenic EBF2 reporter mouse model we have been able to prospectively isolate an MSC like cell directly from the adult mouse BM largely increasing the possibilities to investigate phenotypic and molecular characteristics of the BM primitive mesenchymal progenitor cells ex vivo. Disclosures: No relevant conflicts of interest to declare.
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Pasqualini, Catherine, Dominique Guivarc’h, Jean-Vianney Barnier, Bernard Guibert, Jean-Didier Vincent, and Philippe Vernier. "Differential Subcellular Distribution and Transcriptional Activity of ΣE3, ΣE4, and ΣE3–4 Isoforms of the Rat Estrogen Receptor-α." Molecular Endocrinology 15, no. 6 (June 1, 2001): 894–908. http://dx.doi.org/10.1210/mend.15.6.0642.
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Abstract ΣE3, ΣE4, and ΣE3–4 are naturally occurring estrogen receptor (ER) isoforms, generated through differential splicing of the ERα primary transcript and abundantly expressed in embryonic rat pituitary. Studies in COS cells transfected with full-length ERα or its three splice variants fused to green fluorescent protein (GFP), revealed a different subcellular localization for each isoform. In the absence of estradiol, full-length ERα-GFP was predominantly nuclear, and ΣE3-GFP and ΣE4-GFP were present both in cytoplasm and nucleus, whereas ΣE3–4-GFP was predominantly cytoplasmic. Upon hormone treatment, a dramatic redistribution of full-length ERα-GFP and ΣE3-GFP, from a diffuse to punctate pattern, occurred within the nucleus. In contrast, the distribution of ΣE4-GFP and ΣE3–4-GFP was unaffected. Nuclear fractionation studies showed that full-length ER-α and ΣE3 displayed the same hormone-induced ability to tether to nuclear matrix, whereas nuclear ΣE4 appeared to remain loosely associated to functional nuclear constituents. When cotransfected with an estrogen-inducible reporter plasmid (VIT-TK-CAT) in ER-negative (CHO k1) and ER-positive pituitary (GH4 C1) cells, ΣE3–4 exhibited a very weak estrogen-dependent transactivation activity, whereas ΣE3 had an inhibitory effect on full-length ER action. Conversely, ΣE4 displayed estrogen-independent transcriptional activity in ER-negative cells, and in ER-positive cells, enhanced the estrogen-induced gene expression as efficiently as full-length ERα. In a gel mobility shift assay, phosphorylated ΣE4 was able to form a specific complex with a consensus ERE, while ΣE3 and ΣE3–4 never did bind by themselves. The observed inhibitory action of ΣE3 on estrogen-dependent transcription would rather involve protein-protein interactions such as formation of heterodimers with full-length ERα, as suggested by immunoprecipitation followed by Western blotting. These data suggest that ΣE3 and ΣE4 may play a physiologically relevant role as negative or constitutively positive modulators of transcription, in the developing rat pituitary.
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Majer, Christine, Changzheng Xu, Kenneth W. Berendzen, and Frank Hochholdinger. "Molecular interactions of ROOTLESS CONCERNING CROWN AND SEMINAL ROOTS, a LOB domain protein regulating shoot-borne root initiation in maize ( Zea mays L.)." Philosophical Transactions of the Royal Society B: Biological Sciences 367, no. 1595 (June 5, 2012): 1542–51. http://dx.doi.org/10.1098/rstb.2011.0238.
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Rootless concerning crown and seminal roots ( Rtcs ) encodes a LATERAL ORGAN BOUNDARIES domain (LBD) protein that regulates shoot-borne root initiation in maize ( Zea mays L.). GREEN FLUORESCENT PROTEIN (GFP)-fusions revealed RTCS localization in the nucleus while its paralogue RTCS-LIKE (RTCL) was detected in the nucleus and cytoplasm probably owing to an amino acid exchange in a nuclear localization signal. Moreover, enzyme-linked immunosorbent assay (ELISA) experiments demonstrated that RTCS primarily binds to LBD DNA motifs. RTCS binding to an LBD motif in the promoter of the auxin response factor (ARF) ZmArf34 and reciprocally, reciprocal ZmARF34 binding to an auxin responsive element motif in the promoter of Rtcs was shown by electrophoretic mobility shift assay experiments. In addition, comparative qRT-PCR of wild-type versus rtcs coleoptilar nodes suggested RTCS-dependent activation of ZmArf34 expression. Consistently, luciferase reporter assays illustrated the capacity of RTCS, RTCL and ZmARF34 to activate downstream gene expression. Finally, RTCL homo- and RTCS/RTCL hetero-interaction were demonstrated in yeast-two-hybrid and bimolecular fluorescence complementation experiments, suggesting a role of these complexes in downstream gene regulation. In summary, the data provide novel insights into the molecular interactions resulting in crown root initiation in maize.
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Yu, Z., CH Lee, C. Chinpaisal, and LN Wei. "A constitutive nuclear localization signal from the second zinc-finger of orphan nuclear receptor TR2." Journal of Endocrinology 159, no. 1 (October 1, 1998): 53–60. http://dx.doi.org/10.1677/joe.0.1590053.
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The orphan nuclear receptor TR2 and its truncated isoform deleted in the ligand binding domain (LBD) were localized exclusively in the nuclei as revealed by two methods of detection. An anti-hemagglutinin (HA) antibody detected specific nuclear localization of HA-tagged receptors and the green fluorescent protein (GFP)-tagged receptors were found to be distributed in the nuclei of living cells. By deletion analyses, the sequence responsible for targeting this receptor into the nucleus was defined. A stretch of 20 amino acid residues (KDCVINKHHRNRCQYCRLQR) within the second zinc-finger of this receptor is required for its nuclear localization and this signal is constitutively active. No nuclear localization signal was found in the N-terminus or the LBD. The GFP-tagged receptor remained biologically active, as evidenced by its repressive activity on the reporter that carried a binding site for this receptor, a direct repeat-5 (DR5). An electrophoretic mobility shift assay was performed to characterize the binding property of TR2 and its truncated isoform. TR2 bound to the DR5 as dimers whereas its truncated isoform bound as monomers.