scholarly journals New Reporter Cell Line To Evaluate the Sequential Emergence of Multiple Human Cytomegalovirus Mutations during In Vitro Drug Exposure

2005 ◽  
Vol 49 (12) ◽  
pp. 4860-4866 ◽  
Author(s):  
C. Gilbert ◽  
G. Boivin
Keyword(s):  
Cell Line ◽  
Human Cytomegalovirus ◽  
Drug Exposure ◽  
Luciferase Reporter ◽  
Reporter Cell Line ◽  
Reporter Cell ◽  
Luciferase Reporter Gene ◽  
Selection Of

ABSTRACT We developed a new reporter cell line for human cytomegalovirus (HCMV) drug susceptibility testing. This cell line was obtained by incorporating the luciferase reporter gene under the control of an HCMV-specific promoter into the genome of astrocytoma cells (U373MG). We then used our reporter cell line to evaluate phenotypic changes conferred by the sequential emergence of HCMV UL54 and UL97 mutations following long-term drug exposure. The laboratory strain AD169 was passaged in the presence of increasing concentrations of ganciclovir (one viral line) or foscarnet (two viral lines). Resistant viruses were plaque purified at five different concentrations of ganciclovir and at three different concentrations of foscarnet. In addition to the previously described M460I and L595S UL97 mutations and the L545S and V812L UL54 mutations, exposition to ganciclovir (up to 3,000 μM) resulted in the selection of two unreported UL54 mutations (P829S and D879G). Passages in the presence of foscarnet (up to 3,000 μM) resulted in the selection of seven not previously described UL54 mutations (K500N, T552N, S585A, N757K, L802V, L926V, and L957F) in addition to the N408D mutation that has been associated with ganciclovir and cidofovir resistance. Long-term exposure of HCMV to either ganciclovir or foscarnet ultimately resulted in the selection of multiple UL54 mutations that conferred high levels of resistance to all approved HCMV DNA polymerase inhibitors, i.e., ganciclovir, cidofovir, and foscarnet. Emergence of each viral mutation conferred stepwise increases in drug 50% inhibitory concentrations that could be objectively measured with the new reporter cell assay.

scholarly journals Development and Validation of a Reporter-Cell-Line-Based Bioassay for Therapeutic Soluble gp130-Fc

Molecules ◽  
2019 ◽  
Vol 24 (21) ◽  
pp. 3845
Author(s):  
Lei Yu ◽  
Chuncui Jia ◽  
Wenrong Yao ◽  
Dening Pei ◽  
Xi Qin ◽  
...  
Keyword(s):  
Cell Line ◽  
Dose Response ◽  
Inflammatory Diseases ◽  
Luciferase Reporter ◽  
Reporter Cell Line ◽  
Reporter Cell ◽  
Luciferase Reporter Gene ◽  
Detection Range ◽  
Significant Difference ◽  
Dose Response Effect

Soluble glycoprotein 130 kDa (sgp130)-Fc fusion protein, an innovative therapeutic bio-macromolecular drug specifically targeting IL-6 trans-signaling, proved to have good potential for application in the treatment of chronic inflammatory diseases. A simple and quick bioassay for sgp130-Fc was developed in this study. First, a stable reporter cell line was obtained by transfecting CHO-K1 cells with a sis-inducible element (SIE)-driving luciferase reporter gene (CHO/SIE-Luc). Sgp130-Fc could inhibit the expression of luciferase induced by IL-6/sIL-6Rα complex, and the dose–response curve fitted the four-parameter logistic model, with 50% inhibitive concentration (IC50) being about 500 ng/mL and detection range between 40 and 5000 ng/mL. Both the intra-assay and inter-assay coefficient of variation (CV) were below 10.0%, and the accuracy estimates ranged from 94.1% to 106.2%. The assay indicated a good linearity (R² = 0.99) in the range of 50% to 150% of optimized initial concentration. No significant difference was found between the test results of new assay and BAF3/gp130 proliferation assay (unpaired t test, p = 0.4960, n = 6). The dose-response effect and copy number of the luciferase gene was basically unchanged after long-term culture (up to passage 60), demonstrating the stability of CHO/SIE-Luc cells. These results suggested that the new reporter assay was suited to routine potency determination of therapeutic sgp130-Fc.

scholarly journals A new reporter cell line to monitor HIV infection and drug susceptibility in vitro

1997 ◽  
Vol 94 (9) ◽  
pp. 4653-4658 ◽  
Author(s):  
A. Gervaix ◽  
D. West ◽  
L. M. Leoni ◽  
D. D. Richman ◽  
F. Wong-Staal ◽  
...  
Keyword(s):  
Hiv Infection ◽  
Cell Line ◽  
Drug Susceptibility ◽  
Reporter Cell Line ◽  
Reporter Cell

scholarly journals Development of a Cell-Based High-Throughput Screening Assay to Identify Porcine Host Defense Peptide-Inducing Compounds

2018 ◽  
Vol 2018 ◽  
pp. 1-13 ◽  
Author(s):  
Zhuo Deng ◽  
Jing Wang ◽  
Wentao Lyu ◽  
Xuwen Wieneke ◽  
Robert Matts ◽  
...  
Keyword(s):  
Cell Line ◽  
High Throughput ◽  
High Throughput Screening ◽  
Hdac Inhibitors ◽  
Luciferase Reporter ◽  
Reporter Cell Line ◽  
Reporter Cell ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
A Cell

Novel alternatives to antibiotics are needed for the swine industry, given increasing restrictions on subtherapeutic use of antibiotics. Augmenting the synthesis of endogenous host defense peptides (HDPs) has emerged as a promising antibiotic-alternative approach to disease control and prevention. To facilitate the identification of HDP inducers for swine use, we developed a stable luciferase reporter cell line, IPEC-J2/PBD3-luc, through permanent integration of a luciferase reporter gene driven by a 1.1 kb porcine β-defensin 3 (PBD3) gene promoter in porcine IPEC-J2 intestinal epithelial cells. Such a stable reporter cell line was employed in a high-throughput screening of 148 epigenetic compounds and 584 natural products, resulting in the identification of 41 unique hits with a minimum strictly standardized mean difference (SSMD) value of 3.0. Among them, 13 compounds were further confirmed to give at least a 5-fold increase in the luciferase activity in the stable reporter cell line, with 12 being histone deacetylase (HDAC) inhibitors. Eight compounds were subsequently observed to be comparable to sodium butyrate in inducing PBD3 mRNA expression in parental IPEC-J2 cells in the low micromolar range. Six HDAC inhibitors including suberoylanilide hydroxamine (SAHA), HC toxin, apicidin, panobinostat, SB939, and LAQ824 were additionally found to be highly effective HDP inducers in a porcine 3D4/31 macrophage cell line. Besides PBD3, other HDP genes such as PBD2 and cathelicidins (PG1–5) were concentration-dependently induced by those compounds in both IPEC-J2 and 3D4/31 cells. Furthermore, the antibacterial activities of 3D4/31 cells were augmented following 24 h exposure to HDAC inhibitors. In conclusion, a cell-based high-throughput screening assay was developed for the discovery of porcine HDP inducers, and newly identified HDP-inducing compounds may have potential to be developed as alternatives to antibiotics for applications in swine and possibly other animal species.

scholarly journals Development and Characterisation of a Novel NF-κB Reporter Cell Line for Investigation of Neuroinflammation

2017 ◽  
Vol 2017 ◽  
pp. 1-10 ◽  
Author(s):  
Marie-Theres Zeuner ◽  
Thomas Vallance ◽  
Sakthivel Vaiyapuri ◽  
Graeme S. Cottrell ◽  
Darius Widera
Keyword(s):  
Cell Line ◽  
Glioma Cell Line ◽  
Neural Cells ◽  
Reporter Cell Line ◽  
Reporter Cell ◽  
Necrosis Factor Alpha ◽  
In Vitro Drug Screening ◽  
Anti Inflammatory ◽  
Cost Efficient

Aberrant activation of the transcription factor NF-κB, as well as uncontrolled inflammation, has been linked to autoimmune diseases, development and progression of cancer, and neurological disorders like Alzheimer’s disease. Reporter cell lines are a valuable state-of-the art tool for comparative analysis of in vitro drug screening. However, a reporter cell line for the investigation of NF-κB-driven neuroinflammation has not been available. Thus, we developed a stable neural NF-κB-reporter cell line to assess the potency of proinflammatory molecules and peptides, as well as anti-inflammatory pharmaceuticals. We used lentivirus to transduce the glioma cell line U251-MG with a tandem NF-κB reporter construct containing GFP and firefly luciferase allowing an assessment of NF-κB activity via fluorescence microscopy, flow cytometry, and luminometry. We observed a robust activation of NF-κB after exposure of the reporter cell line to tumour necrosis factor alpha (TNFα) and amyloid-β peptide [1-42] as well as to LPS derived from Salmonella minnesota and Escherichia coli. Finally, we demonstrate that the U251-NF-κB-GFP-Luc reporter cells can be used for assessing the anti-inflammatory potential of pharmaceutical compounds using Bay11-7082 and IMD0354. In summary, our newly generated cell line is a robust and cost-efficient tool to study pro- and anti-inflammatory potential of drugs and biologics in neural cells.

scholarly journals An In Vitro Test to Screen Skin Sensitizers Using a Stable THP-1–Derived IL-8 Reporter Cell Line, THP-G8

2011 ◽  
Vol 124 (2) ◽  
pp. 359-369 ◽  
Author(s):  
Toshiya Takahashi ◽  
Yutaka Kimura ◽  
Rumiko Saito ◽  
Yoshihiro Nakajima ◽  
Yoshihiro Ohmiya ◽  
...  
Keyword(s):  
Cell Line ◽  
In Vitro Test ◽  
Reporter Cell Line ◽  
Reporter Cell ◽  
Vitro Test

scholarly journals SUN-267 Dissecting Type 2 CRH Receptor Signaling Characteristics in the Hypothalamic Cell Line MHYPOA-2/30

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Viridiana Alcantara-Alonso ◽  
Patricia de Gortari ◽  
Robert Dallmann ◽  
Dimitris Grammatopoulos
Keyword(s):  
Gene Expression ◽  
Plasma Membrane ◽  
Protein Expression ◽  
Cell Line ◽  
Cellular Localization ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Luciferase Reporter Gene

Abstract The stress peptides coticotropin-releasing hormone (CRH) and urocortins (Ucns) exert anorectic effects acting mainly through the type 2 CRH receptor (CRH-R2) in the hypothalamus. Impairment of CRH-R2 signaling in chronically stressed rats has been linked with the development of hyperphagia (Alcantara-Alonso et al. Neuropeptides, 2017) however the exact mechanisms and molecular defects are unknown. In the present study we used the mHypoA-2/30, a hypothalamic immortalized cell line derived from adult mice (Belsham et al. FASEB J, 2009) to further explore the signaling molecules mediating the anorexigenic effect of the CRH-R2 cognate agonist urocortin 2 (Ucn2). Specifically, we investigated mRNA, protein expression and cellular localization of CRH-R2 in the mHypoA-2/30 neurons. Additionally, we examined the effects of Ucn2 on the phosphorylation of CREB and AMPK, as well as its transcriptional effects on genes of feeding-related peptides and molecules involved in modulation of circadian rhythms. Both CRH-R2 mRNA and protein expression were detected in mHypoA-2/30; indirect immunoflourescence experiments using a specific CRH-R2 antibody demonstrated widespread localization in the plasma membrane and cytoplasm. Moreover, the receptor sub-cellular localization was redistributed in response to activation by Ucn2 (100 nM), as the plasma membrane immunofluorescent signal was decreased after 4h of agonist treatment, suggesting CRH-R2 homologous internalization. We also observed a 50% increase in the phosphorylation of CREB associated with a concomitant decrease in AMPK phosphorylation after 30 min of Ucn2 treatment. Among the panel of hypothalamic genes analyzed, we identified after 24h of Ucn2 treatment increases in the gene expression of the anorexigenic peptides neurotensin and proopiomelanocortin. Interestingly, sustained CRH-R2 activation also led to an increase in the mRNA levels of Aryl Hydrocarbon Receptor Nuclear Translocator Like (ARNTL), a protein involved in the control of circadian rhythm. A luciferase reporter gene analysis of ARNTL showed that the mHypoA-2/30 cells also exhibit circadian patterns of expression and that the treatment with Ucn2 enhanced circadian amplitude of ARNTL reporter on these cells, which in turn may be involved in glucocorticoid release in circadian cycles and stimulating appetite during the activity phase of the animals. In conclusion, we found that the mHypoA-2/30 cell line expresses endogenous functional CRH-R2 that is linked to downstream regulation of anorexigenic gene expression. This cell line appears to be a useful in vitro tool to study hypothalamic CRH-R2 signaling machinery involved in central control of food intake and circadian cycles.

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