scholarly journals Generation of a novel HEK293 luciferase reporter cell line by CRISPR/Cas9-mediated site-specific integration in the genome to explore the transcriptional regulation of the PGRN gene

Bioengineered ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 98-107 ◽  
Author(s):  
Yanqing Li ◽  
Sai Li ◽  
Yan Li ◽  
Haibin Xia ◽  
Qinwen Mao
Keyword(s):  
Transcriptional Regulation ◽  
Cell Line ◽  
Luciferase Reporter ◽  
Reporter Cell Line ◽  
Reporter Cell ◽  
Site Specific ◽  
Site Specific Integration

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 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 cell-based screen identifies HDAC inhibitors and PLK inhibitor as activators of RIG-I signaling

2021 ◽  
Author(s):  
Eugenia Fraile-Bethencourt ◽  
Marie H Foss ◽  
Dylan Nelson ◽  
Sanjay V Malhotra ◽  
Sudarshan Anand
Keyword(s):  
Nucleic Acid ◽  
Cell Line ◽  
High Throughput Screening ◽  
Hdac Inhibitors ◽  
Luciferase Reporter ◽  
Reporter Cell Line ◽  
Reporter Cell ◽  
High Throughput Screening Assay ◽  
A Cell ◽  
Acid Sensors

Enhancing the immune microenvironment in cancer by targeting the nucleic acid sensors is becoming a potent therapeutic strategy. Among the nucleic acid sensors, activation of the RNA sensor Retinoic Acid-inducible Gene (RIG-I) using small hairpin RNAs has been shown to elicit powerful innate and adaptive immune responses. Given the challenges inherent in pharmacokinetics and delivery of RNA based agonists, we set out to discover small molecule agonists of RIG-I using a cell-based assay. To this end, we established and validated a robust high throughput screening assay based on a commercially available HEK293 reporter cell line with a luciferase reporter downstream of tandem interferon stimulated gene 54 (ISG54) promoter elements. We first confirmed that the luminescence in this cell line is dependent on RIG-I and the interferon receptor using a hairpin RNA RIG-I agonist. We established a 96-well and a 384-well format HTS based on this cell line and performed a proof-of-concept screen using an FDA approved drug library of 1200 compounds. Surprisingly, we found two HDAC inhibitors Entinostat, Mocetinostat and the PLK1 inhibitor Volasertib significantly enhanced ISG-luciferase activity. This luminescence was substantially diminished in the null reporter cell line indicating the increase in signaling was dependent on RIG-I expression. Treatment of tumor cell lines with Entinostat, Mocetinostat or Volasertib induced interferon signature genes and increased RIG-I induced cell death in a mammary carcinoma cell line. Taken together, our data indicates an unexpected role for HDAC1,-3 inhibitors and PLK1 inhibitors in enhancing RIG-I signaling and highlight potential opportunities for therapeutic combinations.

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 Generation of a NKX2.1 – p63 double transgenic knock-in reporter cell line from human induced pluripotent stem cells (MHHi006-A-4)

2020 ◽  
Vol 42 ◽  
pp. 101659
Author(s):  
Nora Drick ◽  
Anais Sahabian ◽  
Praeploy Pongpamorn ◽  
Sylvia Merkert ◽  
Gudrun Göhring ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Cell Line ◽  
Pluripotent Stem Cells ◽  
Reporter Cell Line ◽  
Reporter Cell ◽  
Induced Pluripotent
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