scholarly journals Generation of a Sleeping Beauty transposon-based cellular system for rapid and sensitive identification of SARS-CoV-2 host dependency and restriction factors

2021 ◽  
Author(s):  
Marek Widera ◽  
Alexander Wilhelm ◽  
Tuna Toptan ◽  
Johanna M. Raffel ◽  
Eric Kowarz ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Line ◽  
High Throughput ◽  
A549 Cells ◽  
Sleeping Beauty ◽  
Cellular System ◽  
Reporter Cell Line ◽  
Restriction Factors ◽  
Reporter Cell ◽  
Sleeping Beauty Transposon

SummaryThe severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. The common methods to monitor and quantitate SARS-CoV-2 infectivity in cell culture are so far time-consuming and labor-intensive. Using the Sleeping Beauty transposase system, we generated a robust and versatile reporter cell system that allows SARS-CoV-2 infection experiments compatible for high-throughput and live cell imaging. The reporter cell is based on lung derived A549 cells, which show a profound interferon response and convenient cell culture characteristics. ACE2 and TMPRSS2 were introduced for constitutive expression in A549 cells. Subclones with varying levels of ACE2/TMPRSS2 were screened for optimal SARS-CoV2 susceptibility. Furthermore, extensive evaluation demonstrated that SARS-CoV-2 infected reporter cells were distinguishable from mock-infected cells and already showed approximately 12 h post infection a clear signal to noise ratio in terms of cell roughness, fluorescence and a profound visible cytopathic effect. Moreover, due to the high transfection efficiency and proliferation capacity, Sleeping Beauty transposase-based overexpression cell lines with a second inducible fluorescence reporter cassette (eGFP) can be generated in a very short time, enabling the investigation of host and restriction factors in a doxycycline-inducible manner. Thus, the novel reporter cell line allows rapid and sensitive detection of SARS-CoV-2 infection and the screening for host factors essential for viral replication.Highlights- Sleeping Beauty transposon-based cellular system was used to generate a highly susceptible cell line for monitoring SARS-CoV-2 infection- The versatile reporter cell line A549-AT is suitable for rapid and sensitive high-throughput assays- Additional gene specific expression cassettes allow the identification of SARS-CoV-2 host dependency and restriction factors

scholarly journals Development and utilization of human decidualization reporter cell line uncovers new modulators of female fertility

2019 ◽  
Vol 116 (39) ◽  
pp. 19541-19551
Author(s):  
Meade Haller ◽  
Yan Yin ◽  
Liang Ma
Keyword(s):  
Cell Line ◽  
High Throughput ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Patient Specific ◽  
Reporter Cell Line ◽  
Reporter Cell ◽  
A Genome ◽  
Decidual Cells

Failure of embryo implantation accounts for a significant percentage of female infertility. Exquisitely coordinated molecular programs govern the interaction between the competent blastocyst and the receptive uterus. Decidualization, the rapid proliferation and differentiation of endometrial stromal cells into decidual cells, is required for implantation. Decidualization defects can cause poor placentation, intrauterine growth restriction, and early parturition leading to preterm birth. Decidualization has not yet been systematically studied at the genetic level due to the lack of a suitable high-throughput screening tool. Herein we describe the generation of an immortalized human endometrial stromal cell line that uses yellow fluorescent protein under the control of the prolactin promoter as a quantifiable visual readout of the decidualization response (hESC-PRLY cells). Using this cell line, we performed a genome-wide siRNA library screen, as well as a screen of 910 small molecules, to identify more than 4,000 previously unrecognized genetic and chemical modulators of decidualization. Ontology analysis revealed several groups of decidualization modulators, including many previously unappreciated transcription factors, sensory receptors, growth factors, and kinases. Expression studies of hits revealed that the majority of decidualization modulators are acutely sensitive to ovarian hormone exposure. Gradient treatment of exogenous factors was used to identify EC50 values of small-molecule hits, as well as verify several growth factor hits identified by the siRNA screen. The high-throughput decidualization reporter cell line and the findings described herein will aid in the development of patient-specific treatments for decidualization-based recurrent pregnancy loss, subfertility, and infertility.

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 From c-Photina® Mouse Embryonic Stem Cells to High-Throughput Screening of Differentiated Neural Cells via an Intermediate Step Enriched in Neural Precursor Cells

2010 ◽  
Vol 15 (9) ◽  
pp. 1132-1143 ◽  
Author(s):  
Silvia Cainarca ◽  
Simone Fenu ◽  
Silvia Bovolenta ◽  
Patrizia Arioli ◽  
Andrea Menegon ◽  
...  
Keyword(s):  
Cell Line ◽  
High Throughput ◽  
High Throughput Screening ◽  
Embryonic Stem ◽  
Precursor Cells ◽  
Neural Precursor Cells ◽  
Neural Precursor ◽  
Intermediate Step ◽  
Reporter Cell Line ◽  
Reporter Cell

The use of engineered mouse embryonic stem (mES) cells in high-throughput screening (HTS) can offer new opportunities for studying complex targets in their native environment, increasing the probability of discovering more meaningful hits. The authors have generated and developed a mouse embryonic stem cell line called c-Photina® mES stably expressing a Ca2+-activated photoprotein as a reporter gene. This reporter cell line retains the ability to differentiate into any cell lineage and can be used for miniaturized screening processes in 384-well microplates. The c-Photina® mES cell line is particularly well suited for the study of the pharmacological modulation of target genes that induce Ca2+ mobilization. The authors differentiated this mES reporter cell line into neuronal cells and screened the LOPAC1280™ library monitoring the agonistic or antagonistic activities of compounds. They also demonstrate the possibility to generate and freeze bulk preparations of cells at an intermediate stage of differentiation and enriched in neural precursor cells, which retain the ability to form fully functional neural networks once thawed. The proposed cell model is of high value for HTS purposes because it offers a more physiological environment to the targets of interest and the possibility of using frozen batches of neural precursor cells.

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

scholarly journals Generation of a Jurkat-based fluorescent reporter cell line to evaluate lipid antigen interaction with the human iNKT cell receptor

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Piotr Humeniuk ◽  
Sabine Geiselhart ◽  
Claire Battin ◽  
Tonya Webb ◽  
Peter Steinberger ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Receptor ◽  
Inkt Cell ◽  
Reporter Cell Line ◽  
Reporter Cell ◽  
Fluorescent Reporter ◽  
Lipid Antigen ◽  
Antigen Interaction
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