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 A Genome Scale CRISPR Screen Identifies the ER Cargo Receptor That Facilitates the Efficient Secretion of Erythropoietin

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 340-340
Author(s):  
Zesen Lin ◽  
Greggory Myers ◽  
Beth McGee ◽  
Richard King ◽  
Vi Tang ◽  
...  
Keyword(s):  
Cell Line ◽  
Hek293t Cell ◽  
High Population ◽  
Reporter Cell Line ◽  
Intracellular Accumulation ◽  
Reporter Cell ◽  
Physiological Level ◽  
Genome Wide ◽  
A Genome ◽  
Cargo Receptor

Abstract Erythropoietin (EPO) is a plasma glycoprotein that binds erythroid progenitors in the bone marrow and stimulates their proliferation and differentiation. EPO is secreted into the circulation by specialized kidney peritubular fibroblasts. Though the transcriptional regulation of EPO production has been well studied, the intracellular regulation of EPO trafficking remains poorly understood. In an effort to identify genes involved in EPO secretion, we developed a genome-wide functional screen that provides a quantifiable and selectable readout of intracellular EPO accumulation. In order to perform such a screen, we generated a reporter HEK293T cell line stably expressing EPO fused to GFP and as an internal control, alpha-1-antitrypsin (A1AT) fused to mCherry. We showed that both EPO and A1AT are efficiently secreted from the cell and that treatment with Brefeldin A (which disrupts endoplasmic reticulum [ER] to Golgi transport) results in intracellular accumulation of EPO and A1AT. These findings demonstrate that the machinery required for the efficient secretion of EPO via the classical secretory pathway is intact in this cell line. To identify genes that affect EPO secretion, we mutagenized the reporter cell line with a CRISPR/Cas9 knock-out library (GeCKO-v2), which delivers SpCas9, a puromycin resistance cassette, and a pooled collection of 123,411 single guide RNAs (sgRNAs) that include six independent sgRNAs targeting nearly every coding gene in the human genome. Transduction was performed at low multiplicity of infection (MOI ~0.3), such that most infected cells receive 1 sgRNA to mutate 1 gene in the genome. Puromycin selection was applied from days 1-4 post-transduction. After 14 days, cells with normal mCherry but increased (top 7%) or decreased (bottom 7%) GFP fluorescence were isolated. Integrated sgRNAs sequences were quantified by deep sequencing and analyzed for their enrichment in the GFP high compared to the GFP low population. This strategy allows the identification of genes that affect EPO but not A1AT levels, therefore ruling out genes that affect global secretion. This screen, performed in triplicates, identified that the sgRNAs targeting surfeit locus protein 4 (SURF4) are the mostly enriched sgRNAs (at the gene level) in the GFP high population: 5 out of 6 sgRNAs targeting SURF4 were enriched in the GFP high population, at a genome-wide statistical level. To validate these results, we generated a sgRNA targeting SURF4 and demonstrated that SURF4 deletion results in intra-cellular accumulation of EPO with no effect on A1AT. We confirmed these results in several independent reporter cell line clones, excluding an artifact unique to the original reporter clone used in the screen. Additionally, the intracellular EPO accumulation in SURF4 deficient cells was rescued by SURF4 cDNA, ruling out an off-target sgRNA effect. We next showed that SURF4 interacts with EPO (by co-immunoprecipitation) and that EPO accumulates in the ER of SURF4 deleted cells (using endo-H and fluorescent confocal microscopy). In contrast to EPO, we found that SURF4 deletion does not result in the intracellular accumulation of a related glycoprotein, thrombopoietin. To examine if SURF4 facilitates the secretion of EPO when expressed at a more physiological level, we deleted SURF4 in HEP3B cells induced to express EPO from its endogenous locus and found that SURF4 also mediates the secretion of EPO under these conditions. Taken together, the studies summarized above demonstrate that SURF4 is the ER cargo receptor that promotes the efficient secretion of EPO. Additional work is currently ongoing to further characterize the role of SURF4 in the secretion of EPO. Disclosures No relevant conflicts of interest to declare.

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 Inducible APOBEC3G-Vif Double Stable Cell Line as a High-Throughput Screening Platform To Identify Antiviral Compounds

2009 ◽  
Vol 54 (1) ◽  
pp. 78-87 ◽  
Author(s):  
Boris Nowotny ◽  
Thomas Schneider ◽  
Gabriele Pradel ◽  
Tanja Schirmeister ◽  
Axel Rethwilm ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Cell Line ◽  
High Throughput ◽  
High Throughput Screening ◽  
Yellow Fluorescent Protein ◽  
26S Proteasome ◽  
Stable Cell Line ◽  
Antiviral Compound ◽  
Compound Libraries ◽  
Antiviral Compounds

ABSTRACT Inhibition of the interaction of the human cytidine-deaminase APOBEC3G (A3G) with the human immunodeficiency virus (HIV) type 1-specific viral infectivity factor (Vif) represents a novel therapeutic approach in which a cellular factor with potent antiviral activity (A3G) plays a key role. In HIV-infected cells, the interaction of Vif with A3G leads to the subsequent degradation of A3G by the 26S proteasome via the ubiquitin pathway and to the loss of antiviral activity. To establish a stable and convenient cellular testing platform for the high-throughput screening of potential antiviral compound libraries, we engineered a double transgenic cell line constitutively expressing an enhanced yellow fluorescent protein expressor (EYFP-A3G) fusion as well as a Tet-Off controllable Vif protein. With this cell line, we were able to measure precisely the Vif-induced degradation of A3G in the presence of potential antiviral compounds in an easy-to-handle, robust, and practical high-throughput multiwell plate format with an excellent screening window coefficient (Z factor) of 0.67.

scholarly journals A human VE-cadherin-tdTomato and CD43-green fluorescent protein dual reporter cell line for study endothelial to hematopoietic transition

2016 ◽  
Vol 17 (2) ◽  
pp. 401-405 ◽  
Author(s):  
Ho Sun Jung ◽  
Gene Uenishi ◽  
Akhilesh Kumar ◽  
Mi Ae Park ◽  
Matt Raymond ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Cell Line ◽  
Fluorescent Protein ◽  
Reporter Cell Line ◽  
Reporter Cell ◽  
Dual Reporter ◽  
Green Fluorescent

scholarly journals Optimizing the Expression of Recombinant αβγ GABAA Receptors in HEK293 Cells for High-Throughput Screening

2008 ◽  
Vol 14 (1) ◽  
pp. 86-91 ◽  
Author(s):  
Daniel Gilbert ◽  
Abolghasem Esmaeili ◽  
Joseph W. Lynch
Keyword(s):  
High Throughput ◽  
Gabaa Receptors ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Effective Means ◽  
Hek293 Cells ◽  
Gaba A ◽  
Protein Assay ◽  
Lead Compounds

Despite being important clinical targets, it is not straightforward to reliably express recombinant trimeric αβγ GABA-A receptors (GABAARs) for high-throughput screening. This study therefore sought to devise a simple and reliable means of transiently expressing α1β1γ1 and α1β1γ2 GABAARs in HEK293 cells. Expression efficiencies resulting from 5 different transfection strategies were assessed by flow cytometry and pharmacological analysis using an anion-sensitive yellow fluorescent protein-based assay. PolyFect™ and Effectene™, employed according to the manufacturers' instructions, conferred the strongest and most reliable expression of trimeric αβγ GABAARs. Functional analysis via the yellow fluorescent protein assay revealed dramatic differences in the pharmacological properties of γ1- and γ2-containing receptors, consistent with previous electrophysiological characterizations. The authors conclude that this method of expressing and screening recombinant GABAARs provides an effective means of discovering novel GABAAR modulators for use as therapeutic lead compounds and pharmacological probes. ( Journal of Biomolecular Screening 2009:86-91)

scholarly journals Mycobacterial Shuttle Vectors Designed for High-Level Protein Expression in Infected Macrophages

2012 ◽  
Vol 78 (19) ◽  
pp. 6829-6837 ◽  
Author(s):  
Jennifer L. Eitson ◽  
Jennifer J. Medeiros ◽  
Ashley R. Hoover ◽  
Shashikant Srivastava ◽  
Kole T. Roybal ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Protein Expression ◽  
Cell Line ◽  
Fluorescent Protein ◽  
Reporter Cell Line ◽  
Reporter Cell ◽  
Content Type ◽  
Shuttle Vectors ◽  
E Coli ◽  
High Level

ABSTRACTMycobacterial shuttle vectors contain dual origins of replication for growth in bothEscherichia coliand mycobacteria. One such vector, pSUM36, was re-engineered for high-level protein expression in diverse bacterial species. The modified vector (pSUM-kan-MCS2) enabled green fluorescent protein expression inE. coli,Mycobacterium smegmatis, andM. aviumat levels up to 50-fold higher than that detected with the parental vector, which was originally developed with alacZα promoter. This high-level fluorescent protein expression allowed easy visualization ofM. smegmatisandM. aviumin infected macrophages. TheM. tuberculosisgeneesat-6was cloned in place of the green fluorescence protein gene (gfp) to determine the impact of ESAT-6 on the innate inflammatory response. The modified vector (pSUM-kan-MCS2) yielded high levels of ESAT-6 expression inM. smegmatis. The ability of ESAT-6 to suppress innate inflammatory pathways was assayed with a novel macrophage reporter cell line, designed with an interleukin-6 (IL-6) promoter-driven GFP cassette. This stable cell line fluoresces in response to diverse mycobacterial strains and stimuli, such as lipopolysaccharide.M. smegmatisclones expressing high levels of ESAT-6 failed to attenuate IL-6-driven GFP expression. Pure ESAT-6, produced inE. coli, was insufficient to suppress a strong inflammatory response elicited byM. smegmatisor lipopolysaccharide, with ESAT-6 itself directly activating the IL-6 pathway. In summary, a pSUM-protein expression vector and a mammalian IL-6 reporter cell line provide new tools for understanding the pathogenic mechanisms deployed by various mycobacterial species.

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