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Source: Journal of Biomolecular Screening
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Total 17 results found since Jan 2013.
Activation of Yap-Directed Transcription by Knockdown of Conserved Cellular Functions
The Yap-Hippo pathway has a significant role in regulating cell proliferation and growth, thus controlling organ size and regeneration. The Hippo pathway regulates two highly conserved, transcription coactivators, YAP and TAZ. The upstream regulators of the Yap-Hippo pathway have not been fully characterized. The aim of this study was to use a siRNA screen, in a liver biliary cell line, to identify regulators of the Yap-Hippo pathway that allow activation of the YAP transcription coactivator at high cell density. Activation of the YAP transcription coactivator was monitored using a high-content, image-based assay that meas...
Source: Journal of Biomolecular Screening - February 19, 2016 Category: Molecular Biology Authors: Agarinis, C., Orsini, V., Megel, P., Abraham, Y., Yang, H., Mickanin, C., Myer, V., Bouwmeester, T., Tchorz, J. S., Parker, C. N. Tags: Original Research Source Type: research
Phenotypic Screening for Friedreich Ataxia Using Random shRNA Selection
Friedreich ataxia (FRDA) is an autosomal recessive neuro- and cardio-degenerative disorder for which there are no proven effective treatments. FRDA is caused by decreased expression and/or function of the protein frataxin. Frataxin chaperones iron in the mitochondrial matrix and regulates the iron–sulfur cluster (ISC) assembly complex. ISCs are prosthetic groups critical for the function of the Krebs cycle and the mitochondrial electron transport chain. Decreased expression of frataxin is associated with decreased ISC assembly, mitochondrial iron accumulation, and increased oxidative stress, all of which contribute t...
Source: Journal of Biomolecular Screening - September 18, 2015 Category: Molecular Biology Authors: Cotticelli, M. G., Acquaviva, F., Xia, S., Kaur, A., Wang, Y., Wilson, R. B. Tags: Original Research Source Type: research
Electroporation Knows No Boundaries: The Use of Electrostimulation for siRNA Delivery in Cells and Tissues
The discovery of RNA interference (RNAi) has enabled several breakthrough discoveries in the area of functional genomics. The RNAi technology has emerged as one of the major tools for drug target identification and has been steadily improved to allow gene manipulation in cell lines, tissues, and whole organisms. One of the major hurdles for the use of RNAi in high-throughput screening has been delivery to cells and tissues. Some cell types are refractory to high-efficiency transfection with standard methods such as lipofection or calcium phosphate precipitation and require different means. Electroporation is a powerful and...
Source: Journal of Biomolecular Screening - August 19, 2015 Category: Molecular Biology Authors: Luft, C., Ketteler, R. Tags: Articles Source Type: research
RNAi Screening with Self-Delivering, Synthetic siRNAs for Identification of Genes That Regulate Primary Human T Cell Migration
This study therefore demonstrates the ease and benefits of conducting siRNA library screens in primary human T cells using self-delivering, chemically modified siRNAs, and it emphasizes the feasibility and potential of this approach for elucidating the signaling pathways that regulate T cell function.
Source: Journal of Biomolecular Screening - August 19, 2015 Category: Molecular Biology Authors: Freeley, M., Derrick, E., Dempsey, E., Hoff, A., Davies, A., Leake, D., Vermeulen, A., Kelleher, D., Long, A. Tags: Articles Source Type: research
Acoustic Liquid Handling for Rapid siRNA Transfection Optimization
In this study, we describe a methodology to utilize the flexibility and low-volume range of the Echo acoustic liquid handler to rapidly screen a matrix of transfection conditions. The matrix includes six different transfection lipids from three separate vendors across a broad range of concentrations. Our results validate acoustic liquid transfer for the delivery of siRNAs and transfection reagents. Finally, this methodology is applied to rapidly optimize transfection conditions across many tissue culture cell lines derived from various originating tissues.
Source: Journal of Biomolecular Screening - August 19, 2015 Category: Molecular Biology Authors: Xiao, A. S., Lightcap, E. S., Bouck, D. C. Tags: Articles Source Type: research
MicroRNA Screening and the Quest for Biologically Relevant Targets
This article provides an overview of cell-based screenings for miRNA function that were performed in different biological contexts. The advantages and limitations of computational and experimental approaches commonly used to identify miRNA targets are also discussed.
Source: Journal of Biomolecular Screening - August 19, 2015 Category: Molecular Biology Authors: Eulalio, A., Mano, M. Tags: Articles Source Type: research
Targeting Human Long Noncoding Transcripts by Endoribonuclease-Prepared siRNAs
We present a compendium of lncRNA expression data for 11 human cancer cell lines. Furthermore, we show that the resource is suitable for combined knockdown and localization analysis. We discuss challenges in sequence annotation of lncRNAs with respect to their often low and cell type–specific expression and specify esiRNAs that are suitable for targeting lncRNAs in commonly used human cell lines.
Source: Journal of Biomolecular Screening - August 19, 2015 Category: Molecular Biology Authors: Theis, M., Paszkowski-Rogacz, M., Weisswange, I., Chakraborty, D., Buchholz, F. Tags: Articles Source Type: research
High-Throughput Silencing Using the CRISPR-Cas9 System: A Review of the Benefits and Challenges
The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system has been seized upon with a fervor enjoyed previously by small interfering RNA (siRNA) and short hairpin RNA (shRNA) technologies and has enormous potential for high-throughput functional genomics studies. The decision to use this approach must be balanced with respect to adoption of existing platforms versus awaiting the development of more "mature" next-generation systems. Here, experience from siRNA and shRNA screening plays an important role, as issues such as targeting efficiency, pooling strategies, and off-target effects with those tec...
Source: Journal of Biomolecular Screening - August 19, 2015 Category: Molecular Biology Authors: Wade, M. Tags: Articles Source Type: research
A Combination of Screening and Computational Approaches for the Identification of Novel Compounds That Decrease Mast Cell Degranulation
High-content screening of compound libraries poses various challenges in the early steps in drug discovery such as gaining insights into the mode of action of the selected compounds. Here, we addressed these challenges by integrating two biological screens through bioinformatics and computational analysis. We screened a small-molecule library enriched in amphiphilic compounds in a degranulation assay in rat basophilic leukemia 2H3 (RBL-2H3) cells. The same library was rescreened in a high-content image-based endocytosis assay in HeLa cells. This assay was previously applied to a genome-wide RNAi screen that produced quanti...
Source: Journal of Biomolecular Screening - June 19, 2015 Category: Molecular Biology Authors: McShane, M. P., Friedrichson, T., Giner, A., Meyenhofer, F., Barsacchi, R., Bickle, M., Zerial, M. Tags: Original Research Source Type: research
Genome-Engineering Tools to Establish Accurate Reporter Cell Lines That Enable Identification of Therapeutic Strategies to Treat Friedreich's Ataxia
Friedreich’s ataxia is a neurodegenerative disease caused by deficiency of the mitochondrial protein frataxin. This deficiency results from expansion of a trinucleotide repeat in the first intron of the frataxin gene. Because this repeat expansion resides in an intron and hence does not alter the amino acid sequence of the frataxin protein, gene reactivation could be of therapeutic benefit. High-throughput screening for frataxin activators has so far met with limited success because current cellular models may not accurately assess endogenous frataxin gene regulation. Here we report the design and validation of genom...
Source: Journal of Biomolecular Screening - June 19, 2015 Category: Molecular Biology Authors: Villasenor, R., Miraglia, L., Romero, A., Tu, B., Punga, T., Knuckles, P., Duss, S., Orth, T., Buhler, M. Tags: Original Research Source Type: research
Impact of RNA-Guided Technologies for Target Identification and Deconvolution
For well over a decade, RNA interference (RNAi) has provided a powerful tool for investigators to query specific gene targets in an easily modulated loss-of-function setting, both in vitro and in vivo. Hundreds of publications have demonstrated the utility of RNAi in arrayed and pooled-based formats, in a wide variety of cell-based systems, including clonal, stem, transformed, and primary cells. Over the years, there have been significant improvements in the design of target-specific small-interfering RNA (siRNA) and short-hairpin RNA (shRNA), expression vectors, methods for mitigating off-target effects, and accurately in...
Source: Journal of Biomolecular Screening - November 20, 2014 Category: Molecular Biology Authors: Fennell, M., Xiang, Q., Hwang, A., Chen, C., Huang, C.-H., Chen, C.-C., Pelossof, R., Garippa, R. J. Tags: Review Article Source Type: research
Global Human-Kinase Screening Identifies Therapeutic Host Targets against Influenza
During viral infection of human cells, host kinases mediate signaling activities that are used by all viruses for replication; therefore, targeting of host kinases is of broad therapeutic interest. Here, host kinases were globally screened during human influenza virus (H1N1) infection to determine the time-dependent effects of virus infection and replication on kinase function. Desthiobiotin-labeled analogs of adenosine triphosphate and adenosine diphosphate were used to probe and covalently label host kinases in infected cell lysates, and probe affinity was determined. Using infected human A549 cells, we screened for time...
Source: Journal of Biomolecular Screening - June 24, 2014 Category: Molecular Biology Authors: Atkins, C., Evans, C. W., Nordin, B., Patricelli, M. P., Reynolds, R., Wennerberg, K., Noah, J. W. Tags: Original Research Source Type: research
Identification of a Selective Agonist for Liver X Receptor {alpha} (LXR{alpha}) via Screening of a Synthetic Compound Library
Liver X receptor α (LXRα) plays an important role in reverse cholesterol transport (RCT), and activation of LXRα could reduce atherosclerosis. In the present study, we developed a screening method to identify new potential LXRα agonists using an LXRα-GAL4 chimera reporter assay. A novel analogue of N,N-disubstituted 2,8-diazaspiro[4.5]decane, IMB-151, was identified as an LXRα agonist by using this method. IMB-151 showed a significant activation effect on LXRα, with an EC50 value of 1.47 µM. IMB-151 also increased the expression of ATP-binding cassette transporter A1 (ABCA1) ...
Source: Journal of Biomolecular Screening - March 7, 2014 Category: Molecular Biology Authors: Li, N., Xu, Y., Feng, T., Liu, C., Li, Y., Wang, X., Si, S. Tags: Original Research Source Type: research
A Novel Microscopy-Based High-Throughput Screening Method to Identify Proteins That Regulate Global Histone Modification Levels
Posttranslational modifications of histones play an important role in the regulation of gene expression and chromatin structure in eukaryotes. The balance between chromatin factors depositing (writers) and removing (erasers) histone marks regulates the steady-state levels of chromatin modifications. Here we describe a novel microscopy-based screening method to identify proteins that regulate histone modification levels in a high-throughput fashion. We named our method CROSS, for Chromatin Regulation Ontology SiRNA Screening. CROSS is based on an siRNA library targeting the expression of 529 proteins involved in chromatin r...
Source: Journal of Biomolecular Screening - January 16, 2014 Category: Molecular Biology Authors: Baas, R., Lelieveld, D., van Teeffelen, H., Lijnzaad, P., Castelijns, B., van Schaik, F. M., Vermeulen, M., Egan, D. A., Timmers, H. T. M., de Graaf, P. Tags: Original Research Source Type: research
A High-Throughput Assay to Identify Modifiers of Premature Chromosome Condensation
Premature chromosome condensation (PCC) is a consequence of early mitotic entry, where mitosis begins before completion of DNA replication. Previously we have identified mutations in MCPH1, a DNA damage response and potential tumor suppressor gene, as a cause of primary microcephaly and PCC. Here we describe a high-throughput assay to identify modifiers of PCC. Reverse transfection of control siRNA followed by a forward transfection of MCPH1 small interfering RNA (siRNA) was performed to induce PCC. Condensin II subunits CAPG2 and CAPH2 were validated as PCC modifiers and therefore positive controls. Cell nuclei were detec...
Source: Journal of Biomolecular Screening - December 16, 2013 Category: Molecular Biology Authors: Adams, M., Cookson, V. J., Higgins, J., Martin, H. L., Tomlinson, D. C., Bond, J., Morrison, E. E., Bell, S. M. Tags: Technical Notes Source Type: research
