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1.  Functional complementation of RNA interference mutants in trypanosomes 
BMC Biotechnology  2005;5:6.
Background
In many eukaryotic cells, double-stranded RNA (dsRNA) triggers RNA interference (RNAi), the specific degradation of RNA of homologous sequence. RNAi is now a major tool for reverse-genetics projects, including large-scale high-throughput screens. Recent reports have questioned the specificity of RNAi, raising problems in interpretation of RNAi-based experiments.
Results
Using the protozoan Trypanosoma brucei as a model, we designed a functional complementation assay to ascertain that phenotypic effect(s) observed upon RNAi were due to specific silencing of the targeted gene. This was applied to a cytoskeletal gene encoding the paraflagellar rod protein 2 (TbPFR2), whose product is essential for flagellar motility. We demonstrate the complementation of TbPFR2, silenced via dsRNA targeting its UTRs, through the expression of a tagged RNAi-resistant TbPFR2 encoding a protein that could be immunolocalized in the flagellum. Next, we performed a functional complementation of TbPFR2, silenced via dsRNA targeting its coding sequence, through heterologous expression of the TbPFR2 orthologue gene from Trypanosoma cruzi: the flagellum regained its motility.
Conclusions
This work shows that functional complementation experiments can be readily performed in order to ascertain that phenotypic effects observed upon RNAi experiments are indeed due to the specific silencing of the targetted gene. Further, the results described here are of particular interest when reverse genetics studies cannot be easily achieved in organisms not amenable to RNAi. In addition, our strategy should constitute a firm basis to elaborate functional-dissection studies of genes from other organisms.
doi:10.1186/1472-6750-5-6
PMCID: PMC549545  PMID: 15703078
2.  Automated Quantitative Live Cell Fluorescence Microscopy 
Advances in microscopy automation and image analysis have given biologists the tools to attempt large scale systems-level experiments on biological systems using microscope image readout. Fluorescence microscopy has become a standard tool for assaying gene function in RNAi knockdown screens and protein localization studies in eukaryotic systems. Similar high throughput studies can be attempted in prokaryotes, though the difficulties surrounding work at the diffraction limit pose challenges, and targeting essential genes in a high throughput way can be difficult. Here we will discuss efforts to make live-cell fluorescent microscopy based experiments using genetically encoded fluorescent reporters an automated, high throughput, and quantitative endeavor amenable to systems-level experiments in bacteria. We emphasize a quantitative data reduction approach, using simulation to help develop biologically relevant cell measurements that completely characterize the cell image. We give an example of how this type of data can be directly exploited by statistical learning algorithms to discover functional pathways.
The diffraction limit makes high-throughput fluorescence microscopy more challenging in prokaryotes, but approaches such as quantitative data reduction now allow systems-level analysis of bacteria by this technique.
doi:10.1101/cshperspect.a000455
PMCID: PMC2908775  PMID: 20591990
3.  RNAi Screening: New Approaches, Understandings and Organisms 
RNA interference (RNAi) leads to sequence-specific knockdown of gene function. The approach can be used in large-scale screens to interrogate function in various model organisms and an increasing number of other species. Genome-scale RNAi screens are routinely performed in cultured or primary cells or in vivo in organisms such as C. elegans. High-throughput RNAi screening is benefitting from the development of sophisticated new instrumentation and software tools for collecting and analyzing data, including high-content image data. The results of large-scale RNAi screens have already proved useful, leading to new understandings of gene function relevant to topics such as infection, cancer, obesity and aging. Nevertheless, important caveats apply and should be taken into consideration when developing or interpreting RNAi screens. Some level of false discovery is inherent to high-throughput approaches and specific to RNAi screens, false discovery due to off-target effects (OTEs) of RNAi reagents remains a problem. The need to improve our ability to use RNAi to elucidate gene function at large scale and in additional systems continues to be addressed through improved RNAi library design, development of innovative computational and analysis tools and other approaches.
doi:10.1002/wrna.110
PMCID: PMC3249004  PMID: 21953743
RNAi; high-throughput screens; high-content imaging; cell-based assays
4.  Scale-up and Validation of a MALDI-MS-based Inhibitor Screening Technology 
Recently, mass spectrometry (MS)-based readout has been demonstrated to be a highly effective for high throughput screening (HTS) assays. The major advantages compared to the most common fluorescence readout are the paucity of false readouts, reduced reagent costs, and the ability to multiplex assays such that multiple therapeutic targets can be screened for inhibitor hits with one pass through the compound repository. Previously, we have developed MS-based methods for rapid and accurate compound screening for inhibitors to therapeutic targets. However, the limited use of MS-based methods with small test libraries has been insufficient to validate the overall utility of this readout for large screening campaigns. Thus, in this report, the MS-based readout technology was scaled to include a library of 30,400 compounds to systematically validate the reliability of MALDI-MS readout head-to-head versus a traditional methods of HTS. The target enzyme for these comparative assays is PKC-iota, which plays a role cancer cell survival, tumor growth and potentially invasion. First the MS-based assay was fully integrated into an automated workflow on a PerkinElmer Plate:Explorer HTS system in a 384-well format. Then, the primary screen of 30,400 compounds with both the MS and fluorescence-based readouts yielded a hit rate of 0.3% and 0.9% for the two methods, respectively. Only 29% of the MS-based hits confirmed in triplicate assays; however, 95% of those confirmed hits validated as concentration-dependent inhibitors with IC50 value ranging from low nM to high μM inhibitors. By contrast, 58% of the fluorescence hits were deemed as false positives since they produced fluorescence inhibition even in the absences of PKC-iota. Overall the data validate the utility of the MS-based readout in terms of sensitivity, reproducibility and minimal reagent cost. We are now investigating ways to incorporate screening technologies as an additional service and revenue stream for our core laboratory.
PMCID: PMC3635425
5.  A cell spot microarray method for production of high density siRNA transfection microarrays 
BMC Genomics  2011;12:162.
Background
High-throughput RNAi screening is widely applied in biological research, but remains expensive, infrastructure-intensive and conversion of many assays to HTS applications in microplate format is not feasible.
Results
Here, we describe the optimization of a miniaturized cell spot microarray (CSMA) method, which facilitates utilization of the transfection microarray technique for disparate RNAi analyses. To promote rapid adaptation of the method, the concept has been tested with a panel of 92 adherent cell types, including primary human cells. We demonstrate the method in the systematic screening of 492 GPCR coding genes for impact on growth and survival of cultured human prostate cancer cells.
Conclusions
The CSMA method facilitates reproducible preparation of highly parallel cell microarrays for large-scale gene knockdown analyses. This will be critical towards expanding the cell based functional genetic screens to include more RNAi constructs, allow combinatorial RNAi analyses, multi-parametric phenotypic readouts or comparative analysis of many different cell types.
doi:10.1186/1471-2164-12-162
PMCID: PMC3073923  PMID: 21443765
6.  Multiplexing siRNAs to compress RNAi-based screen size in human cells 
Nucleic Acids Research  2007;35(8):e57.
Here we describe a novel strategy using multiplexes of synthetic small interfering RNAs (siRNAs) corresponding to multiple gene targets in order to compress RNA interference (RNAi) screen size. Before investigating the practical use of this strategy, we first characterized the gene-specific RNAi induced by a large subset (258 siRNAs, 129 genes) of the entire siRNA library used in this study (∼800 siRNAs, ∼400 genes). We next demonstrated that multiplexed siRNAs could silence at least six genes to the same degree as when the genes were targeted individually. The entire library was then used in a screen in which randomly multiplexed siRNAs were assayed for their affect on cell viability. Using this strategy, several gene targets that influenced the viability of a breast cancer cell line were identified. This study suggests that the screening of randomly multiplexed siRNAs may provide an important avenue towards the identification of candidate gene targets for downstream functional analyses and may also be useful for the rapid identification of positive controls for use in novel assay systems. This approach is likely to be especially applicable where assay costs or platform limitations are prohibitive.
doi:10.1093/nar/gkm141
PMCID: PMC1885663  PMID: 17392344
7.  Single-cell analysis of population context advances RNAi screening at multiple levels 
A large set of high-content RNAi screens investigating mammalian virus infection and multiple cellular activities is analysed to reveal the impact of population context on phenotypic variability and to identify indirect RNAi effects.
Cell population context determines phenotypes in RNAi screens of multiple cellular activities (including virus infection, cell size regulation, endocytosis, and lipid homeostasis), which can be accounted for by a combination of novel image analysis and multivariate statistical methods.Accounting for cell population context-mediated effects strongly changes the reproducibility and consistency of RNAi screens across cell lines as well as of siRNAs targeting the same gene.Such analyses can identify the perturbed regulation of population context dependent cell-to-cell variability, a novel perturbation phenotype.Overall, these methods advance the use of large-scale RNAi screening for a systems-level understanding of cellular processes.
Isogenic cells in culture show strong variability, which arises from dynamic adaptations to the microenvironment of individual cells. Here we study the influence of the cell population context, which determines a single cell's microenvironment, in image-based RNAi screens. We developed a comprehensive computational approach that employs Bayesian and multivariate methods at the single-cell level. We applied these methods to 45 RNA interference screens of various sizes, including 7 druggable genome and 2 genome-wide screens, analysing 17 different mammalian virus infections and four related cell physiological processes. Analysing cell-based screens at this depth reveals widespread RNAi-induced changes in the population context of individual cells leading to indirect RNAi effects, as well as perturbations of cell-to-cell variability regulators. We find that accounting for indirect effects improves the consistency between siRNAs targeted against the same gene, and between replicate RNAi screens performed in different cell lines, in different labs, and with different siRNA libraries. In an era where large-scale RNAi screens are increasingly performed to reach a systems-level understanding of cellular processes, we show that this is often improved by analyses that account for and incorporate the single-cell microenvironment.
doi:10.1038/msb.2012.9
PMCID: PMC3361004  PMID: 22531119
cell-to-cell variability; image analysis; population context; RNAi; virus infection
8.  Automated microscopy for high-content RNAi screening 
The Journal of Cell Biology  2010;188(4):453-461.
Fluorescence microscopy is one of the most powerful tools to investigate complex cellular processes such as cell division, cell motility, or intracellular trafficking. The availability of RNA interference (RNAi) technology and automated microscopy has opened the possibility to perform cellular imaging in functional genomics and other large-scale applications. Although imaging often dramatically increases the content of a screening assay, it poses new challenges to achieve accurate quantitative annotation and therefore needs to be carefully adjusted to the specific needs of individual screening applications. In this review, we discuss principles of assay design, large-scale RNAi, microscope automation, and computational data analysis. We highlight strategies for imaging-based RNAi screening adapted to different library and assay designs.
doi:10.1083/jcb.200910105
PMCID: PMC2828931  PMID: 20176920
9.  A Direct Phenotypic Comparison of siRNA Pools and Multiple Individual Duplexes in a Functional Assay 
PLoS ONE  2009;4(12):e8471.
Background
RNAi is a prominent tool for the identification of novel regulatory elements within complex cellular pathways. In invertebrates, RNAi is a relatively straightforward process, where large double-stranded RNA molecules initiate sequence-specific transcript destruction in target cells. In contrast, RNAi in mammalian cell culture assays requires the delivery of short interfering RNA duplexes to target cells. Due to concerns over off-target phenotypes and extreme variability in duplex efficiency, investigators typically deliver and analyze multiple duplexes per target. Currently, duplexes are delivered and analyzed either individually or as a pool of several independent duplexes. A choice between experiments based on siRNA pools or multiple individual duplexes has considerable implications for throughput, reagent requirements and data analysis in genome-wide surveys, yet there are relatively few data that directly compare the efficiency of the two approaches.
Methodology/Principal Findings
To address this critical issue, we conducted a direct comparison of siRNA pools and multiple single siRNAs that target all human phosphatases in a robust functional assay. We determined the frequency with which both approaches uncover loss-of-function phenotypes and compared the phenotypic severity for siRNA pools and the constituent individual duplexes.
Conclusions/Significance
Our survey indicates that screens with siRNA pools have several significant advantages over identical screens with the corresponding individual siRNA duplexes. Of note, we frequently observed greater phenotypic penetrance for siRNA pools than for the parental individual duplexes. Thus, our data indicate that experiments with siRNA pools have a greater likelihood of generating loss-of-function phenotypes than individual siRNA duplexes.
doi:10.1371/journal.pone.0008471
PMCID: PMC2793519  PMID: 20041186
10.  Genomic Screening with RNAi: Results and Challenges 
RNA interference (RNAi) is an effective tool for genome-scale, high-throughput analysis of gene function. In the past five years, a number of genome-scale RNAi high-throughput screens (HTSs) have been done in both Drosophila and mammalian cultured cells to study diverse biological processes, including signal transduction, cancer biology, and host cell responses to infection. Results from these screens have led to the identification of new components of these processes and, importantly, have also provided insights into the complexity of biological systems, forcing new and innovative approaches to understanding functional networks in cells. Here, we review the main findings that have emerged from RNAi HTS and discuss technical issues that remain to be improved, in particular the verification of RNAi results and validation of their biological relevance. Furthermore, we discuss the importance of multiplexed and integrated experimental data analysis pipelines to RNAi HTS.
doi:10.1146/annurev-biochem-060408-092949
PMCID: PMC3564595  PMID: 20367032
bioinformatics; cell biology; Drosophila; high-throughput screening
11.  Statistical Methods for Analysis of High-Throughput RNA Interference Screens 
Nature methods  2009;6(8):569-575.
RNA interference (RNAi) has become a powerful technique for reverse genetics and drug discovery and, in both of these areas, large-scale high-throughput RNAi screens are commonly performed. The statistical techniques used to analyze these screens are frequently borrowed directly from small-molecule screening; however small-molecule and RNAi data characteristics differ in meaningful ways. We examine the similarities and differences between RNAi and small-molecule screens, highlighting particular characteristics of RNAi screen data that must be addressed during analysis. Additionally, we provide guidance on selection of analysis techniques in the context of a sample workflow.
doi:10.1038/nmeth.1351
PMCID: PMC2789971  PMID: 19644458
12.  Genome-wide screens for effective siRNAs through assessing the size of siRNA effects 
BMC Research Notes  2008;1:33.
Background
RNA interference (RNAi) has been seen as a revolution in functional genomics and system biology. Genome-wide RNAi research relies on the development of RNAi high-throughput screening (HTS) assays. One of the most fundamental challenges in RNAi HTS is to glean biological significance from mounds of data, which relies on the development of effective analytic methods for selecting effective small interfering RNAs (siRNAs).
Findings
Based on a recently proposed parameter, strictly standardized mean difference (SSMD), I propose an analytic method for genome-wide screens of effective siRNAs through assessing and testing the size of siRNA effects. Central to this method is the capability of SSMD in quantifying siRNA effects. This method has relied on normal approximation, which works only in the primary screens but not in the confirmatory screens. In this paper, I explore the non-central t-distribution property of SSMD estimates and use this property to extend the SSMD-based method so that it works effectively in either primary or confirmatory screens as well as in any HTS screens with or without replicates. The SSMD-based method maintains a balanced control of false positives and false negatives.
Conclusion
The central interest in genome-wide RNAi research is the selection of effective siRNAs which relies on the development of analytic methods to measure the size of siRNA effects. The new analytic method for hit selection provided in this paper offers a good analytic tool for selecting effective siRNAs, better than current analytic methods, and thus may have broad utility in genome-wide RNAi research.
doi:10.1186/1756-0500-1-33
PMCID: PMC2526086  PMID: 18710486
13.  Screensaver: an open source lab information management system (LIMS) for high throughput screening facilities 
BMC Bioinformatics  2010;11:260.
Background
Shared-usage high throughput screening (HTS) facilities are becoming more common in academe as large-scale small molecule and genome-scale RNAi screening strategies are adopted for basic research purposes. These shared facilities require a unique informatics infrastructure that must not only provide access to and analysis of screening data, but must also manage the administrative and technical challenges associated with conducting numerous, interleaved screening efforts run by multiple independent research groups.
Results
We have developed Screensaver, a free, open source, web-based lab information management system (LIMS), to address the informatics needs of our small molecule and RNAi screening facility. Screensaver supports the storage and comparison of screening data sets, as well as the management of information about screens, screeners, libraries, and laboratory work requests. To our knowledge, Screensaver is one of the first applications to support the storage and analysis of data from both genome-scale RNAi screening projects and small molecule screening projects.
Conclusions
The informatics and administrative needs of an HTS facility may be best managed by a single, integrated, web-accessible application such as Screensaver. Screensaver has proven useful in meeting the requirements of the ICCB-Longwood/NSRB Screening Facility at Harvard Medical School, and has provided similar benefits to other HTS facilities.
doi:10.1186/1471-2105-11-260
PMCID: PMC3001403  PMID: 20482787
14.  web cellHTS2: A web-application for the analysis of high-throughput screening data 
BMC Bioinformatics  2010;11:185.
Background
The analysis of high-throughput screening data sets is an expanding field in bioinformatics. High-throughput screens by RNAi generate large primary data sets which need to be analyzed and annotated to identify relevant phenotypic hits. Large-scale RNAi screens are frequently used to identify novel factors that influence a broad range of cellular processes, including signaling pathway activity, cell proliferation, and host cell infection. Here, we present a web-based application utility for the end-to-end analysis of large cell-based screening experiments by cellHTS2.
Results
The software guides the user through the configuration steps that are required for the analysis of single or multi-channel experiments. The web-application provides options for various standardization and normalization methods, annotation of data sets and a comprehensive HTML report of the screening data analysis, including a ranked hit list. Sessions can be saved and restored for later re-analysis. The web frontend for the cellHTS2 R/Bioconductor package interacts with it through an R-server implementation that enables highly parallel analysis of screening data sets. web cellHTS2 further provides a file import and configuration module for common file formats.
Conclusions
The implemented web-application facilitates the analysis of high-throughput data sets and provides a user-friendly interface. web cellHTS2 is accessible online at http://web-cellHTS2.dkfz.de. A standalone version as a virtual appliance and source code for platforms supporting Java 1.5.0 can be downloaded from the web cellHTS2 page. web cellHTS2 is freely distributed under GPL.
doi:10.1186/1471-2105-11-185
PMCID: PMC3098057  PMID: 20385013
15.  Unsupervised automated high throughput phenotyping of RNAi time-lapse movies 
BMC Bioinformatics  2013;14:292.
Background
Gene perturbation experiments in combination with fluorescence time-lapse cell imaging are a powerful tool in reverse genetics. High content applications require tools for the automated processing of the large amounts of data. These tools include in general several image processing steps, the extraction of morphological descriptors, and the grouping of cells into phenotype classes according to their descriptors. This phenotyping can be applied in a supervised or an unsupervised manner. Unsupervised methods are suitable for the discovery of formerly unknown phenotypes, which are expected to occur in high-throughput RNAi time-lapse screens.
Results
We developed an unsupervised phenotyping approach based on Hidden Markov Models (HMMs) with multivariate Gaussian emissions for the detection of knockdown-specific phenotypes in RNAi time-lapse movies. The automated detection of abnormal cell morphologies allows us to assign a phenotypic fingerprint to each gene knockdown. By applying our method to the Mitocheck database, we show that a phenotypic fingerprint is indicative of a gene’s function.
Conclusion
Our fully unsupervised HMM-based phenotyping is able to automatically identify cell morphologies that are specific for a certain knockdown. Beyond the identification of genes whose knockdown affects cell morphology, phenotypic fingerprints can be used to find modules of functionally related genes.
doi:10.1186/1471-2105-14-292
PMCID: PMC3851277  PMID: 24090185
16.  Intelligent Interfaces for Mining Large-Scale RNAi-HCS Image Databases 
Recently, High-content screening (HCS) has been combined with RNA interference (RNAi) to become an essential image-based high-throughput method for studying genes and biological networks through RNAi-induced cellular phenotype analyses. However, a genome-wide RNAi-HCS screen typically generates tens of thousands of images, most of which remain uncategorized due to the inadequacies of existing HCS image analysis tools. Until now, it still requires highly trained scientists to browse a prohibitively large RNAi-HCS image database and produce only a handful of qualitative results regarding cellular morphological phenotypes. For this reason we have developed intelligent interfaces to facilitate the application of the HCS technology in biomedical research. Our new interfaces empower biologists with computational power not only to effectively and efficiently explore large-scale RNAi-HCS image databases, but also to apply their knowledge and experience to interactive mining of cellular phenotypes using Content-Based Image Retrieval (CBIR) with Relevance Feedback (RF) techniques.
doi:10.1109/BIBE.2007.4375742
PMCID: PMC3028207  PMID: 21278820
17.  A case study of the reproducibility of transcriptional reporter cell-based RNAi screens in Drosophila 
Genome Biology  2007;8(9):R203.
A second generation dsRNA library was used to re-assess factors that influence the outcome of transcriptional reporter-based whole-genome RNAi screens for the Wnt/Wingless (wg) and Hedgehog (hh)-signaling pathways.
Off-target effects have been demonstrated to be a major source of false-positives in RNA interference (RNAi) high-throughput screens. In this study, we re-assess the previously published transcriptional reporter-based whole-genome RNAi screens for the Wingless and Hedgehog signaling pathways using second generation double-stranded RNA libraries. Furthermore, we investigate other factors that may influence the outcome of such screens, including cell-type specificity, robustness of reporters, and assay normalization, which determine the efficacy of RNAi-knockdown of target genes.
doi:10.1186/gb-2007-8-9-r203
PMCID: PMC2375041  PMID: 17903264
18.  Constraint factor graph cut–based active contour method for automated cellular image segmentation in RNAi screening 
Journal of microscopy  2008;230(Pt 2):177-191.
Summary
Image-based, high throughput genome-wide RNA interference (RNAi) experiments are increasingly carried out to facilitate the understanding of gene functions in intricate biological processes. Automated screening of such experiments generates a large number of images with great variations in image quality, which makes manual analysis unreasonably time-consuming. Therefore, effective techniques for automatic image analysis are urgently needed, in which segmentation is one of the most important steps. This paper proposes a fully automatic method for cells segmentation in genome-wide RNAi screening images. The method consists of two steps: nuclei and cytoplasm segmentation. Nuclei are extracted and labelled to initialize cytoplasm segmentation. Since the quality of RNAi image is rather poor, a novel scale-adaptive steerable filter is designed to enhance the image in order to extract long and thin protrusions on the spiky cells. Then, constraint factor GCBAC method and morphological algorithms are combined to be an integrated method to segment tight clustered cells. Compared with the results obtained by using seeded watershed and the ground truth, that is, manual labelling results by experts in RNAi screening data, our method achieves higher accuracy. Compared with active contour methods, our method consumes much less time. The positive results indicate that the proposed method can be applied in automatic image analysis of multi-channel image screening data.
doi:10.1111/j.1365-2818.2008.01974.x
PMCID: PMC2839415  PMID: 18445146
active contour; automatic image segmentation; constraint factor; fluorescent microscopy; genome-wide screening; graph cut; morphological algorithm; RNAi
19.  Integrated Functional, Gene Expression and Genomic Analysis for the Identification of Cancer Targets 
PLoS ONE  2009;4(4):e5120.
The majority of new drug approvals for cancer are based on existing therapeutic targets. One approach to the identification of novel targets is to perform high-throughput RNA interference (RNAi) cellular viability screens. We describe a novel approach combining RNAi screening in multiple cell lines with gene expression and genomic profiling to identify novel cancer targets. We performed parallel RNAi screens in multiple cancer cell lines to identify genes that are essential for viability in some cell lines but not others, suggesting that these genes constitute key drivers of cellular survival in specific cancer cells. This approach was verified by the identification of PIK3CA, silencing of which was selectively lethal to the MCF7 cell line, which harbours an activating oncogenic PIK3CA mutation. We combined our functional RNAi approach with gene expression and genomic analysis, allowing the identification of several novel kinases, including WEE1, that are essential for viability only in cell lines that have an elevated level of expression of this kinase. Furthermore, we identified a subset of breast tumours that highly express WEE1 suggesting that WEE1 could be a novel therapeutic target in breast cancer. In conclusion, this strategy represents a novel and effective strategy for the identification of functionally important therapeutic targets in cancer.
doi:10.1371/journal.pone.0005120
PMCID: PMC2663812  PMID: 19357772
20.  Regulators of Trypanosoma brucei Cell Cycle Progression and Differentiation Identified Using a Kinome-Wide RNAi Screen 
PLoS Pathogens  2014;10(1):e1003886.
The African trypanosome, Trypanosoma brucei, maintains an integral link between cell cycle regulation and differentiation during its intricate life cycle. Whilst extensive changes in phosphorylation have been documented between the mammalian bloodstream form and the insect procyclic form, relatively little is known about the parasite's protein kinases (PKs) involved in the control of cellular proliferation and differentiation. To address this, a T. brucei kinome-wide RNAi cell line library was generated, allowing independent inducible knockdown of each of the parasite's 190 predicted protein kinases. Screening of this library using a cell viability assay identified ≥42 PKs that are required for normal bloodstream form proliferation in culture. A secondary screen identified 24 PKs whose RNAi-mediated depletion resulted in a variety of cell cycle defects including in G1/S, kinetoplast replication/segregation, mitosis and cytokinesis, 15 of which are novel cell cycle regulators. A further screen identified for the first time two PKs, named repressor of differentiation kinase (RDK1 and RDK2), depletion of which promoted bloodstream to procyclic form differentiation. RDK1 is a membrane-associated STE11-like PK, whilst RDK2 is a NEK PK that is essential for parasite proliferation. RDK1 acts in conjunction with the PTP1/PIP39 phosphatase cascade to block uncontrolled bloodstream to procyclic form differentiation, whilst RDK2 is a PK whose depletion efficiently induces differentiation in the absence of known triggers. Thus, the RNAi kinome library provides a valuable asset for functional analysis of cell signalling pathways in African trypanosomes as well as drug target identification and validation.
Author Summary
The African trypanosome, which is transmitted by the tsetse fly, causes the usually fatal disease Sleeping Sickness in humans and a wasting disease, called Nagana, in livestock in sub-Saharan Africa. There are no vaccines available against the diseases, and various problems are associated with current drug treatments (including toxicity to the patient and parasite drug resistance). Thus, it is important to identify essential parasite proteins that could be targeted by novel drugs. Protein kinases (PKs) are important cell signalling molecules, and are generally considered to have potential as drug targets. Here we report the construction of a library of trypanosome cell lines that allows us to specifically deplete each of the trypanosome's 190 PKs individually and analyse their function. Using this library, we show that ≥42 PKs are essential for proliferation of the mammalian-infective bloodstream form of the parasite (and thus have potential as drug targets), and demonstrate that 24 of these play important roles in coordinating cell division. We also shed light on how the parasite develops during its life cycle as it passes from the mammalian bloodstream form to the tsetse fly gut by identifying the first two PKs that regulate this life cycle developmental step.
doi:10.1371/journal.ppat.1003886
PMCID: PMC3894213  PMID: 24453978
21.  A high-throughput screening system for barley/powdery mildew interactions based on automated analysis of light micrographs 
BMC Plant Biology  2008;8:6.
Background
To find candidate genes that potentially influence the susceptibility or resistance of crop plants to powdery mildew fungi, an assay system based on transient-induced gene silencing (TIGS) as well as transient over-expression in single epidermal cells of barley has been developed. However, this system relies on quantitative microscopic analysis of the barley/powdery mildew interaction and will only become a high-throughput tool of phenomics upon automation of the most time-consuming steps.
Results
We have developed a high-throughput screening system based on a motorized microscope which evaluates the specimens fully automatically. A large-scale double-blind verification of the system showed an excellent agreement of manual and automated analysis and proved the system to work dependably. Furthermore, in a series of bombardment experiments an RNAi construct targeting the Mlo gene was included, which is expected to phenocopy resistance mediated by recessive loss-of-function alleles such as mlo5. In most cases, the automated analysis system recorded a shift towards resistance upon RNAi of Mlo, thus providing proof of concept for its usefulness in detecting gene-target effects.
Conclusion
Besides saving labor and enabling a screening of thousands of candidate genes, this system offers continuous operation of expensive laboratory equipment and provides a less subjective analysis as well as a complete and enduring documentation of the experimental raw data in terms of digital images. In general, it proves the concept of enabling available microscope hardware to handle challenging screening tasks fully automatically.
doi:10.1186/1471-2229-8-6
PMCID: PMC2262080  PMID: 18215267
22.  Generation of RNAi Libraries for High-Throughput Screens 
The completion of the genome sequencing for several organisms has created a great demand for genomic tools that can systematically analyze the growing wealth of data. In contrast to the classical reverse genetics approach of creating specific knockout cell lines or animals that is time-consuming and expensive, RNA-mediated interference (RNAi) has emerged as a fast, simple, and cost-effective technique for gene knockdown in large scale. Since its discovery as a gene silencing response to double-stranded RNA (dsRNA) with homology to endogenous genes in Caenorhabditis elegans (C elegans), RNAi technology has been adapted to various high-throughput screens (HTS) for genome-wide loss-of-function (LOF) analysis. Biochemical insights into the endogenous mechanism of RNAi have led to advances in RNAi methodology including RNAi molecule synthesis, delivery, and sequence design. In this article, we will briefly review these various RNAi library designs and discuss the benefits and drawbacks of each library strategy.
doi:10.1155/JBB/2006/45716
PMCID: PMC1559919  PMID: 17057364
23.  High-throughput RNAi screening identifies a role for TNK1 in growth and survival of pancreatic cancer cells 
Molecular cancer research : MCR  2011;9(6):724-732.
In order to identify novel targets in pancreatic cancer cells, we utilized high-throughput RNAi (HT-RNAi) to identify genes that, when silenced, would decrease viability of pancreatic cancer cells. The HT-RNAi screen involved reverse transfecting the pancreatic cancer cell line BxPC3 with a siRNA library targeting 572 kinases. From replicate screens, approximately thirty-two kinases were designated as hits, of which twenty-two kinase targets were selected for confirmation and validation. One kinase identified as a hit from this screen was tyrosine kinase non-receptor 1 (TNK1), a kinase previously identified as having tumor suppressor-like properties in embryonic stem cells. Silencing of TNK1 with siRNA showed reduced proliferation in a panel of pancreatic cancer cell lines. Furthermore, we demonstrated that silencing of TNK1 led to increased apoptosis through a caspase-dependent pathway and that targeting TNK1 with siRNA can synergize with gemcitabine treatment. Despite previous reports that TNK1 affects Ras and NFκB signaling, we did not find similar correlations with these pathways in pancreatic cancer cells. Our results suggest that TNK1 in pancreatic cancer cells does not possess the same tumor suppressor properties seen in embryonic cells, but appears to be involved in growth and survival. The application of functional genomics using HT-RNAi screens has allowed us to identify TNK1 as a growth-associated kinase in pancreatic cancer cells.
doi:10.1158/1541-7786.MCR-10-0436
PMCID: PMC3137903  PMID: 21536687
HT-RNAi; TNK1; gemcitabine; pancreatic cancer
24.  Insights to transcriptional networks by using high throughput RNAi strategies 
RNA interference (RNAi) is a powerful method to unravel the role of a given gene in eukaryotic cells. The development of high throughput assay platforms such as fluorescence plate readers and high throughput microscopy has allowed the design of genome wide RNAi screens to systemically discern members of regulatory networks around various cellular processes. Here we summarize the different strategies employed in RNAi screens to reveal regulators of transcriptional networks. We focus our discussion in experimental approaches designed to uncover regulatory interactions modulating transcription factor activity.
doi:10.1093/bfgp/elp046
PMCID: PMC3097102  PMID: 19952073
RNAi screen; transcription factor; reporter; localization; post-translational modification
25.  Emergence of Chemical Biology Approaches to the RNAi/miRNA Pathway 
Chemistry & biology  2010;17(6):584-589.
Summary
RNA interference (RNAi) is a well-conserved mechanism that uses small noncoding RNAs to silence gene expression posttranscriptionally. Gene regulation by RNAi is now recognized as one of the major regulatory pathways in eukaryotic cells. Although the main components of the RNAi/miRNA pathway have been identified, the molecular mechanisms regulating the activity of the RNAi/miRNA pathway have only begun to emerge within the last couple of years. Recently, high-throughput reporter assays to monitor the activity of the RNAi/miRNA pathway have been developed and used for proof-of-concept pilot screens. Both inhibitors and activators of the RNAi/miRNA pathway have been found. Although still in its infancy, a chemical biology approach using high-throughput chemical screens should open up a new avenue for dissecting the RNAi/miRNA pathway, as well as developing novel RNAi- or miRNA-based therapeutic interventions.
doi:10.1016/j.chembiol.2010.05.014
PMCID: PMC2901241  PMID: 20609408

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