Systems biology aims to describe the complex interplays between cellular building blocks which, in their concurrence, give rise to the emergent properties observed in cellular behaviors and responses. This approach tries to determine the molecular players and the architectural principles of their interactions within the genetic networks that control certain biological processes. Large-scale loss-of-function screens, applicable in various different model systems, have begun to systematically interrogate entire genomes to identify the genes that contribute to a certain cellular response. In particular, RNA interference (RNAi)-based high-throughput screens have been instrumental in determining the composition of regulatory systems and paired with integrative data analyses have begun to delineate the genetic networks that control cell biological and developmental processes. Through the creation of tools for both, in vitro and in vivo genome-wide RNAi screens, Drosophila melanogaster has emerged as one of the key model organisms in systems biology research and over the last years has massively contributed to and hence shaped this discipline. WIREs Syst Biol Med 2011 3 471-478 DOI: 10.1002/wsbm.127
We provide a detailed protocol for the mass culturing of primary cells dissociated from Drosophila embryos. The advantage of this protocol over others is that we have optimized it for a robust large-scale performance that is suitable for screening. More importantly, we further present conditions to treat these cells with double stranded (ds) RNAs for gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. This method provides the basis for functional genomic screens in differentiated cells, such as neurons and muscles, using RNAi or small molecules. The entire protocol takes approximately 14 d, whereas the preparation of primary cells from Drosophila embryos only requires 2-4 h.
Defects in miRNA biogenesis or activity are associated to development abnormalities and diseases. In Drosophila, miRNAs are predominantly loaded in Argonaute-1, which they guide for silencing of target RNAs. The miRNA pathway overlaps the RNAi pathway in this organism, as miRNAs may also associate with Argonaute-2, the mediator of RNAi. We set up a gene construct in which a single inducible promoter directs the expression of the GFP protein as well as two miRNAs perfectly matching the GFP sequences. We show that self-silencing of the resulting automiG gene requires Drosha, Pasha, Dicer-1, Dicer-2 and Argonaute-2 loaded with the anti-GFP miRNAs. In contrast, self-silencing of the automiG gene does not involve Argonaute-1. Thus, automiG reports in vivo for both miRNA biogenesis and Ago-2 mediated silencing, providing a powerful biosensor to identify situations where miRNA or siRNA pathways are impaired. As a proof of concept, we used automiG as a biosensor to screen a chemical library and identified 29 molecules that strongly inhibit miRNA silencing, out of which 5 also inhibit RNAi triggered by long double-stranded RNA. Finally, the automiG sensor is also self-silenced by the anti-GFP miRNAs in HeLa cells and might be easily used to identify factors involved in miRNA biogenesis and silencing guided by perfect target complementarity in mammals.
RNA interference (RNAi) in tissue culture cells has emerged as an excellent methodology for identifying gene functions systematically and in an unbiased manner. Here, we describe how RNAi high-throughput screening (HTS) in Drosophila cells are currently being performed and emphasize the strengths and weaknesses of the approach. Further, to demonstrate the versatility of the technology, we provide examples of the various applications of the method to problems in signal transduction and cell and developmental biology. Finally, we discuss emerging technological advances that will extend RNAi-based screening methods.
To identify Huntington's Disease therapeutics, we conducted high-content small molecule and RNAi suppressor screens using a Drosophila primary neural culture Huntingtin model. Drosophila primary neurons offer a sensitive readout for neurotoxicty, as their neurites develop dysmorphic features in the presence of mutant polyglutamine-expanded Huntingtin compared to nonpathogenic Huntingtin. By tracking the subcellular distribution of mRFP-tagged pathogenic Huntingtin and assaying neurite branch morphology via live-imaging, we identified suppressors that could reduce Huntingtin aggregation and/or prevent the formation of dystrophic neurites. The custom algorithms we used to quantify neurite morphologies in complex cultures provide a useful tool for future high-content screening approaches focused on neurodegenerative disease models. Compounds previously found to be effective aggregation inhibitors in mammalian systems were also effective in Drosophila primary cultures, suggesting translational capacity between these models. However, we did not observe a direct correlation between the ability of a compound or gene knockdown to suppress aggregate formation and its ability to rescue dysmorphic neurites. Only a subset of aggregation inhibitors could revert dysmorphic cellular profiles. We identified lkb1, an upstream kinase in the mTOR/Insulin pathway, and four novel drugs, Camptothecin, OH-Camptothecin, 18β-Glycyrrhetinic acid, and Carbenoxolone, that were strong suppressors of mutant Huntingtin-induced neurotoxicity. Huntingtin neurotoxicity suppressors identified through our screen also restored viability in an in vivo Drosophila Huntington's Disease model, making them attractive candidates for further therapeutic evaluation.
Biological networks are highly complex systems, consisting largely of enzymes that act as molecular switches to activate/inhibit downstream targets via post-translational modification. Computational techniques have been developed to perform signaling network inference using some high-throughput data sources, such as those generated from transcriptional and proteomic studies, but comparable methods have not been developed to use high-content morphological data, which are emerging principally from large-scale RNAi screens, to these ends. Here, we describe a systematic computational framework based on a classification model for identifying genetic interactions using high-dimensional single-cell morphological data from genetic screens, apply it to RhoGAP/GTPase regulation in Drosophila, and evaluate its efficacy. Augmented by knowledge of the basic structure of RhoGAP/GTPase signaling, namely, that GAPs act directly upstream of GTPases, we apply our framework for identifying genetic interactions to predict signaling relationships between these proteins. We find that our method makes mediocre predictions using only RhoGAP single-knockdown morphological data, yet achieves vastly improved accuracy by including original data from a double-knockdown RhoGAP genetic screen, which likely reflects the redundant network structure of RhoGAP/GTPase signaling. We consider other possible methods for inference and show that our primary model outperforms the alternatives. This work demonstrates the fundamental fact that high-throughput morphological data can be used in a systematic, successful fashion to identify genetic interactions and, using additional elementary knowledge of network structure, to infer signaling relations.
Gene silencing through sequence-specific targeting of mRNAs by RNAi has enabled genome-wide functional screens in cultured cells and in vivo in model organisms. These screens have resulted in the identification of new cellular pathways and potential drug targets. Considerable progress has been made to improve the quality of RNAi screen data through the development of new experimental and bioinformatics approaches. The recent availability of genome-editing strategies, such as the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system, when combined with RNAi, could lead to further improvements in screen data quality and follow-up experiments, thus promoting our understanding of gene function and gene regulatory networks.
This protocol describes the various steps and considerations involved in planning and carrying out RNA interference (RNAi) genome-wide screens in cultured Drosophila cells. We focus largely on the procedures that have been modified as a result of our experience over the past 3 years and of our better understanding of the underlying technology. Specifically, our protocol offers a set of suggestions and considerations for screen optimization and a step-by-step description of the procedures successfully used at the Drosophila RNAi Screening Center for screen implementation, data collection and analysis to identify potential hits. In addition, this protocol briefly covers postscreen analysis approaches that are often needed to finalize the hit list. Depending on the scope of the screen and subsequent analysis and validation involved, the full protocol can take anywhere from 3 months to 2 years to complete.
The pairing of homologous chromosomes is a fundamental feature of the meiotic cell. In addition, a number of species exhibit homolog pairing in nonmeiotic, somatic cells as well, with evidence for its impact on both gene regulation and double-strand break (DSB) repair. An extreme example of somatic pairing can be observed in Drosophila melanogaster, where homologous chromosomes remain aligned throughout most of development. However, our understanding of the mechanism of somatic homolog pairing remains unclear, as only a few genes have been implicated in this process. In this study, we introduce a novel high-throughput fluorescent in situ hybridization (FISH) technology that enabled us to conduct a genome-wide RNAi screen for factors involved in the robust somatic pairing observed in Drosophila. We identified both candidate "pairing promoting genes" and candidate "anti-pairing genes," providing evidence that pairing is a dynamic process that can be both enhanced and antagonized. Many of the genes found to be important for promoting pairing are highly enriched for functions associated with mitotic cell division, suggesting a genetic framework for a long-standing link between chromosome dynamics during mitosis and nuclear organization during interphase. In contrast, several of the candidate anti-pairing genes have known interphase functions associated with S-phase progression, DNA replication, and chromatin compaction, including several components of the condensin II complex. In combination with a variety of secondary assays, these results provide insights into the mechanism and dynamics of somatic pairing.