Genes required for
            <i>Drosophila</i>
            nervous system development identified by RNA interference

Ivanov, A.; Rovescalli, A; Pozzi, Paola; Yoo, Siuk; Mozer, Brian A.; Li, Hsi-Ping; Yu, Shuhua; Higashida, Haruhiro; Guo, Vicky; Spencer, Michael; Nirenberg, Marshall W.

doi:10.1073/pnas.0407188101

Cited by 55 publications

(48 citation statements)

References 50 publications

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“…Genes involved in the embryonic development of the Drosophila nervous system were identified by RNAi, which extends the results of our previous study (4). Double-stranded RNA was injected into preblastoderm embryos, and the embryos were incubated to allow development to proceed to about stage 15-16.…”

Section: Resultsmentioning

confidence: 49%

“…We also observed decreases in the size of embryos resulting from segment polarity defects (data not shown). This phenotype resembles the shotgun RNAi-dependent phenotype shown in our previous study (4). arr is an essential factor in the Wg/Wnt signaling pathway functioning in embryonic segmentation, limb development, and CNS organization (54).…”

Section: Genes With Previously Uncharacterized Nervous System Phenotymentioning

confidence: 62%

“…RNAi has been used successfully to screen the genomes of various species to identify genes involved in biological processes (3). Previously, we used an RNAi-based in vivo screen to identify genes required for the development of the embryonic nervous system in Drosophila (4). From a library of double-stranded (ds) RNAs corresponding to approximately one-fourth of the fruit fly genome, we identified 43 genes including 18 novel genes whose roles in embryonic nervous system development had not been described.…”

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confidence: 99%

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RNA interference screen to identify genes required for Drosophila embryonic nervous system development

Koizumi

Higashida

Yoo

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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RNA interference (RNAi) has been shown to be a powerful method to study the function of genes in vivo by silencing endogenous mRNA with double-stranded (ds) RNA. Previously, we performed in vivo RNAi screening and identified 43 Drosophila genes, including 18 novel genes required for the development of the embryonic nervous system. In the present study, 22 additional genes affecting embryonic nervous system development were found. Novel RNAiinduced phenotypes affecting nervous system development were found for 16 of the 22 genes. Seven of the genes have unknown functions. Other genes found encode transcription factors, a chromatin-remodeling protein, membrane receptors, signaling molecules, and proteins involved in cell adhesion, RNA binding, and ion transport. Human orthologs were identified for proteins encoded by 16 of the genes. The total number of dsRNAs that we have tested for an RNAi-induced phenotype affecting the embryonic nervous system, including our previous study, is 7,312, which corresponds to Ϸ50% of the genes in the Drosophila genome.high-throughput screen ͉ neural development ͉ neural mutants

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Section: Resultsmentioning

confidence: 49%

Section: Genes With Previously Uncharacterized Nervous System Phenotymentioning

confidence: 62%

mentioning

confidence: 99%

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RNA interference screen to identify genes required for Drosophila embryonic nervous system development

Koizumi

Higashida

Yoo

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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“…RNAi screens in intact Drosophila embryos also have been used to identify genes that play roles in neural development (14)(15) and cardiac development (16).…”

Section: G Enome-wide Rnai Screens Using Clonal Lines Of Culturedmentioning

confidence: 99%

Silencing of genes in cultured Drosophila neurons by RNA interference

Sharma

Nirenberg²

2007

Proc. Natl. Acad. Sci. U.S.A.

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Cultures of neuroblasts that generate abundant neurons were established from Drosophila embryos to study silencing of genes by RNA interference (RNAi). Cultured cells expressed ELAV, a marker of neurons, Futsch, a marker of neurites, and Synapsin, Synaptobrevin, and Synaptogamin, proteins involved in neurotransmitter secretion. Conditions were found for efficient transfection of cells with siRNAs for ELAV or the insulin-like receptor, which resulted in marked decreases in neurons that express ELAV and Futsch. Cells also were successfully transfected with long-chain Sox-Neuro dsRNA resulting in a 55% reduction of neurons expressing Futsch. The results suggest that this cultured neural cell system can be used to study RNAi-dependent silencing of genes involved in many kinds of neural functions.ELAV ͉ insulin receptor ͉ neurites G enome-wide RNAi screens using clonal lines of cultured Drosophila cells have been used to identify genes involved in cell growth and viability (1), signaling pathways (2-12), and factors required for bacterial infection (13). RNAi screens in intact Drosophila embryos also have been used to identify genes that play roles in neural development (14-15) and cardiac development (16).We have been using RNAi to find genes that are involved in the assembly of the Drosophila embryonic nervous system. It is much easier and faster to screen tissue culture cells by RNAi by transfecting cells with double-stranded oligoribonucleotides (siRNA) or long-chain dsRNA than to laboriously inject embryos with RNA preparations. However, well differentiated, well characterized clonal lines of Drosophila neural cells are needed.As an alternative, we have dissociated neuroblasts (NBs) from Drosophila embryos and cultured them under conditions that result in an enriched population of NBs that generate abundant neurons in vitro and that can be transfected with siRNA or long-chain dsRNA with relatively high efficiency. Cultures enriched in NBs and neurons provide a means of studying molecular and cellular events during nervous system development (17). NB cultures have been used successfully to study gene expression during NB lineage development (18) and to explore mechanisms controlling asymmetric cell division and protein localization (19). Cultured Drosophila motor neurons also have been shown to form synapses with striated muscle cells (20). Specific cells have been removed from embryos with capillaries and have been grown in culture to study their development state (21), ion channels and responses to neurotransmitters, neuronal morphology, and action potential-dependent synaptic activity (22). K. Sepp also has screened cultured fluorescent Drosophila embryo neurons by RNAi (http://flyrnai.org).Our main objective in this study has been to devise conditions that enable mass cultures of Drosophila neural cells to be used for RNAi-induced silencing of genes. Results and DiscussionDrosophila embryos Ϸ5 h after fertilization (stage 10) were used to establish highly enriched NB cultures by a slight modification of publi...

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“…An alternative approach to that taken in generating the random siRNA library (14), would be to restriction digest cDNA to generate a library of long dsRNAs. Long dsRNA libraries have been used to analyze gene function in Drosophila (23,24) but potential off-target effects and induction of the stress response are issues for mammalian gene silencing. Thus, the focus of this study was to analyze the efficiency, selectivity and toxicity of long dsRNAs in mammalian cells and to determine if long dsRNAs lead to the desired biological response in these cells, as a first step to generate long dsRNA libraries.…”

Section: Introductionmentioning

confidence: 99%

Development of RNAi Libraries for Target Validation and Therapeutics

Giordano¹

2006

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Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information Operations and Reports (0704-0188), 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to any penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. REPORT DATE SPONSOR/MONITOR'S REPORT NUMBER(S) DISTRIBUTION / AVAILABILITY STATEMENTApproved for Public Release; Distribution Unlimited SUPPLEMENTARY NOTESOriginal contains colored plates: ALL DTIC reproductions will be in black and white. ABSTRACTA number of groups have developed libraries of siRNAs to identify genes through functional genomics. While these studies have validated the approach of making functional RNAi libraries to understand fundamental cellular mechanisms, they require information and knowledge of existing sequences since the RNAi sequences are generated synthetically. An alternative strategy would be to create an RNAi library from cDNA. Unfortunately, the complexity of such a library of siRNAs would make screening difficult. To reduce the complexity, longer dsRNAs could be used; however, concerns of induction of the interferon response and off-target effects of long dsRNAs have prevented their use. As a first step in creating such libraries, long dsRNA was expressed in mammalian cells. The 250-nt dsRNAs were capable of efficiently silencing a luciferase reporter gene that was stably transfected in MDA-MB-231 cells without inducing the interferon response or off-target effects any more than reported for siRNAs. In addition, a long dsRNA expressed in the same cell line was capable of silencing endogenous c-met expression and inhibited cell migration, whereas the dsRNA against luciferase had no effect on c-met or cell migration. The studies suggest that large dsRNA libraries are feasible and that functional selection of genes will be possible. SUBJECT TERMS IntroductionThe focus of this Concept Award was to develop methods of generating large dsRNA libraries to silence gene expression in cells. If large dsRNA can be used to effectively silence gene expression, a library could be generated from a cancer cell possessing a phenotype of interest, in this case metastasis, and the library transfected back into the cancer cells. Essential genes that are silenced would be toxic and the cells would be lost; genes silenced that are not involved in metastasis would not prevent cell migration; only those in...

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Genes required for Drosophila nervous system development identified by RNA interference

Cited by 55 publications

References 50 publications

RNA interference screen to identify genes required for Drosophila embryonic nervous system development

RNA interference screen to identify genes required for Drosophila embryonic nervous system development

Silencing of genes in cultured Drosophila neurons by RNA interference

Development of RNAi Libraries for Target Validation and Therapeutics

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