Advances in RNA interference (RNAi)-based methodologies and tools in recent years have provided unprecedented opportunities to systematically interrogate the function of all genes relevant to a particular process. In Drosophila, arguably the best-understood multicellular organism and a proven model system for studying human diseases, conditional and tissue-specific expression of RNAi constructs using the Gal4/UAS system has emerged as the method of choice to determine the functions of all genes. To facilitate in vivo RNAi studies, a number of large-scale resources have been developed that rely on long double- stranded RNAs (dsRNAs) as the active RNAi reagents. These long dsRNAs are problematic as they do not work in the female germline and many have poor knockdown efficiencies. This application is to request continued funding for the Transgenic RNAi Project (TRiP;://www.flyrnai.org) at Harvard Medical School. The goals of the TRiP, funded by NIH/NIGMS R01-GM084947 three years ago, were to improve methods of transgenic RNAi and to generate RNAi lines for the community. During this funding period we have optimized vectors for transgenic RNAi and shown that small hairpin RNAs (shRNAs) are much better reagents than long dsRNAs for in vivo RNAi as they are more effective in somatic tissues and work in both the male and female germlines. Based on the growing need by the community for shRNA fly lines, we have already generated more than 3000 fly lines that are distributed by the Bloomington Drosophila Stock Center. As we move forward, we propose to continue expanding the collection of TRiP shRNA lines (Aim 1), which we will accomplish by generating lines ourselves as well as coordinating the production of lines by a number of outside groups (the Japanese stock center, the Tsinghua University Chinese stock center, and individual laboratories) that are interested in helping build the resource. In addition as information on the efficacy of RNAi lines is currently not being tracked, we will establish the """"""""Digital Red Book of RNAi"""""""" (Aim 2) by collecting the increasing amount of information available on existing lines and performing a number of validation experiments to evaluate the performance of existing and new lines. Well-organized public availability of this information will not only help individual researchers select the best lines for their experiments but also help us identify which additional shRNA lines need to be produced. Together, these Aims will provide the Drosophila community with an eagerly awaited, comprehensive in vivo RNAi resource that will foster studies at the single gene level and facilitate larger-scale screens. This is criticall important as both approaches make it possible for researchers to address fundamental biological questions that will ultimately lead to much-needed understandings of gene function and vital translational projects.

Public Health Relevance

This project is seeking continued support for the Transgenic RNAi Project (TRiP) at Harvard Medical School. We will continue to provide the community with state-of-the-art reagents for in vivo transgenic RNAi in Drosophila and generate a public database of information regarding the quality of the RNAi reagents.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM084947-05
Application #
8293913
Study Section
Genomics, Computational Biology and Technology Study Section (GCAT)
Program Officer
Bender, Michael T
Project Start
2008-09-01
Project End
2016-07-31
Budget Start
2012-08-01
Budget End
2013-07-31
Support Year
5
Fiscal Year
2012
Total Cost
$810,331
Indirect Cost
$330,963
Name
Harvard University
Department
Genetics
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
Wang, Liqun; Hagemann, Tracy L; Messing, Albee et al. (2016) An In Vivo Pharmacological Screen Identifies Cholinergic Signaling as a Therapeutic Target in Glial-Based Nervous System Disease. J Neurosci 36:1445-55
Ma, Meifang; Zhao, Hang; Zhao, Hanfei et al. (2016) Wildtype adult stem cells, unlike tumor cells, are resistant to cellular damages in Drosophila. Dev Biol 411:207-16
Günther, Maximilian N; Nettesheim, Guilherme; Shubeita, George T (2016) Quantifying and predicting Drosophila larvae crawling phenotypes. Sci Rep 6:27972
Das, Arunika; Shah, Shital J; Fan, Bensen et al. (2016) Spindle Assembly and Chromosome Segregation Requires Central Spindle Proteins in Drosophila Oocytes. Genetics 202:61-75
Vinayagam, Arunachalam; Kulkarni, Meghana M; Sopko, Richelle et al. (2016) An Integrative Analysis of the InR/PI3K/Akt Network Identifies the Dynamic Response to Insulin Signaling. Cell Rep 16:3062-74
Hartley, Paul S; Motamedchaboki, Khatereh; Bodmer, Rolf et al. (2016) SPARC-Dependent Cardiomyopathy in Drosophila. Circ Cardiovasc Genet 9:119-29
Musashe, Derek T; Purice, Maria D; Speese, Sean D et al. (2016) Insulin-like Signaling Promotes Glial Phagocytic Clearance of Degenerating Axons through Regulation of Draper. Cell Rep 16:1838-50
Tan, William; Schauder, Curtis; Naryshkina, Tatyana et al. (2016) Zfrp8 forms a complex with fragile-X mental retardation protein and regulates its localization and function. Dev Biol 410:202-12
Lai, Yen-Wei; Chu, Sao-Yu; Wei, Jia-Yi et al. (2016) Drosophila microRNA-34 Impairs Axon Pruning of Mushroom Body γ Neurons by Downregulating the Expression of Ecdysone Receptor. Sci Rep 6:39141
Ando, Kanae; Maruko-Otake, Akiko; Ohtake, Yosuke et al. (2016) Stabilization of Microtubule-Unbound Tau via Tau Phosphorylation at Ser262/356 by Par-1/MARK Contributes to Augmentation of AD-Related Phosphorylation and Aβ42-Induced Tau Toxicity. PLoS Genet 12:e1005917

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