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.
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.
|Branch, Audrey; Zhang, Yiwen; Shen, Ping (2017) Genetic and Neurobiological Analyses of the Noradrenergic-like System in Vulnerability to Sugar Overconsumption Using a Drosophila Model. Sci Rep 7:17642|
|Onodera, Koun; Baba, Shumpei; Murakami, Akira et al. (2017) Small conductance Ca2+-activated K+ channels induce the firing pause periods during the activation of Drosophila nociceptive neurons. Elife 6:|
|Kim, Kevin; Hung, Ruei-Jiun; Perrimon, Norbert (2017) miR-263a Regulates ENaC to Maintain Osmotic and Intestinal Stem Cell Homeostasis in Drosophila. Dev Cell 40:23-36|
|Feng, Lijuan; Shi, Zhen; Chen, Xin (2017) Enhancer of polycomb coordinates multiple signaling pathways to promote both cyst and germline stem cell differentiation in the Drosophila adult testis. PLoS Genet 13:e1006571|
|Beaven, Robin; Bastos, Ricardo Nunes; Spanos, Christos et al. (2017) 14-3-3 regulation of Ncd reveals a new mechanism for targeting proteins to the spindle in oocytes. J Cell Biol 216:3029-3039|
|Koehler, Christopher L; Perkins, Guy A; Ellisman, Mark H et al. (2017) Pink1 and Parkin regulate Drosophila intestinal stem cell proliferation during stress and aging. J Cell Biol 216:2315-2327|
|Heer, Natalie C; Miller, Pearson W; Chanet, Soline et al. (2017) Actomyosin-based tissue folding requires a multicellular myosin gradient. Development 144:1876-1886|
|Artoni, Filippo; Kreipke, Rebecca E; Palmeira, Ondina et al. (2017) Loss of foxo rescues stem cell aging in Drosophila germ line. Elife 6:|
|Bhattacharya, Tamanash; Newton, Irene L G; Hardy, Richard W (2017) Wolbachia elevates host methyltransferase expression to block an RNA virus early during infection. PLoS Pathog 13:e1006427|
|Lim, Angeline; Rechtsteiner, Andreas; Saxton, William M (2017) Two kinesins drive anterograde neuropeptide transport. Mol Biol Cell 28:3542-3553|
Showing the most recent 10 out of 276 publications