Abstract
Thetransforming growth factor-β (TGF-β) family of cytokines figures prominently in regulation of embryonic development and adult tissue homeostasis from Drosophila to mammals. Genetic defects affecting TGF-β signaling underlie developmental disorders and diseases such as cancer in human. Therefore, delineating the molecular mechanism by which TGF-β regulates cell biology is critical for understanding normal biology and disease mechanisms. Forward genetic screens in model organisms and biochemical approaches in mammalian tissue culture were instrumental in initial characterization of the TGF-β signal transduction pathway. With complete sequence information of the genomes and the advent of RNA interference (RNAi) technology, genome-wide RNAi screening emerged as a powerful functional genomics approach to systematically delineate molecular components of signal transduction pathways. Here, we describe a protocol for image-based whole-genome RNAi screening aimed at identifying molecules required for TGF-β signaling into the nucleus. Using this protocol we examined >90 % of annotated Drosophila open reading frames (ORF) individually and successfully uncovered several novel factors serving critical roles in the TGF-β pathway. Thus cell-based high-throughput functional genomics can uncover new mechanistic insights on signaling pathways beyond what the classical genetics had revealed.
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Acknowledgements
We would like to thank the DRSC at the Harvard Medical School for providing critical reagents, instrumentations, technical advice, and other resources that greatly facilitated our screening. The authors work was funded by the NIH (RO1 CA108509).
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Chen, X., Xu, L. (2016). Genome-Wide RNAi Screening to Dissect the TGF-β Signal Transduction Pathway. In: Feng, XH., Xu, P., Lin, X. (eds) TGF-β Signaling. Methods in Molecular Biology, vol 1344. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2966-5_24
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DOI: https://doi.org/10.1007/978-1-4939-2966-5_24
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2965-8
Online ISBN: 978-1-4939-2966-5
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