title: pWormgatePro enables promoter-driven knockdown by hairpin RNA interference of muscle and neuronal gene products in Caenorhabditis elegans.
abstract: Recent advances in genome research and RNA interference (RNAi) technology have accelerated the adoption of genome-wide experimental approaches for determining gene function in the model organism Caenorhabditis elegans. Despite recent successes, the application of RNAi is limited when gene knockdown causes complex phenotypes or embryonic lethality. Recently, the high-throughput pWormgate cloning system has been introduced as a tool to efficiently generate heat-shock-inducible hairpin RNA constructs using the Gateway recombination technology. We have modified pWormgate into a versatile hairpin cloning plasmid, pWormgatePro, which facilitates temporally and spatially inducible hairpin RNAi using constitutively active, tissue-specific promoters. To demonstrate its utility we knocked down unc-22 in body wall muscles as well as the axon guidance gene unc-5 in the nervous system indicating that promoter-driven hairpins can overcome the neuronal resistance to RNAi. Using pWormgatePro we also show that RNAi in the nervous system of C. elegans is non-autonomous and that spreading of the RNAi signal from neurons to muscle is substantially reduced but not abolished in spreading-defective sid-1 mutant animals. Our findings illustrate the effectiveness of pWormgatePro for gene silencing in muscle cells and neurons and bring forward the possibility of applying tissue-specific RNAi on a genome-wide scale.
authors.affiliation: MRC Functional Genetics Unit, Department of Human Anatomy and Genetics, University of Oxford, South Parks Road, OX1 3QX, Oxford, UK.
authors.forename: Michael
authors.initials: M
authors.lastname: Briese
authors.forename: Behrooz
authors.initials: B
authors.lastname: Esmaeili
authors.forename: Nicholas M
authors.initials: NM
authors.lastname: Johnson
authors.forename: David B
authors.initials: DB
authors.lastname: Sattelle
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics*; D029742:Caenorhabditis elegans Proteins / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D020868:Gene Silencing; D009132:Muscles / Q000502:physiology*; D009474:Neurons / Q000502:physiology*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D034622:RNA Interference
chemical_list: D029742:Caenorhabditis elegans Proteins