title: A Fusion PCR Method for Expressing Genetic Tools in C. elegans.
abstract: C. elegans offer a unique opportunity for understanding computation in neural networks. This is largely due to their relatively compact neural network for which a wiring diagram is available. Recent advances in genetic tools for interrogating neural activity (e.g., optogenetics) make C. elegans particularly compelling as they can be expressed in many different combinations in target individual neurons. Thus, the prospect to decipher principles underlying functionality in neural networks largely depends on the ease by which transgenic animals can be generated. Traditionally, to generate transgenic animals one would inject a plasmid containing the gene of interest under the regulation of the cell- or lineage-specific promoter. This often requires laborious cloning steps of both the gene and the promoter. The Hobert lab has developed a simpler protocol in which linear PCR fragments can be injected to generate transgenic animals. Relying on this PCR fusion-based method, here we provide a detailed protocol that we have optimized for expressing various genetically encoded calcium indicators and optogenetic tools in individual or sets of neurons. We use these simple procedures to generate multiple constructs within a very short time frame (typically 1-2 days).
authors.affiliation: Genetics Department, Silberman Life Sciences Institute, The Hebrew University of Jerusalem, Jerusalem, 91904, Israel.
authors.forename: Yifat
authors.initials: Y
authors.lastname: Eliezer
authors.affiliation: Genetics Department, Silberman Life Sciences Institute, The Hebrew University of Jerusalem, Jerusalem, 91904, Israel. alonzas@mail.huji.ac.il.
authors.forename: Alon
authors.initials: A
authors.lastname: Zaslaver
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics*; D003001:Cloning, Molecular; D015870:Gene Expression; D017930:Genes, Reporter; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics*
chemical_list: D011993:Recombinant Fusion Proteins
keywords: C. elegans; Fluorescent reporters; Fusion PCR; Genetic ablations; Transgenics