title: A conditional knockout toolkit for Caenorhabditis elegans based on the Cre/loxP recombination.
abstract: Conditional knockout (cKO) based on site-specific recombination (SSR) technology is a powerful approach for estimating gene functions in a spatially and temporally specific manner in many model animals. In Caenorhabditis elegans (C. elegans), spatial- and temporal-specific gene functions have been largely determined by mosaic analyses, rescue experiments and feeding RNAi methods. To develop a systematic and stable cKO system in C. elegans, we generated Cre recombinase expression vectors that are driven by various tissue-specific or heat-shock promoters. Validation using Cre-mediated fluorescence protein inactivation or activation systems demonstrated successful Cre-dependent loxP excision. We established a collection of multi-copy Cre transgenic strains for each evaluated vector. To evaluate our Cre/loxP-based cKO system, we generated sid-1 deletion mutants harboring floxed sid-1 single-copy integration (SCI) using ultraviolet trimethylpsoralen (UV/TMP) methods. sid-1 mutants that were rescued by the floxed sid-1 SCI were then crossed with the Pdpy-7::Cre strain for cKO in the hypodermis. The sid-1 cKO animals were resistant to bli-3 RNAi, which causes the Bli-phenotyple in the hypodermis, but they were sensitive to unc-22 RNAi, which leads to twitching of the body wall muscle. Our system, which is based on the combination of a transgenic Cre collection, pre-existing deletion mutants, and UV/TMP SCI methods, provided a systematic approach for cKO in C. elegans.
authors.affiliation: Department of Physiology, Tokyo Women's Medical University School of Medicine, Tokyo, Japan; The OCU Advanced Research Institute for Natural Science and Technology, Osaka City University, Osaka, Japan.
authors.forename: Eriko
authors.initials: E
authors.lastname: Kage-Nakadai
authors.affiliation: Department of Physiology, Tokyo Women's Medical University School of Medicine, Tokyo, Japan.
authors.forename: Rieko
authors.initials: R
authors.lastname: Imae
authors.affiliation: Department of Physiology, Tokyo Women's Medical University School of Medicine, Tokyo, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Suehiro
authors.affiliation: Department of Physiology, Tokyo Women's Medical University School of Medicine, Tokyo, Japan.
authors.forename: Sawako
authors.initials: S
authors.lastname: Yoshina
authors.affiliation: Department of Physiology, Tokyo Women's Medical University School of Medicine, Tokyo, Japan.
authors.forename: Sayaka
authors.initials: S
authors.lastname: Hori
authors.affiliation: Department of Physiology, Tokyo Women's Medical University School of Medicine, Tokyo, Japan.
authors.forename: Shohei
authors.initials: S
authors.lastname: Mitani
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D017173:Caenorhabditis elegans / Q000235:genetics*; D055786:Gene Knockout Techniques / Q000379:methods*; D005818:Genetic Engineering; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D019426:Integrases / Q000235:genetics; D009928:Organ Specificity; D034622:RNA Interference
chemical_list: D049452:Green Fluorescent Proteins; C045073:Cre recombinase; D019426:Integrases