A Conditional Knockout Toolkit for <i>Caenorhabditis elegans</i> Based on the <i>Cre/loxP</i> Recombination

<div><p>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 <i>Caenorhabditis elegans (C. elegans)</i>, 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 <i>C. elegans</i>, 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 <i>loxP</i> excision. We established a collection of multi-copy Cre transgenic strains for each evaluated vector. To evaluate our Cre/<i>loxP</i>-based cKO system, we generated <i>sid-1</i> deletion mutants harboring floxed <i>sid-1</i> single-copy integration (SCI) using ultraviolet trimethylpsoralen (UV/TMP) methods. <i>sid-1</i> mutants that were rescued by the floxed <i>sid-1</i> SCI were then crossed with the <i>Pdpy-7::Cre</i> strain for cKO in the hypodermis. The <i>sid-1</i> cKO animals were resistant to <i>bli-3</i> RNAi, which causes the Bli-phenotyple in the hypodermis, but they were sensitive to <i>unc-22</i> 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 <i>C. elegans</i>.</p>