A conditional knockout toolkit for Caenorhabditis elegans based on the Cre/loxP recombination

PLoS One. 2014 Dec 4;9(12):e114680. doi: 10.1371/journal.pone.0114680. eCollection 2014.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Genetically Modified
  • Caenorhabditis elegans / genetics*
  • Gene Knockout Techniques / methods*
  • Genetic Engineering
  • Genetic Vectors
  • Green Fluorescent Proteins / biosynthesis
  • Green Fluorescent Proteins / genetics
  • Integrases / genetics
  • Organ Specificity
  • RNA Interference

Substances

  • Green Fluorescent Proteins
  • Cre recombinase
  • Integrases

Grant support

This study was supported by Grants-in-Aid for Scientific Research from Japan Society for the promotion of Science (JSPS) (to E.K.-N. and S.M.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.