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A Conditional Knockout Toolkit for Caenorhabditis elegans Based on the Cre/loxP Recombination
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A Conditional Knockout Toolkit for Caenorhabditis elegans Based on the Cre/loxP Recombination
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A Conditional Knockout Toolkit for Caenorhabditis elegans Based on the Cre/loxP Recombination
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Journal Article
A Conditional Knockout Toolkit for Caenorhabditis elegans Based on the Cre/loxP Recombination
2014
Overview
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.
Publisher
Public Library of Science,Public Library of Science (PLoS)
Subject
/ Animals, Genetically Modified
/ Caenorhabditis elegans - genetics
/ Cloning
/ CRISPR
/ Gene Knockout Techniques - methods
/ Genomes
/ Green Fluorescent Proteins - biosynthesis
/ Green Fluorescent Proteins - genetics
/ Heat
/ Medicine
/ Methods
/ Muscles
/ Mutants
/ Mutation
/ Plasmids
/ Proteins
/ Worms
