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.
Highlights
The spatial and temporal control of gene inactivation is an essential approach for assessing gene functions in multicellular organisms
Products of the 200 bp were observed selectively in Pges-1::Cre and Pscm::Cre transgenic animals (Fig. 1C, D), and the results indicated that tissue-specific Cre/ loxP-mediated gene inactivation occurred
We provide an approach for C. elegans Conditional knockout (cKO) based on the combination of a Cre-transgenic collection, pre-existing deletion mutants, and ultraviolet trimethylpsoralen (UV/TMP) single-copy integration (SCI) methods
Summary
The spatial and temporal control of gene inactivation is an essential approach for assessing gene functions in multicellular organisms. A cKO resource that covers 9,000 targeted alleles has been produced in mouse embryonic stem cells [1], and it has promoted systematic studies of mouse genes. In Caenorhabditis elegans (C. elegans), various genetic approaches such as mosaic analyses using extrachromosomal arrays [2], transgenic rescue driven by cell-type specific or heat-shock promoters, and feeding RNAi methods [3], have been largely used to investigate spatial- and temporal-specific gene functions. Conditional genome editing methods using the TALENs or CRISPR-cas systems have been developed [4,5,6]. These somatic genome editing methods are, heterogeneous and not heritable
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