Kage-Nakadai Eriko, Imae Rieko, Suehiro Yuji, Yoshina Sawako, Hori Sayaka, Mitani Shohei
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.
Department of Physiology, Tokyo Women's Medical University School of Medicine, Tokyo, Japan.
PLoS One. 2014 Dec 4;9(12):e114680. doi: 10.1371/journal.pone.0114680. eCollection 2014.
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.
基于位点特异性重组（SSR）技术的条件性基因敲除（cKO）是一种在许多模式动物中以空间和时间特异性方式评估基因功能的强大方法。在秀丽隐杆线虫（C. elegans）中，空间和时间特异性基因功能很大程度上已通过镶嵌分析、拯救实验和喂食RNA干扰方法得以确定。为了在秀丽隐杆线虫中开发一个系统且稳定的cKO系统，我们构建了由各种组织特异性或热休克启动子驱动的Cre重组酶表达载体。使用Cre介导的荧光蛋白失活或激活系统进行系统进行验证，结果表明成功实现了依赖Cre的loxP位点切除。对于每个评估的载体，我们建立了多拷贝Cre转基因菌株库。为了评估我们基于Cre/loxP的cKO系统，我们使用紫外线三甲基补骨脂素（UV/TMP）方法构建了携带floxed sid-1单拷贝整合（SCI）的sid-1缺失突变体。然后将由floxed sid-1 SCI拯救的sid-1突变体与Pdpy-7::Cre菌株杂交，以在皮下组织中进行cKO。sid-1 cKO动物对bli-3 RNA干扰具有抗性，bli-3 RNA干扰会在皮下组织中导致Bli表型，但它们对unc-22 RNA干扰敏感，unc-22 RNA干扰会导致体壁肌肉抽搐。我们的系统基于转基因Cre菌株库、现有的缺失突变体和UV/TMP SCI方法的组合，为秀丽隐杆线虫的cKO提供了一种系统方法。
