A conditional knockout toolkit for Caenorhabditis elegans based on the Cre/loxP recombination.
A conditional knockout toolkit for Caenorhabditis elegans based on the Cre/loxP recombination.
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DOI：
10.1371/journal.pone.0114680
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Mitani S
中科院分区：
文献类型：
--
作者：
Kage-Nakadai E;Imae R;Suehiro Y;Yoshina S;Hori S;Mitani S
基于位点特异性重组(SSR)技术的条件性基因敲除(CKO)是在许多动物模型中以空间和时间特异性方式估计基因功能的一种有效方法。在秀丽线虫(C.elegans)中，空间和时间特异的基因功能在很大程度上是通过镶嵌分析、救援实验和RNAi方法确定的。为了在线虫中建立一个系统和稳定的CKO系统，我们构建了由各种组织特异性启动子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的验证表明，Cre依赖的loxP切除成功。我们为每个评估载体建立了多拷贝Cre转基因菌株集合。为了评估我们基于Cre/loxP的CKO系统，我们使用紫外三甲基补骨脂素(UV/TMP)方法获得了含有花状SID-1单拷贝整合(SCI)的SID-1缺失突变体。SID-1突变体经SID-1 SCI解救后，与Pdpy-7：：Cre品系杂交，检测皮下CKO。SID-1 CKO动物对BLI-3RNAi具有抵抗力，可引起皮下蓝表型，但对UNC-22RNAi敏感，可导致体壁肌肉抽动。我们的系统基于转基因Cre收集、预先存在的缺失突变体和UV/TMP SCI方法的组合，为线虫CKO提供了一种系统的方法。
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.
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影响因子：
64.8
作者：
Skarnes WC;Rosen B;West AP;Koutsourakis M;Bushell W;Iyer V;Mujica AO;Thomas M;Harrow J;Cox T;Jackson D;Severin J;Biggs P;Fu J;Nefedov M;de Jong PJ;Stewart AF;Bradley A
通讯作者：
Bradley A
影响因子：
3.5
作者：
Kage-Nakadai E;Kobuna H;Funatsu O;Otori M;Gengyo-Ando K;Yoshina S;Hori S;Mitani S
通讯作者：
Mitani S
DOI：
10.1006/bbrc.2000.2260
发表时间：
2000-03-05
影响因子：
3.1
作者：
Gengyo-Ando, K;Mitani, S
通讯作者：
Mitani, S
DOI：
10.1016/j.ymeth.2014.05.007
发表时间：
2014-08-01
期刊：
Methods (San Diego, Calif.)
影响因子：
--
作者：
Hubbard EJ
通讯作者：
Hubbard EJ
影响因子：
12.3
作者：
通讯作者：
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