The auxin-inducible degron 2 technology provides sharp degradation control in yeast, mammalian cells, and mice.
The auxin-inducible degron 2 technology provides sharp degradation control in yeast, mammalian cells, and mice.
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DOI：
10.1038/s41467-020-19532-z
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发表时间：
2020-11-11
影响因子：
16.6
通讯作者：
Kanemaki MT
中科院分区：
文献类型：
--
作者：
Yesbolatova A;Saito Y;Kitamoto N;Makino-Itou H;Ajima R;Nakano R;Nakaoka H;Fukui K;Gamo K;Tominari Y;Takeuchi H;Saga Y;Hayashi KI;Kanemaki MT
使用生长素诱导降解决定子（AID）技术进行蛋白质敲除对于研究活细胞中的蛋白质功能很有用，因为它会诱导快速消耗，从而可以观察到立即的表型。然而，目前的AID系统有两个主要缺点：泄漏降解和需要高剂量的生长素。这些负面特征使得难以精确控制活细胞中感兴趣的蛋白质的表达水平，并且难以将该方法应用于小鼠。在这里，我们克服了这些问题，利用凹凸和孔的方法来建立AID版本2（AID 2）系统。AID 2采用OsTIR 1（F74 G）突变体和配体5-Ph-IAA，没有显示出可检测的泄漏降解，需要670倍低的配体浓度，并且实现了比常规AID更快的降解。我们证明了人类细胞突变体的基因，以前很难处理的成功一代，并表明艾滋病2实现快速目标消耗不仅在酵母和哺乳动物细胞，而且在小鼠。生长素诱导的降解决定子系统可能是泄漏的，需要高剂量的生长素。在这里，作者建立了AID 2，它使用OsTIR 1突变体和配体5-Ph-IAA来克服这些问题，并在小鼠中建立了AIDS介导的靶点消除。
Protein knockdown using the auxin-inducible degron (AID) technology is useful to study protein function in living cells because it induces rapid depletion, which makes it possible to observe an immediate phenotype. However, the current AID system has two major drawbacks: leaky degradation and the requirement for a high dose of auxin. These negative features make it difficult to control precisely the expression level of a protein of interest in living cells and to apply this method to mice. Here, we overcome these problems by taking advantage of a bump-and-hole approach to establish the AID version 2 (AID2) system. AID2, which employs an OsTIR1(F74G) mutant and a ligand, 5-Ph-IAA, shows no detectable leaky degradation, requires a 670-times lower ligand concentration, and achieves even quicker degradation than the conventional AID. We demonstrate successful generation of human cell mutants for genes that were previously difficult to deal with, and show that AID2 achieves rapid target depletion not only in yeast and mammalian cells, but also in mice. Auxin-inducible degron systems can be leaky and require high doses of auxin. Here the authors establish AID2 which uses an OsTIR1 mutant and the ligand 5-Ph-IAA to overcome these problems and establish AID-mediated target depletion in mice.
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影响因子：
10.5
作者：
Natsume T;Nishimura K;Minocherhomji S;Bhowmick R;Hickson ID;Kanemaki MT
通讯作者：
Kanemaki MT
影响因子：
14.9
作者：
Frankish A;Diekhans M;Ferreira AM;Johnson R;Jungreis I;Loveland J;Mudge JM;Sisu C;Wright J;Armstrong J;Barnes I;Berry A;Bignell A;Carbonell Sala S;Chrast J;Cunningham F;Di Domenico T;Donaldson S;Fiddes IT;García Girón C;Gonzalez JM;Grego T;Hardy M;Hourlier T;Hunt T;Izuogu OG;Lagarde J;Martin FJ;Martínez L;Mohanan S;Muir P;Navarro FCP;Parker A;Pei B;Pozo F;Ruffier M;Schmitt BM;Stapleton E;Suner MM;Sycheva I;Uszczynska-Ratajczak B;Xu J;Yates A;Zerbino D;Zhang Y;Aken B;Choudhary JS;Gerstein M;Guigó R;Hubbard TJP;Kellis M;Paten B;Reymond A;Tress ML;Flicek P
通讯作者：
Flicek P
影响因子：
4
作者：
Buckley DL;Raina K;Darricarrere N;Hines J;Gustafson JL;Smith IE;Miah AH;Harling JD;Crews CM
通讯作者：
Crews CM
影响因子：
14.8
作者：
Nabet B;Roberts JM;Buckley DL;Paulk J;Dastjerdi S;Yang A;Leggett AL;Erb MA;Lawlor MA;Souza A;Scott TG;Vittori S;Perry JA;Qi J;Winter GE;Wong KK;Gray NS;Bradner JE
通讯作者：
Bradner JE
DOI：
10.1073/pnas.1216880109
发表时间：
2012-12-04
影响因子：
11.1
作者：
Holland, Andrew J.;Fachinetti, Daniele;Cleveland, Don W.
通讯作者：
Cleveland, Don W.