Cybulski Nadine, Zinzalla Vittoria, Hall Michael N
Biozentrum, University of Basel, Basel, Switzerland.
Methods Mol Biol. 2012;821:267-78. doi: 10.1007/978-1-61779-430-8_16.
The mammalian Target of Rapamycin (mTOR) kinase functions within two structurally and functionally distinct multiprotein complexes termed mTOR complex 1 (mTORC1) and mTORC2. The immunosuppressant and anticancer drug rapamycin is commonly used in basic research as a tool to study mTOR signaling. However, rapamycin inhibits only, and only incompletely, mTORC1, and no mTORC2-specific inhibitor is available. Hence, a full understanding of mTOR signaling in vivo, including the function of both complexes, requires genetic inhibition in addition to pharmacological inhibition. Taking advantage of the Cre/LoxP system, we generated inducible knockout mouse embryonic fibroblasts (MEFs) deficient for either the mTORC1-specific component raptor (iRapKO) or the mTORC2-specific component rictor (iRicKO). Inducibility of the knockout was important because mTOR complex components are essential. Induction of either raptor or rictor knockout eliminated raptor or rictor expression, respectively, and impaired the corresponding mTOR signaling branch. The described knockout MEFs are a valuable tool to study the full function of the two mTOR complexes individually.
哺乳动物雷帕霉素靶蛋白（mTOR）激酶在两种结构和功能不同的多蛋白复合物中发挥作用，这两种复合物分别称为mTOR复合物1（mTORC1）和mTOR复合物2（mTORC2）。免疫抑制剂和抗癌药物雷帕霉素在基础研究中常用作研究mTOR信号传导的工具。然而，雷帕霉素仅抑制mTORC1，且抑制不完全，目前尚无mTORC2特异性抑制剂。因此，要全面了解体内的mTOR信号传导，包括两种复合物的功能，除了药理学抑制外，还需要基因抑制。利用Cre/LoxP系统，我们构建了可诱导敲除mTORC1特异性组分雷帕霉素靶蛋白结合蛋白（raptor）的小鼠胚胎成纤维细胞（iRapKO）或mTORC2特异性组分rictor的小鼠胚胎成纤维细胞（iRicKO）。敲除的可诱导性很重要，因为mTOR复合物组分是必不可少的。诱导raptor或rictor敲除分别消除了raptor或rictor的表达，并损害了相应的mTOR信号传导分支。所描述的敲除小鼠胚胎成纤维细胞是单独研究两种mTOR复合物完整功能的宝贵工具。
