A Dual-Function "TRE-Lox" System for Genetic Deletion or Reversible, Titratable, and Near-Complete Downregulation of Cathepsin D.
A Dual-Function "TRE-Lox" System for Genetic Deletion or Reversible, Titratable, and Near-Complete Downregulation of Cathepsin D.
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DOI：
10.3390/ijms24076745
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发表时间：
2023-04-04
影响因子：
5.6
通讯作者：
Leissring, Malcolm A. A.
中科院分区：
文献类型：
--
作者：
Terron, Heather M. M.;Maranan, Derek S. S.;Burgard, Luke A. A.;LaFerla, Frank M. M.;Lane, Shelley;Leissring, Malcolm A. A.
关键词：
用于可逆破坏基因表达的常用方法，例如基于RNAi或CRISPRi的那些方法，很少能够实现>80-90%的下调，使得它们不适合靶向需要更完全破坏以引发表型的基因。另一方面，基因缺失虽然能够完全破坏靶基因，但通常会产生不希望的不可逆后果，如细胞毒性或细胞死亡。在这里，我们描述了一个双功能的“TRE-Lox”系统的设计，开发和详细表征，用于实现（a）多西环素（Dox）介导的下调或（B）靶基因-溶酶体氨酰蛋白酶组织蛋白酶D（CatD）的遗传缺失-基于靶向插入四环素反应元件（TRE）和两个LoxP位点到内源性CatD基因（CTSD）的5 '端。使用一个优化的反向四环素反式阻遏物（rtTR）变体融合的Krüppel相关框（KRAB）结构域，我们表明，CatD的表达可以破坏多达98%的小鼠胚胎成纤维细胞（MEFs）。该系统对Dox高度敏感（IC 50 = 1.46 ng/mL），并导致CatD的快速（t1/2 = 0.57 d）和可滴定下调。值得注意的是，即使几乎完全破坏的CatD表达完全逆转的Dox撤出。如预期的，Cre重组酶的瞬时表达导致CTSD基因的完全缺失。这种新系统的双重功能将有助于未来研究CatD在各种疾病中的参与，特别是那些可归因于CatD功能部分丧失的疾病。此外，TRE-Lox方法应该适用于需要比传统方法更完全破坏的其他靶基因的调节。
Commonly employed methods for reversibly disrupting gene expression, such as those based on RNAi or CRISPRi, are rarely capable of achieving >80–90% downregulation, making them unsuitable for targeting genes that require more complete disruption to elicit a phenotype. Genetic deletion, on the other hand, while enabling complete disruption of target genes, often produces undesirable irreversible consequences such as cytotoxicity or cell death. Here we describe the design, development, and detailed characterization of a dual-function “TRE-Lox” system for effecting either (a) doxycycline (Dox)-mediated downregulation or (b) genetic deletion of a target gene—the lysosomal aspartyl protease cathepsin D (CatD)—based on targeted insertion of a tetracycline-response element (TRE) and two LoxP sites into the 5′ end of the endogenous CatD gene (CTSD). Using an optimized reverse-tetracycline transrepressor (rtTR) variant fused with the Krüppel-associated box (KRAB) domain, we show that CatD expression can be disrupted by as much as 98% in mouse embryonic fibroblasts (MEFs). This system is highly sensitive to Dox (IC50 = 1.46 ng/mL) and results in rapid (t1/2 = 0.57 d) and titratable downregulation of CatD. Notably, even near-total disruption of CatD expression was completely reversed by withdrawal of Dox. As expected, transient expression of Cre recombinase results in complete deletion of the CTSD gene. The dual functionality of this novel system will facilitate future studies of the involvement of CatD in various diseases, particularly those attributable to partial loss of CatD function. In addition, the TRE-Lox approach should be applicable to the regulation of other target genes requiring more complete disruption than can be achieved by traditional methods.
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