Institute for Memory Impairments and Neurological Disorders, University of California, Irvine, Irvine, CA 92697, USA.
Department of Neurobiology and Behavior, University of California, Irvine, Irvine, CA 92697, USA.
Int J Mol Sci. 2023 Apr 4;24(7):6745. doi: 10.3390/ijms24076745.
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 (). 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 (IC = 1.46 ng/mL) and results in rapid (t = 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 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.
常用的可逆基因表达抑制方法，如基于 RNAi 或 CRISPRi 的方法，很少能够达到 >80-90%的下调水平，因此不适合针对需要更完全抑制才能引起表型的基因。另一方面，遗传缺失虽然能够完全抑制靶基因，但通常会产生不可逆转的不良后果，如细胞毒性或细胞死亡。在这里，我们描述了一种双功能“TRE-Lox”系统的设计、开发和详细特征，该系统可用于（a）强力霉素（Dox）介导的下调，或（b）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（）的 5'端，对目标基因天冬氨酸蛋白酶 CatD 进行基因删除。使用优化的反向四环素转录阻遏物（rtTR）变体融合 Krüppel 相关盒（KRAB）结构域，我们表明 CatD 表达可以在小鼠胚胎成纤维细胞（MEFs）中被抑制高达 98%。该系统对 Dox 高度敏感（IC = 1.46ng/mL），并导致 CatD 的快速（t = 0.57d）和可滴定下调。值得注意的是，即使是 CatD 表达的近乎完全抑制，也可以通过停止 Dox 处理来完全逆转。正如预期的那样，Cre 重组酶的瞬时表达会导致完全删除 基因。该新型系统的双重功能将有助于研究 CatD 在各种疾病中的作用，特别是那些归因于 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抑制的其他目标基因的调控。
