Abstract

Tools for tuning transcription in mammalian cells have broad applications, from basic biological discovery to human gene therapy. While precise control over target gene transcription via dosing with small molecules (drugs) is highly sought, the design of such inducible systems that meets required performance metrics poses a great challenge in mammalian cell synthetic biology. Important characteristics include tight and tunable gene expression with a low background, minimal drug toxicity, and orthogonality. Here, we review small-molecule-inducible transcriptional control devices that have demonstrated success in mammalian cells and mouse models. Most of these systems employ natural or designed ligand-binding protein domains to directly or indirectly communicate with transcription machinery at a target sequence, via carefully constructed fusions. Example fusions include those to transcription activator-like effectors (TALEs), DNA-targeting proteins (e.g. dCas systems) fused to transactivating domains, and recombinases. Similar to the architecture of Type I nuclear receptors, many of the systems are designed such that the transcriptional controller is excluded from the nucleus in the absence of an inducer. Techniques that use ligand-induced proteolysis and antibody-based chemically induced dimerizers are also described. Collectively, these transcriptional control devices take advantage of a variety of recently developed molecular biology tools and cell biology insights and represent both proof of concept (e.g. targeting reporter gene expression) and disease-targeting studies.

摘要

在哺乳动物细胞中调节转录的工具具有广泛的应用，从基本的生物学发现到人类基因治疗。同时通过对靶基因转录的精确控制小分子（药物）剂量受到高度追捧，但满足所需性能指标的此类诱导系统的设计对哺乳动物细胞合成生物学提出了巨大挑战。重要特征包括具有低背景、最小药物毒性和正交性的紧密和可调基因表达。在这里，我们回顾了在哺乳动物细胞和小鼠模型中取得成功的小分子诱导转录控制装置。这些系统中的大多数采用天然或设计的配体结合蛋白结构域直接或间接与靶序列的转录机制进行通信，通过精心构建的融合。示例融合包括与转录激活因子样效应子 (TALE)、与反式激活结构域融合的 DNA 靶向蛋白（例如 dCas 系统）和重组酶的融合。与 I 型核受体的结构相似，许多系统的设计使得在没有诱导剂的情况下，转录控制器被排除在细胞核之外。还描述了使用配体诱导的蛋白水解和基于抗体的化学诱导二聚体的技术。总的来说，这些转录控制装置利用了各种最近开发的分子生物学工具和细胞生物学见解，代表了概念证明（例如靶向报告基因表达）和疾病靶向研究。