Genome-Edited Cell Lines for High-Throughput Screening.
Genome-Edited Cell Lines for High-Throughput Screening.
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用于高通量筛选的基因组编辑细胞系。
DOI：
10.1007/978-1-4939-7724-6_1
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发表时间：
2018
期刊：
影响因子：
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通讯作者：
Svaren,John
中科院分区：
文献类型：
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作者：
Dranchak,Patricia;Moran,JohnJ;MacArthur,Ryan;Lopez-Anido,Camila;Inglese,James;Svaren,John
用于高通量筛选的基因表达测量是一种越来越多使用的技术，其不仅已开发用于由疾病相关拷贝数变异引起的基因剂量紊乱，而且还用于诱导/抑制调节疾病表型严重程度的基因。传统方法采用瞬时或稳定转染报告构建体，其中单个报告由来自候选基因的选定调控元件驱动。然而，单个调控元件固有地不能捕获内源基因座处的多个增强子的整合调控，并且随机报告子插入可导致影响报告子的生理响应性的邻近效应。因此，我们概述了一种采用基因组编辑将报告基因插入候选基因的3′ UTR的一般方法，该方法已成功用于我们对与Charcot-Marie-Tooth病相关的Pmp 22基因的研究。该方法采用基因组编辑来插入两个非同源报告基因，其通过小分子筛选中的一致响应来最大化生物活性分子的鉴定效率。我们包括这些报告基因检测的设计和构建的许多方面，这些方面将适用于在各种细胞类型中创建类似的检测。
Measurement of gene expression for high-throughput screening is an increasingly used technique that has been developed for not only gene dosage disorders resulting from disease-associated copy number variations, but also for induction/repression of genes modulating the severity of a disease phenotype. Traditional methods have employed transient or stable transfection of reporter constructs in which a single reporter is driven by selected regulatory elements from the candidate gene. However, individual regulatory elements are inherently unable to capture the integrated regulation of multiple enhancers at the endogenous locus, and random reporter insertion can result in neighborhood effects that impact the physiological responsiveness of the reporter. Therefore, we outline a general method of employing genome editing to insert reporters into the 3′ UTR of a candidate gene, which has been used successfully in our studies of thePmp22gene associated with Charcot–Marie–Tooth disease. The method employs genome editing to insert two nonhomologous reporters that maximize the efficiency of identification of biologically active molecules through concordant responses in small molecule screening. We include a number of aspects of the design and construction of these reporter assays that will be applicable to creation of similar assays in a variety of cell types.