Department of Oral Biology and Diagnostic Sciences, Dental College of Georgia, Augusta University, Augusta, GA, USA.
Georgia Cancer Center, Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta University, Augusta, GA, USA.
Methods Mol Biol. 2020;2138:159-166. doi: 10.1007/978-1-0716-0471-7_8.
Transgene-based reporter gene assays have been used for discovery of inhibitors targeting vital gene transcription. In traditional assays, the reporter gene is commonly fused with a cloned promoter and integrated into a random genomic location. This has been widely applied but significantly dampened by disadvantages, including incomplete cis-acting elements, the influence of foreign epigenetic environments, and generation of false hits that disrupt the luciferase reporter activity. Therefore, there is a need to develop novel strategies for developing in situ reporter assays closely mimicking endogenous gene expression without disrupting its function. By employing the CRISPR-Cas9 system, we developed an effective in situ coincidence reporter system with a selection marker in the endogenous locus of ATAD3A, which provides a means of screening for transcription-targeted lead compounds with high confidence.
基于转基因的报告基因检测已被用于发现针对重要基因转录的抑制剂。在传统的检测中，报告基因通常与克隆的启动子融合，并整合到随机的基因组位置。这种方法已经得到了广泛的应用，但也存在一些缺点，包括不完全的顺式作用元件、外源表观遗传环境的影响以及产生破坏荧光素酶报告基因活性的假阳性。因此，需要开发新的策略来开发与内源性基因表达紧密模拟的原位报告基因检测，而不干扰其功能。我们利用 CRISPR-Cas9 系统，在 ATAD3A 的内源性基因座上开发了一种有效的原位符合报告系统，该系统带有一个选择标记，为筛选具有高置信度的转录靶向先导化合物提供了一种手段。
