Nooti Sunil, Naylor Madison, Long Trevor, Groll Brayden
Department of Biology, University of North Dakota, Grand Forks, ND, United States of America.
PLoS One. 2023 Oct 4;18(10):e0292317. doi: 10.1371/journal.pone.0292317. eCollection 2023.
Reporter assays, in which the expression of an inert protein is driven by gene regulatory elements such as promoters and enhancers, are a workhorse for investigating gene regulation. Techniques for measuring reporter gene expression vary from single-cell or single-molecule approaches having low throughput to bulk Luciferase assays that have high throughput. We developed a Luciferase Reporter Assay using Flow-Cytometry (LucFlow), which measures reporter expression in single cells immunostained for Luciferase. We optimized and tested LucFlow with a murine cell line that can be differentiated into neutrophils, into which promoter-reporter and enhancer-promoter-reporter constructs have been integrated in a site-specific manner. The single-cell measurements are comparable to bulk ones but we found that dead cells have no detectable Luciferase protein, so that bulk assays underestimate reporter expression. LucFlow is able to achieve a higher accuracy than bulk methods by excluding dead cells during flow cytometry. Prior to fixation and staining, the samples are spiked with stained cells that can be discriminated during flow cytometry and control for tube-to-tube variation in experimental conditions. Computing fold change relative to control cells allows LucFlow to achieve a high level of precision. LucFlow, therefore, enables the accurate and precise measurement of reporter expression in a high throughput manner.
报告基因检测是研究基因调控的常用方法，在这种检测中，启动子和增强子等基因调控元件驱动惰性蛋白的表达。测量报告基因表达的技术多种多样，从通量较低的单细胞或单分子方法到通量较高的批量荧光素酶检测。我们开发了一种利用流式细胞术的荧光素酶报告基因检测方法（LucFlow），该方法可测量针对荧光素酶进行免疫染色的单细胞中的报告基因表达。我们用一种可分化为中性粒细胞的小鼠细胞系对LucFlow进行了优化和测试，该细胞系已通过位点特异性方式整合了启动子-报告基因和增强子-启动子-报告基因构建体。单细胞测量结果与批量测量结果具有可比性，但我们发现死细胞检测不到荧光素酶蛋白，因此批量检测会低估报告基因的表达。通过在流式细胞术中排除死细胞，LucFlow能够比批量方法获得更高的准确性。在固定和染色之前，将经过染色的细胞加入样本中，这些细胞在流式细胞术中可被区分出来，用于控制实验条件下管与管之间的差异。计算相对于对照细胞的倍数变化可使LucFlow达到高精度水平。因此，LucFlow能够以高通量方式准确、精确地测量报告基因的表达。
