Department of Biochemistry, Purdue University, West Lafayette, Indiana, USA.
Department of Agriculture and Biological Engineering, Purdue University, West Lafayette, Indiana, USA.
Fly (Austin). 2021 Dec;15(1):73-88. doi: 10.1080/19336934.2021.1915683.
Binary expression systems are a powerful tool for tissue- and cell-specific research. Many of the currently available eye-specific drivers have not been systematically characterized for their expression level and cell-type specificity in the adult eye or during development. Here, we used a luciferase reporter to measure expression levels of different drivers in the adult eye, and characterized the cell type-specificity of each driver using a fluorescent reporter in live 10-day-old adult males. We also further characterized the expression pattern of these drivers in various developmental stages. We compared several Gal4 drivers from the Bloomington Stock Center (BDSC) including and with newly developed Gal4 and QF2 drivers that are specific to different cell types in the adult eye. In addition, we generated drug-inducible lines and compared their induced expression with an available line. Although both lines had significant induction of gene expression measured by luciferase activity, was expressed at levels below the detection of the fluorescent reporter by confocal microscopy, while showed substantial reporter expression in the absence of drug by microscopy. Overall, our study systematically characterizes and compares a large toolkit of eye- and photoreceptor-specific drivers, while also uncovering some of the limitations of currently available expression systems in the adult eye.
二元表达系统是组织和细胞特异性研究的有力工具。目前许多可用的眼部特异性驱动子尚未系统地对其在成年眼睛或发育过程中的表达水平和细胞类型特异性进行表征。在这里,我们使用荧光素酶报告基因来测量成年眼睛中不同驱动子的表达水平,并使用活体 10 天大的成年雄性中的荧光报告基因来表征每个驱动子的细胞类型特异性。我们还进一步表征了这些驱动子在不同发育阶段的表达模式。我们比较了几种来自 Bloomington Stock Center (BDSC) 的 Gal4 驱动子,包括 和 ,以及新开发的 Gal4 和 QF2 驱动子,它们针对成年眼睛中的不同细胞类型具有特异性。此外,我们还生成了可诱导的 线,并将其诱导表达与可用的 线进行了比较。尽管这两条线的基因表达诱导都通过荧光素酶活性进行了显著测量,但通过共聚焦显微镜, 的表达水平低于荧光报告基因的检测下限,而 在没有药物的情况下通过显微镜观察到大量报告基因的表达。总的来说,我们的研究系统地表征和比较了一大组眼部和光感受器特异性驱动子,同时也揭示了成年眼睛中一些现有表达系统的局限性。