Biomedical Engineering Department, Stony Brook University; Laufer Center for Physical and Quantitative Biology, Stony Brook University; Stony Brook Medical Scientist Training Program;
Laufer Center for Physical and Quantitative Biology, Stony Brook University.
J Vis Exp. 2021 Jul 6(173). doi: 10.3791/62109.
Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, regardless of the method used. Toward this goal, optogenetic gene expression systems have gained much attention over the past decade for spatiotemporal control of protein levels in mammalian cells. However, most existing circuits controlling light-induced gene expression vary in architecture, are expressed from plasmids, and utilize variable optogenetic equipment, creating a need to explore characterization and standardization of optogenetic components in stable cell lines. Here, the study provides an experimental pipeline of reliable gene circuit construction, integration, and characterization for controlling light-inducible gene expression in mammalian cells, using a negative feedback optogenetic circuit as a case example. The protocols also illustrate how standardizing optogenetic equipment and light regimes can reliably reveal gene circuit features such as gene expression noise and protein expression magnitude. Lastly, this paper may be of use for laboratories unfamiliar with optogenetics who wish to adopt such technology. The pipeline described here should apply for other optogenetic circuits in mammalian cells, allowing for more reliable, detailed characterization and control of gene expression at the transcriptional, proteomic, and ultimately phenotypic level in mammalian cells.
为了可靠地控制哺乳动物细胞中的基因表达，无论采用何种方法，都需要具有高倍数变化、低噪声和确定的输入-输出传递函数的工具。为此，在过去的十年中，光遗传学基因表达系统因其可在哺乳动物细胞中对蛋白质水平进行时空控制而备受关注。然而，大多数现有的控制光诱导基因表达的电路在结构上有所不同，它们由质粒表达，并利用可变的光遗传学设备，这就需要探索在稳定细胞系中对光遗传学元件进行特征描述和标准化。在这里，该研究提供了一种可靠的基因电路构建、整合和特征描述的实验流程，用于控制哺乳动物细胞中的光诱导基因表达，使用负反馈光遗传学电路作为案例。该方案还说明了如何标准化光遗传学设备和光照条件，可以可靠地揭示基因电路的特征，如基因表达噪声和蛋白质表达幅度。最后，本文可能对不熟悉光遗传学但希望采用该技术的实验室有用。这里描述的流程应适用于哺乳动物细胞中的其他光遗传学电路，从而可以在转录组、蛋白质组学以及最终的表型水平上更可靠、更详细地对基因表达进行特征描述和控制。
