Zhang Zhiwen, Stickney Zachary, Duong Natalie, Curley Kevin, Lu Biao
Department of Bioengineering, Santa Clara University, 500 El Camino Real, Santa Clara, CA 95053 USA.
J Biol Eng. 2017 Jun 5;11:18. doi: 10.1186/s13036-017-0060-9. eCollection 2017.
High-throughput methods based on molecular reporters have greatly advanced our knowledge of cell signaling in mammalian cells. However, their ability to monitor various types of cells is markedly limited by the inefficiency of reporter gene delivery. Recombinant adeno-associated virus (AAV) vectors are efficient tools widely used for delivering and expressing transgenes in diverse animal cells in vitro and in vivo. Here we present the design, construction and validation of a novel AAV-based dual-reporter circuit that can be used to monitor and quantify cell signaling in living human cells.
We first design and construct the AAV-based reporter system. We then validate the versatility and specificity of this system in monitoring and quantifying two important cell signaling pathways, inflammation (NFκB) and cell growth and differentiation (AP-1), in cultured HEK293 and MCF-7 cells. Our results demonstrate that the AAV reporter system is both specific and versatile, and it can be used in two common experimental protocols including transfection with plasmid DNA and transduction with packaged viruses. Importantly, this system is efficient, with a high signal-to-background noise ratio, and can be easily adapted to monitor other common signaling pathways.
The AAV-based system extends the dual-reporter technology to more cell types, allowing for cost-effective and high throughput applications.
基于分子报告基因的高通量方法极大地推进了我们对哺乳动物细胞信号传导的认识。然而，报告基因递送效率低下显著限制了它们监测各类细胞的能力。重组腺相关病毒（AAV）载体是广泛用于在体外和体内多种动物细胞中递送和表达转基因的有效工具。在此，我们展示一种新型基于AAV的双报告基因电路的设计、构建及验证，该电路可用于监测和量化活的人类细胞中的细胞信号传导。
我们首先设计并构建基于AAV的报告系统。然后我们在培养的HEK293和MCF-7细胞中验证该系统在监测和量化两条重要细胞信号通路（炎症（NFκB）以及细胞生长与分化（AP-1））方面的通用性和特异性。我们的结果表明，AAV报告系统既具有特异性又具有通用性，并且可用于两种常见实验方案，包括用质粒DNA转染和用包装病毒转导。重要的是，该系统效率高，具有高信噪比，并且能够轻松适用于监测其他常见信号通路。
基于AAV的系统将双报告基因技术扩展到更多细胞类型，实现了经济高效的高通量应用。
