PDF(Pubmed)
Abstract：
The Hypoxia Inducible Factor (HIF) transcription factors are imperative for cell adaption to low oxygen conditions and development; however, they also contribute to ischaemic disease and cancer. To identify novel genetic regulators which target the HIF pathway or small molecules for therapeutic use, cell-based reporter systems are commonly used. Here, we present a new, highly sensitive and versatile reporter system, NanoFIRE: a NanoLuciferase and Fluorescent Integrated Reporter Element. Under the control of a Hypoxic Response Element (HRE-NanoFIRE), this system is a robust sensor of HIF activity within cells and potently responds to both hypoxia and chemical inducers of the HIF pathway in a highly reproducible and sensitive manner, consistently achieving 20 to 150-fold induction across different cell types and a Z\' score > 0.5. We demonstrate that the NanoFIRE system is adaptable via substitution of the response element controlling NanoLuciferase and show that it can report on the activity of the transcriptional regulator Factor Inhibiting HIF, and an unrelated transcription factor, the Progesterone Receptor. Furthermore, the lentivirus-mediated stable integration of NanoFIRE highlights the versatility of this system across a wide range of cell types, including primary cells. Together, these findings demonstrate that NanoFIRE is a robust reporter system for the investigation of HIF and other transcription factor-mediated signalling pathways in cells, with applications in high throughput screening for the identification of novel small molecule and genetic regulators.
摘要：
缺氧诱导因子（HIF）转录因子对于细胞适应低氧条件和发育至关重要；然而，它们也有助于缺血性疾病和癌症。为了鉴定靶向治疗用途的HIF途径或小分子的新型遗传调节因子，通常使用基于细胞的报告系统。这里,我们提出了一个新的，高度敏感和通用的记者系统，NanoFIRE:纳米荧光素酶和荧光整合报告元件。在低氧反应元件（HRE-NanoFIRE）的控制下，该系统是细胞内HIF活性的强大传感器，并以高度可重复和灵敏的方式有效响应HIF途径的缺氧和化学诱导剂，在不同细胞类型中始终实现20至150倍的诱导，并且Z得分>0.5。我们证明了NanoFIRE系统通过取代控制Nano荧光素酶的反应元件是适应性的，并表明它可以报告转录调节因子抑制HIF的活性，和一个无关的转录因子，孕激素受体.此外，慢病毒介导的NanoFIRE稳定整合突出了该系统在多种细胞类型中的多功能性，包括原代细胞。一起,这些发现表明，NanoFIRE是一个强大的报告系统，用于研究细胞中HIF和其他转录因子介导的信号通路，应用于高通量筛选，以鉴定新型小分子和遗传调节因子。
