Stress-responsive gene expression and protein adaptation: analysis with CFX96 real-time PCR detection system

我的实验室研究的是生物化学适应，使动物能够在极端环境中生存。我们分析了惊人的自然现象，包括冬眠，耐寒性，耐寒性，休眠性和耐缺氧性。所有这些都是通过低代谢（过渡到迟钝或休眠状态）来实现在压力条件下的长期生存能力。我们的研究不仅确定了代谢调节的关键原理，这些原理是动物王国中低代谢和延长寿命的基础，而且它在生物医学中具有重要的潜在应用，因为诱导休眠的方法可以大大改善用于移植的人体器官的储存程序。我的实验室（目前有17名学生）的研究主要依赖于两种主要的实验方法——应激反应性基因表达的研究（mRNA转录水平的变化、mRNA翻译的microRNA调控、转录因子激活等）和酶适应的研究（动力学性质、结构稳定性、应激诱导的共价修饰等）。基于pcr的基因表达研究受到了严重的限制，因为我们无法进行定量(Q)-PCR（这是现在发表的分析标准），这需要实时荧光检测系统。本应用程序将通过添加我们的核心分析仪器CFX96实时PCR检测系统（Bio-Rad）来解决这个问题。该仪器将提供最先进的Q-PCR能力，并且非常重要的是，将为我们提供一种新的，高灵敏度的分析酶/蛋白质稳定性的方法：差示扫描荧光法（DSF），以检测使用荧光染料的蛋白质热展开。通过这种方法，我们可以研究一系列对酶稳定性的影响（效应剂、冷冻保护剂、共价修饰），通过微量蛋白质、非常精确的热控制和在单个96孔板中比较多种条件来比较耐受性和不耐受性物种。添加CFX96将真正给我们在分析能力上的定量飞跃，这将有力地帮助我们发现动物如何工作的分子机制的目标。

Research in my lab explores the biochemical adaptations that allow animals to survive in extreme environments. We analyze amazing natural phenomena including hibernation, freeze tolerance, cold hardiness, estivation and anoxia tolerance. All of these are linked by the use of hypometabolism (transitions to torpid or dormant states) to achieve long term viability under stressful conditions. Our research not only identifies key principles of metabolic regulation that underlie hypometabolism and life extension across the animal kingdom but it has important potential applications in biomedicine because methods for inducible dormancy could greatly improve procedures for storage of human organs removed for transplantation. Research by students in my lab (currently 17) relies heavily on two major experimental approaches - studies of stress-responsive gene expression (changes in mRNA transcript levels, microRNA regulation of mRNA translation, transcription factor activation, etc.) and studies of enzyme adaptation (kinetic properties, structural stability, stress-induced covalent modification, etc.). PCR-based gene expression studies have become critically limited by our inability to do quantitative (Q)-PCR (that is now the analytical standard for publication) that requires a real-time fluorescence detection system. The present application will solve this problem with the addition to our core analytical instrumentation of the CFX96 Real-Time PCR Detection System (Bio-Rad). This instrument will provide state-of-the-art Q-PCR capabilities and, very importantly, will give us a new, highly sensitive method for analyzing enzyme/protein stability: differential scanning fluorimetry (DSF) to detect thermal unfolding of proteins using fluorescent dyes. With this method we can study a range of influences on enzyme stability (effectors, cryoprotectants, covalent modification) comparing stress tolerant vs intolerant species with micro-amounts of proteins, very precise thermal control and the ability to compare multiple conditions in a single 96 well plate. Addition of the CFX96 will truly give us a quantitative leap in analytical capacity that will strongly aid our goals of discovering the molecular mechanisms of how animals work.