A mouse model for genetic tracing to study stress responses

描述（由申请人提供）： 人类所处的环境往往含有毒素和应激因子，可诱发各种生物反应。这些都是基因导向的主动过程;因此，细胞如何反应以及基因和环境如何相互作用在疾病发展中起着重要作用。该R21的目标是建立小鼠模型来研究对各种环境压力的生物反应。设计和实用程序：设计是这样的，它将允许研究人员跟踪和研究在收获时处于压力下的细胞 这些组织和细胞在它们过去的压力下--在收获之前很久。小鼠将携带两个转基因等位基因。 1.应激诱导型Cre*：编码重组酶Cre融合体（简称Cre*），其表达由广谱胁迫信号开启，和2. ROSA报告基因：编码黄色荧光蛋白（YFP），前面是Lox-STOP-Lox（LSL）。YFP的产生被LSL阻止，只有当STOP被激活的Cre* 删除时才可能。这一事件在物理上永久改变了DNA，导致YFP的产生（绿色），永远“标记”细胞。这使得人们可以“追踪”细胞并确定它们的“命运”。Cre* 是荧光的（红色）;因此，如果细胞在压力下活跃（产生Cre*），则可以将其标记为红色。通过适当的设计，人们可以区分过去曾经处于压力下的细胞和目前处于压力下的细胞。荧光颜色不仅允许细胞成像，还允许细胞分离（通过分选）用于生物化学和分子分析。目的1：建立应激诱导的Cre* 转基因小鼠并鉴定其生物学特性。转基因构建体将使用BAC重组工程产生并注射到原核中以制备小鼠。然后将表征小鼠在急性和慢性应激范例下Cre* 的表达和半衰期。目的2：建立双转基因小鼠并鉴定其生物学特性。应激诱导型Cre* 小鼠将与YFP报告小鼠（可用）杂交以产生双转基因小鼠。然后分析小鼠以确定系统的紧密程度以及活性Cre* 在去除STOP方面的效率。创新与意义：小鼠模型是新颖的，因为它能够追踪细胞在各种压力下的命运，例如那些诱导肝损伤，心脏病发作，神经元变性和癌症发展的压力。此外，提出的小鼠模型使研究人员能够解决从急性到慢性（或重复）损伤的过渡问题，这是一个重要但尚未完全理解的问题。Cre* 的广泛诱导性使该模型成为研究环境引起的疾病的极好工具-NIEHS特别强调。

DESCRIPTION (provided by applicant): The environment to which humans are exposed often contains toxins and stress factors, which induce a variety of biological responses. These are gene-directed active processes; thus, how the cells respond and how genes and environments interact play an important role in disease development. The goal of this R21 is to establish a mouse model to study the biological responses to various environmental stresses. Designs and Utilities: The design is such that it will allow the researchers to trace and study both the cells under stress at the time of harvesting the tissues and the cells that were under stress in their past - long before harvesting. The mice will carry two transgenic alleles. 1. Stress-inducible Cre*: encodes a recombinase Cre fusion (Cre* for short), whose expression is turned on by a broad spectrum of stress signals, and 2. ROSA-Reporter: encodes Yellow Fluorescent Protein (YFP) preceded by Lox-STOP-Lox (LSL). The production of YFP is blocked by LSL and is possible only if the STOP is removed by active Cre*. This event physically and thus permanently alters the DNA, resulting in YFP production (green) to forever "mark" the cells. This allows one to "trace" the cells and determine their "fate. The Cre* is fluorescent (red); thus, the cells can be labeled red, if they are actively under stres (producing Cre*). With proper designs, one can distinguish cells that were once under stress in their past from those currently under stress. The fluorescent colors allow not only cell imaging but also cell isolation (by sorting) for biochemical and molecular analyses. Aim 1: To generate and characterize the stress-inducible Cre* transgenic mice. The transgenic construct will be generated using BAC recombineering and injected into pronucleus to make the mice. The mice will then be characterized for the expression and half-life of Cre* under acute and chronic stress paradigms. Aim 2: To generate and characterize the double transgenic mice. The stress-inducible Cre* mice will be crossed with the YFP reporter mice (available) to generate the double transgenic mice. The mice will then be analyzed to determine how tight the system is and how efficient the active Cre* is in removing the STOP. Innovation and Significance: The mouse model is novel for its ability to trace the fate of cells under various stresses, such as those induce liver injury, heart attack, neuronal degeneration, and cancer development. In addition, the proposed mouse model allows researchers to address issues regarding the transition from acute to chronic (or repeated) injuries¿an important but not well understood issue. The broad inducibility of Cre* makes the model an excellent tool to study environmentally-induced diseases - a special emphasis of NIEHS.