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，它们会引发各种生物反应。这些都是基因指导的活性过程；因此，细胞如何响应以及基因和环境如何相互作用在疾病的发展中发挥着重要作用。该R21的目标是建立一种小鼠模型，以研究对各种环境压力的生物反应。设计和实用：这种设计允许研究人员追踪和研究收获时处于压力下的两个细胞 过去处于压力下的组织和细胞--在收获之前很久。这些小鼠将携带两个转基因等位基因。1.胁迫诱导的Cre*：编码一种重组酶Cre融合蛋白(简称Cre*)，其表达受一系列胁迫信号的调控；2.ROSA-Reporter：编码黄色荧光蛋白(YFP)，其前面是Lox-Stop-Lox(LSL)。YFP的生产被LSL阻止，并且只有在激活的CRE*移除停止的情况下才有可能。这一事件在物理上并因此永久地改变了DNA，导致YFP的生产(绿色)永久地“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，如果细胞处于活跃的应激状态(产生Cre*)，则可以标记为红色。有了适当的设计，人们可以区分过去曾经处于压力下的细胞和现在处于压力下的细胞。荧光颜色不仅允许细胞成像，还允许细胞分离(通过分选)用于生化和分子分析。目的1：建立并鉴定应激诱导的Cre*转基因小鼠。转基因构建物将通过BAC重组工程产生，并注射到原核中制造小鼠。然后，研究小鼠在急性和慢性应激模式下Cre*的表达和半衰期。目的2：制备并鉴定双转基因小鼠。应激诱导的Cre*小鼠将与YFP报告鼠(可用)杂交，产生双转基因小鼠。然后将对鼠标进行分析，以确定系统有多紧，以及活动的CRE*在移除止动装置方面的效率如何。创新和意义：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，例如那些导致肝脏损伤、心脏病发作、神经元退化和癌症发展的压力。此外，提出的小鼠模型允许研究人员解决从急性到慢性(或反复)损伤的过渡问题，这是一个重要但尚未被很好理解的问题。Cre*的广泛诱导性使该模型成为研究环境引起的疾病的极佳工具--NIEHS特别强调这一点。 公共卫生相关性： R21的目标是建立一种新的小鼠模型，以获得对环境诱导疾病的新理解。这种设计使研究人员能够追踪和研究在收获组织时处于压力下的细胞，以及在收获之前很久就处于压力下的细胞。该模型的成功建立将使我们能够追踪细胞在各种压力下的命运，例如那些导致肝损伤、心脏病发作、神经元退化和癌症发展的压力。

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. PUBLIC HEALTH RELEVANCE: The goal of this R21 is to establish a novel mouse model to obtain new understanding of environmentally- induced diseases. 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 successful establishment of the model will allow us to trace the fate of cells under a variety of stresses, such as those induce liver injury, heart attack, neuronal degeneration, and cancer development.