Identification of Mammalian Genes Promoting Life Extension

描述（由申请人提供）：在无脊椎动物如酵母、果蝇和C.优雅一些长寿的突变小鼠也能抵抗多种形式的压力，包括氧化剂，紫外线和重金属。这种关系同样适用于特别长寿的裸鼹鼠。抗逆选择已被成功地用作鉴定低等生物中长寿突变体的替代标记。基于这些发现，我们开创了新的方法来应用通常仅在微生物系统中可用的方法：使用应激抗性作为选择性替代标记来在小鼠中产生长寿突变体。我们已经开发了新的策略以允许诱变和大量选择小鼠胚胎干（ES）细胞中的应激抗性，并且已经将这些策略与其中这些ES细胞保持应激抗性表型和多能性的方法结合，从而允许我们产生应激抗性的小鼠突变体。这些新的小鼠模型使我们能够批判性地测试这一假设，即细胞抗应激能力的增加减缓了哺乳动物的衰老并延长了寿命和健康寿命。这些都是有价值的模型，我们有许多合作者等待获得这些菌株;来自五个实验室的信件附在本申请中。由于该方法不限于先前鉴定的基因和途径，因此可以无偏地检测影响小鼠多重应激抗性的新基因。在本申请中结合了若干改进 使我们能够检测显性和隐性突变，并快速识别突变基因。为了实现这些新的高通量策略，我们构建了新的转座子载体，并将产生一个全基因组的多应激抗性突变小鼠文库。我们筛选对百草枯有抗性的突变ES细胞克隆，并筛选这些细胞的多重胁迫抗性、胁迫下活性氧的水平、多能性、Nrf 2和其他抗氧化基因的表达水平，以及它们在分化成其他细胞类型后保持胁迫抗性的能力。基因捕获的等位基因赋予抗逆性很容易识别和验证。一组抗应激ES细胞克隆是 因此产生并将作为共享资源存放在公共小区库中。我们将从这些细胞中产生小鼠突变株，用于小鼠衰老的体内研究。将检查细胞抗应激性增加对寿命、总体发育、一般行为、生命末期病理学和年龄相关疾病的影响。重要的是，寿命延长的致病基因也将得到验证;这些基因和途径是药物干预的潜在靶点，以减缓衰老并改善人类多种衰老疾病。总之，这些研究的成功将确定与抗应激有关的新基因，并可能调节哺乳动物的寿命和健康。我们预计，从这些研究中获得的见解将为老龄化研究提供各种新的方向。

DESCRIPTION (provided by applicant): The close relationship between increased resistance to stress and extended life span has been extensively demonstrated in invertebrates such as yeast, the fruit fly, and C. elegans. Several long-lived mouse mutants are also resistant to multiple forms of stress, including oxidants, UV, and heavy metals. This relationship holds equally true for the exceptionally long-lived naked mole rat. Selection for stress-resistance has been successfully employed as a surrogate marker to identify long-lived mutants in lower organisms. Based on these findings, we have pioneered novel approaches to apply methodology usually only available in microbial systems: using stress resistance as a selectable surrogate marker to generate long-lived mutants in the mouse. We have developed novel strategies to allow mutagenesis and mass selection for stress resistance in mouse embryonic stem (ES) cells and have coupled these to a method wherein these ES cells maintain both the stress resistant phenotype and pluripotency, thus allowing us to generate mouse mutants that are stress- resistant. These new mouse models allow us to critically test the hypothesis that increased cellular stress-resistance slows aging and improves life and health span in mammals. These are valuable models and we have numerous collaborators awaiting access to these strains; letters from five labs are attached to this application. Since the procedure is not limite to previously identified genes and pathways, it allows for unbiased detection of novel genes affecting multi-stress resistance in mice. Several refinements are incorporated in this application allowing us to detect both dominant and recessive mutations and to rapidly identify the mutated gene(s). To carry out these novel high-throughput strategies we have constructed novel transposon vectors and will generate a genome-wide library of multi-stress resistant mutant mice. We screen for mutant ES cell clones that are resistant to paraquat and we screen these for multi-stress resistance, levels of reactive oxygen species under stress, pluripotency, expression levels of Nrf2 and other antioxidant genes, and their capability of maintaining stress-resistance after differentiation into other cell types. The gene-trapped alleles conferring stress-resistance are easily identified and verified. A collection of stress- resistant ES cell clones are thus generated and will be deposited in the public cell repository as shared resources. We will generate strains of mouse mutants from these cells for in vivo studies of aging in the mouse. The impact of increased cellular stress-resistance on life span, gross development, general behavior, end-of-life pathology, and age-related diseases will be examined. Importantly, the causative gene(s) for life span extension will also be validated; these genes and pathways are potential targets for pharmacological intervention to slow aging and ameliorate multiple diseases of aging in humans. In summary, the success of these studies will identify new genes implicated in stress-resistance and perhaps modulating mammalian life span and health. We anticipate that the insights gained from these studies will foster a variety of new directions for aging research.