Identification of Mammalian Genes Promoting Life Extension

描述(由申请人提供)：在酵母、果蝇和线虫等无脊椎动物中，已经广泛证明了提高抗逆性和延长寿命之间的密切关系。一些长寿的小鼠突变体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色鼹鼠。抗逆性选择已被成功地用作在低等生物中鉴定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只在微生物系统中可用的方法学：使用抗逆性作为可选择的替代标记，在小鼠中产生长寿突变。我们已经开发出新的策略来允许对小鼠胚胎干细胞(ES)进行抗应激的突变和质量选择，并将这些策略与一种方法相结合，在这种方法中，这些ES细胞既保持抗压表型又保持多能性，从而使我们能够产生抗压的小鼠突变。这些新的小鼠模型使我们能够批判性地检验这一假说，即增强细胞抗应激能力可以减缓哺乳动物的衰老，并提高他们的寿命和健康寿命。这些都是有价值的模型，我们有许多合作者等待访问这些菌株；这份申请附上了来自五个实验室的信件。由于该程序不限于先前识别的基因和途径，它允许无偏见地检测影响小鼠多重应激抗性的新基因。在此应用程序中包含了几个改进 使我们能够同时检测显性和隐性突变，并快速识别突变基因(S)。为了实施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耐多重应激突变小鼠文库。我们筛选抗百草枯的突变ES细胞克隆，并筛选这些克隆的多重抗逆性、应激状态下的活性氧水平、多能性、Nrf2和其他抗氧化基因的表达水平，以及它们在分化为其他类型细胞后保持抗逆性的能力。携带抗逆性基因的等位基因很容易被识别和验证。一组抗应激的ES细胞克隆是 从而产生并将作为共享资源存放在公共单元储存库中。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。本课程将研究细胞抗应激能力增强对寿命、大体发育、一般行为、临终病理和年龄相关疾病的影响。重要的是，延长寿命的致病基因(S)也将得到验证；这些基因和途径是药物干预延缓衰老和改善人类多种衰老疾病的潜在靶点。总而言之，这些研究的成功将发现新的基因，这些基因与抗逆性有关，可能还会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促进衰老研究的各种新方向。

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.