Identification of Mammalian Genes Promoting Life Extension

描述（由申请人提供）：在酵母、果蝇和秀丽隐杆线虫等无脊椎动物中，已经广泛证明了增强的抗应激能力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异常长寿的裸鼹鼠。选择抗逆性已成功地用作替代标记，以识别在低等生物的长寿命突变体。基于这些发现，我们开创了新的方法来应用通常只适用于微生物系统的方法：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体。我们已经开发了新的策略，允许诱变和大量选择小鼠胚胎干细胞（ES）细胞的抗逆性，并将这些策略与一种方法结合起来，使这些胚胎干细胞保持抗逆性表型和多能性，从而使我们能够产生具有抗逆性的小鼠突变体。这些新的小鼠模型使我们能够严格地测试这样的假设，即增加细胞抗压力能力可以减缓哺乳动物的衰老，延长寿命和健康寿命。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；五个实验室的信函附在本申请中。由于该程序不局限于先前确定的基因和途径，因此可以无偏地检测影响小鼠多重应激抗性的新基因。在这个应用程序中包含了几个改进

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.