An episomal marking system for tracking cell proliferation

描述(由申请人提供)：一种用于跟踪发育中的细胞增殖、组织动态平衡和衰老的附体标记系统。摘要大多数成人组织通过用来自成人组织干细胞的年轻细胞替换老化或受损的细胞来维持体内平衡状态。由于干细胞的数量很少，它们需要在每一步分化后扩张，才能构成一个大的组织块。因此，成人组织中的细胞增殖通常可以作为干细胞活性的指标。人们普遍观察到，衰老伴随着干细胞活性的下降。例如，在小鼠模型中的研究表明，衰老小鼠的免疫反应不良与造血干细胞和淋巴祖细胞的扩张能力下降有关。到目前为止，可用来评估活体动物体内干细胞活性所致的动态平衡增殖的方法仍然有限。我们建议建立一种用于跟踪小鼠细胞增殖的上体标记系统。我们将使用Cre/lox重组系统来诱导和跟踪组织，并从重组盒中分阶段形成特定的切除环。由Cre重组酶启动的重组事件将导致两个可见标记的同时激活，一个在染色体上，另一个在切除环上。染色体标记将标记所有的后代细胞。标记的不复制的附体DNA将在每次细胞分裂后被稀释。表达两种标记的细胞与只表达染色体标记的细胞的比率提供了一个简单而定量的群体增殖史读数。我们计划建立携带重组盒的报告小鼠，并通过跟踪淋巴细胞和淋巴祖细胞在发育、稳态和衰老过程中的增殖来测试报告系统的可行性。虽然拟议的研究重点放在淋巴系统上，但报告小鼠应该普遍适用于研究大多数其他组织类型的发育和衰老。 公共卫生相关性：拟议的研究将建立一种新的方法来跟踪小鼠模型中与衰老有关的生物学变化。该实验系统可用于揭示和研究影响衰老的遗传和环境因素。

DESCRIPTION (provided by applicant): An episomal marking system for tracking cell proliferation in development, tissue homeostasis, and aging. Abstract Most adult tissues are maintained in a homeostatic state through replacement of aged or damaged cells with young cells derived from adult tissue stem cells. Because stem cells are present in small numbers, they need to expand following each step of differentiation in order to constitute a large tissue mass. Therefore cell proliferation in adult tissues can often be used as an indicator of stem cell activity. It has been generally observed that aging is accompanied with a decline in stem cell activity. For example, studies in mouse models showed that the ailing immune response in aged mice is correlated with a decline in expansion capacity of hematopoietic stem cells and the lymphoid progenitor cells. Thus far, the methods available to assess homeostatic proliferation due to stem cell activity in live animals are still limited. We propose to establish an episomal marking system for tracking cell proliferation in mice. We will use a Cre/lox recombination system to induce and track tissue and stage specific formation of excision circles from a recombination cassette. The recombination event, initiated by Cre recombinase, will lead to simultaneous activation of two visible markers, one on the chromosome and the other on the excision circle. The chromosomal marker will label all the descendant cells. The marked episomal DNA, which does not replicate, will be diluted out following each cell division. The ratio of cells expressing both markers vs. cells expressing only the chromosomal marker provides a simple and quantitative readout of the proliferative history of the population. We plan to establish reporter mice carrying the recombination cassette and test the feasibility of the reporter system by tracking lymphocyte and lymphoid progenitor proliferation during development, homeostasis, and aging. While the proposed study focuses on the lymphoid system, the reporter mice should be generally applicable for studying development and aging in most other tissue types. PUBLIC HEALTH RELEVANCE: The proposed study will establish a new method for tracking ageing related biological changes in mouse models. The experimental system can be used in revealing and investigating genetic and environmental factors that influence ageing.