An episomal marking system for tracking cell proliferation

描述（由申请人提供）：用于跟踪发育、组织稳态和衰老中的细胞增殖的附加型标记系统。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衰老或受损的细胞来维持体内平衡状态。由于干细胞数量较少，它们需要在分化的每一步后扩增，以构成大的组织块。因此，成人组织中的细胞增殖通常可以用作干细胞活性的指标。人们普遍观察到，衰老伴随着干细胞活性的下降。例如，对小鼠模型的研究表明，老年小鼠的免疫反应与造血干细胞和淋巴祖细胞的扩增能力下降有关。到目前为止，可用于评估活体动物中由于干细胞活性引起的稳态增殖的方法仍然有限。我们建议建立一个附加体标记系统来跟踪小鼠细胞增殖。我们将使用Cre/lox重组系统来诱导和跟踪来自重组盒的切除环的组织和阶段特异性形成。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后代细胞。标记的附加体DNA不会复制，在每次细胞分裂后将被稀释。表达两种标志物的细胞与仅表达染色体标志物的细胞的比率提供了群体增殖历史的简单和定量的读数。我们计划建立携带重组盒的报告小鼠，并通过跟踪发育、稳态和衰老过程中的淋巴细胞和淋巴祖细胞增殖来测试报告系统的可行性。虽然拟议的研究重点是淋巴系统，但报告小鼠应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，用于跟踪小鼠模型中与衰老相关的生物学变化。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.